http://2008.igem.org/wiki/index.php?title=Special:Contributions/Magni&feed=atom&limit=50&target=Magni&year=&month=2008.igem.org - User contributions [en]2024-03-29T09:46:52ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/Team:UNIPV-PaviaTeam:UNIPV-Pavia2009-10-21T13:35:32Z<p>Magni: </p>
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|[[Image:pv_logo_synthbiol.png|500px|center|Synthetic Biology]]<br />
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<div align="center" style="font-size:30px;">University of Pavia IGEM 2008 Team</div><br />
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{|align="center"<br />
|[[Image:unipv_logo.jpg|120px|center|University of Pavia]]<br />
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<div align="justify" style="padding:30px;"><br />
Here in Pavia, a collaboration between bioengineers and biotechnologists gave life to a research consortium, called CIT, dedicated to tissue engineering. We are a small subgroup of this consortium, who were interested in synthetic biology: during this year, thanks to IGEM08 call, we have had the chance to work in this field for the first time and then to introduce this study perspective into our University. <br />
Our team is composed by Lorenzo Pasotti, master student in Bioengineering, and Mattia Quattrocelli, master student in Molecular Biology. We are advised by Daniela Galli, post-doc in Tissue Engineering, and instructed by Paolo Magni, professor of Bioinformatics, and Maria Gabriella Cusella, professor of Anatomy.<br />
<br><br />
We are mixing engineering and biology skills in order to build up original devices, learn new abilities and have a lot of fun during IGEM08 Competition!<br />
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''We are interested in genetic implementation of logic circuits: our goal is to provide a Boolean logic-based processing in biosensors, especially in multi-input ones. This year we are building up genetic networks that mimic a logic circuit function: in particular we are trying to implement Multiplexer and Demultiplexer functions in E. coli.''<br />
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<div style="padding:30px;"><h1>Our sponsors</h1><br />
{|align="center"<br />
|-<br />
|[[Image:logoDIS.gif|80px|left|DIS]]<br />
|[http://dis.unipv.it DIS - Dipartimento di Informatica e Sistemistica (Department of Computer Science and Systems)]<br />
|-<br />
|-<br />
|[[Image:cit_logo.jpg|80px|left|DIS]]<br />
|[http://cit.unipv.it/cit CIT - Centro di Ingegneria Tissutale (Center for Tissue Engineering)]<br />
|-<br />
|}<br />
</div></div>Magnihttp://2008.igem.org/Team:UNIPV-PaviaTeam:UNIPV-Pavia2009-10-21T13:33:16Z<p>Magni: Undo revision 106449 by Lor18 (Talk)</p>
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{|align="center" border="0" width="60%" align="center"<br />
|[[Image:pv_logo_synthbiol.png|500px|center|Synthetic Biology]]<br />
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{|align="center" border="1" width="60%" align="center"<br />
|bgcolor="#eeeeee" height="450px" align="center"|[[Image:bridge.jpg|480px|center|Pavia]]''Covered Bridge & Cathedral - Pavia''<br />
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<div align="center" style="font-size:30px;">University of Pavia IGEM 2008 Team</div><br />
<br><br />
{|align="center"<br />
|[[Image:unipv_logo.jpg|120px|center|University of Pavia]]<br />
|}<br />
<div align="justify" style="padding:30px;"><br />
Here in Pavia, a collaboration between bioengineers and biotechnologists gave life to a research consortium, called CIT, dedicated to tissue engineering. We are a small subgroup of this consortium, who were interested in synthetic biology: during this year, thanks to IGEM08 call, we have had the chance to work in this field for the first time and then to introduce this study perspective into our University. <br />
Our team is composed by Lorenzo Pasotti, master student in Bioengineering, and Mattia Quattrocelli, master student in Molecular Biology. We are advised by Daniela Galli, post-doc in Tissue Engineering, and instructed by Paolo Magni, professor of Bioinformatics, and Maria Gabriella Cusella, professor of Anatomy.<br />
<br><br />
We are mixing engineering and biology skills in order to build up original devices, learn new abilities and have a lot of fun during IGEM08 Competition!<br />
</div><br />
<br />
[[Image:ddd.jpg]]<br />
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''We are interested in genetic implementation of logic circuits: our goal is to provide a Boolean logic-based processing in biosensors, especially in multi-input ones. This year we are building up genetic networks that mimic a logic circuit function: in particular we are trying to implement Multiplexer and Demultiplexer functions in E. coli.''<br />
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|}<br />
<br />
<div style="padding:30px;"><h1>Our sponsors</h1><br />
{|align="center"<br />
|-<br />
|[[Image:logoDIS.gif|80px|left|DIS]]<br />
|[http://dis.unipv.it DIS - Dipartimento di Informatica e Sistemistica (Department of Computer Science and Systems)]<br />
|-<br />
|-<br />
|[[Image:cit_logo.jpg|80px|left|DIS]]<br />
|[http://cit.unipv.it/cit CIT - Centro di Ingegneria Tissutale (Center for Tissue Engineering)]<br />
|-<br />
|}<br />
</div></div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/ModelingTeam:UNIPV-Pavia/Modeling2008-10-27T15:57:26Z<p>Magni: /* Demux */</p>
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<br />
= '''Mathematical modeling page''' =<br />
In this section we explain two dynamic models that can be used to describe the gene networks in our project. After a brief overview about the motivation of a mathematical model, we will illustrate the general formulas we used, we will show the complete ODE models for Mux and Demux gene networks, and then we will report results of some simulations performed with Matlab and Simulink.<br />
<br><br />
<br><br />
<br />
== '''Why writing a mathematical model?''' ==<br />
The purposes of writing mathematical models for gene networks can be:<br />
*'''Prediction''': a good and well identificated model can be used in simulations to predict real system behavior. In particular we could be interested in system output in response to never seen inputs. In this way, the system can be tested 'in silico', without performing real experiments 'in vitro' or 'in vivo'.<br />
*'''Parameter identification''': we already wrote that it is very important to estimate all the parameters involved in the model, in order to perform realistic simulations. Another goal that can be reached with parameter identification is 'network summarization', in fact estimated parameters can be used as 'behavior indexes' for the network (or a part of it). These indexes can be very useful for synthetic biologists to choose and compare BioBrick standard parts for genetic circuits design, just like electronic engineers choose, for example, a Zener diode, knowing its Zener voltage.<br />
<br><br />
<br><br />
<br />
== '''Equations for gene networks''' ==<br />
In this paragraph, mathematical modeling for gene and protein interactions will be described.<br />
<br><br />
<br />
=== '''Binding of a ligand to a molecule: Hill equation''' ===<br />
The fraction of a molecule saturated by a ligand ( = the probability that the molecule is bound to a ligand) can be expressed as a function of ligand concentration using Hill equation:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_hill.jpg|200px|left]]<br />
|}<br />
*'''Prob:''' the probability that the molecule is bound to the ligand<br />
*'''[L]:''' concentration of the ligand<br />
*'''K50:''' dissociation constant. It is the concentration producing a probability of 0.5<br />
*'''n:''' Hill coefficient. It describes binding cooperativity<br />
<br><br />
<br />
=== '''Regulated transcription''' ===<br />
Transcription of a mRNA molecule can be regulated by transcription factors in active form. These factors can be activators or inhibitors: they respectively increase and decrease the probability that RNA polymerase binds the promoter.<br />
This probability must be function of active transcription factor concentration and can be modeled using Hill equation.<br />
If factor in active form activates transcription, we can write:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_acttranscr.jpg|200px|left]]<br />
|}<br />
*'''Prob:''' probability that the gene is transcripted<br />
*'''[T]:''' concentration of the transcription activator in active form<br />
*'''K50:''' dissociation constant. It is the concentration producing a probability of 0.5<br />
*'''n:''' Hill coefficient (n>0)<br />
<br />
while, if factor in active form inhibits transcription, we are interested in unbound promoter and so we can write:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_inhtranscr.jpg|400px|left]]<br />
|}<br />
*'''Prob:''' probability that the gene is transcripted<br />
*'''[T]:''' concentration of the transcription inhibitor in active form<br />
*'''K50:''' dissociation constant. It is the concentration producing a probability of 0.5<br />
*'''n:''' Hill coefficient (n>0)<br />
<br />
The equations show that:<br />
*In activation formula, if [T]=0 the trascription probability is Prob=0, while the maximum probability, Prob=1, is reached asymptotically for [T]->Inf<br />
*In inhibition formula, if [T]=0 the trascription probability is Prob=1, while the minimum probability, Prob=0, is reached asymptotically for [T]->Inf<br />
<br />
These formulas can be included in a differential equation that can describe the dynamic behavior of a mRNA molecule as a function of the factor that regulates its transcription.<br />
<br><br />
<br />
=== ODE for activated transcription ===<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_ode_acttranscr.jpg|400px|left]]<br />
|}<br />
*'''Vmax:''' maximum transcription rate<br />
*'''[m]:''' concentration of mRNA molecule<br />
*'''[T]:''' concentration of the transcription activator in active form<br />
*'''K50:''' dissociation constant<br />
*'''n:''' Hill coefficient (n>0)<br />
*'''delta:''' mRNA degradation constant<br />
*'''a:''' leakage factor that can modellize promoter basic activity. It is a percentage of Vmax<br />
<br><br />
<br />
=== ODE for inhibited transcription ===<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_ode_inhtranscr.jpg|400px|left]]<br />
|}<br />
*'''Vmax:''' maximum transcription rate<br />
*'''[m]:''' concentration of mRNA molecule<br />
*'''[T]:''' concentration of the transcription inhibitor in active form<br />
*'''K50:''' dissociation constant<br />
*'''n:''' Hill coefficient (n>0)<br />
*'''delta:''' mRNA degradation rate<br />
*'''a:''' leakage factor that can modellize promoter basic activity. It is a percentage of Vmax<br />
<br><br />
<br />
=== ODE for constitutive transcription ===<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_odecontranscr.jpg|180px|left]]<br />
|}<br />
*'''c:''' constant transcription rate<br />
*'''[m]:''' concentration of mRNA molecule<br />
*'''delta:''' mRNA degradation rate<br />
<br><br />
<br />
=== '''Transcription factors in active form''' ===<br />
Formulas described so far are dependent on the concentration of transcription factor in active form. But how can we calculate the amount of transcription factor in active form?<br />
Assuming that the transcription factor can be activated or inhibited by a ''inducer'' factor (that can be an exogenous input), we can use Hill equation again to calculate the concentration of bound and unbound transcription factor as a function of the inducer factor:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_inducers.jpg|490px|left]]<br />
|}<br />
*'''[I]:''' concentration of the inducer<br />
*'''[T]t:''' total concentration of the transcription factor<br />
*'''[T]u:''' concentration of unbound transcription factor<br />
*'''[T]b:''' concentration of bound transcription factor<br />
*'''K50:''' dissociation constant<br />
*'''n:''' Hill coefficient (n>0)<br />
<br />
If active form is bound transcripion factor, we are interested in Tb, while if active form is unbound transcription factor, we are interested in Tu.<br />
<br><br />
<br />
=== '''ODE for protein production''' ===<br />
We have to modellize translation of mRNA molecules. We chose to describe it as a differential equation in which protein production and protein degradation are linear:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_protprod.jpg|200px|left]]<br />
|}<br />
*'''[P]:''' concentration of protein<br />
*'''[m]:''' concentration of mRNA molecule<br />
*'''alpha:''' translation rate<br />
*'''beta''' protein degradation rate<br />
<br><br />
<br />
== '''Our model''' ==<br />
We considered Tetracycline, IPTG, Arabinose and GFP respectively as CH0, CH1, SEL and OUT signals for Mux, while we considered IPTG, Tetracycline, RFP and GFP respectively as IN, SEL, OUT0 and OUT1 signals for Demux. For all these signals, logic 1 corresponds to the presence of the molecule.<br />
<br><br />
According to the equations described so far, every protein and mRNA is a state variable for the model. Mux and Demux models are described in the next paragraphs.<br />
<br><br />
We considered full ODE models, in which transcription and translation processes are separated. In many contexts, anyway, we can write only one differential equation to describe a "regulated protein production", in which trascription process is hidden. In this section we preferred to be general and we considered the transcription dynamic because our aim is to provide a mathematical model for future parameter identification. We also decided to neglect AHL synthesis dynamic, considering luxI protein a "direct" activator for luxR.<br />
<br><br />
In the following paragraphs, the complete ODE systems and the Simulink block diagrams will be reported.<br />
=== '''Mux'''===<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_simulink.jpg|200%|left]]<br />
|}<br />
<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_ODEmux.jpg|200%|left]]<br />
|}<br />
=== '''Demux'''===<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_demux_simulink.jpg|200%|left]]<br />
|}<br />
<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_ODEdemux.jpg|200%|left]]<br />
|}<br />
<br><br />
<br />
== '''Simulations''' ==<br />
The written models have been simulated in Matlab and Simulink, in order to perform a preliminary heuristic study of the system behavior and to search for critical parameters. For simulations, we used dimensionless ODEs to avoid numeric errors due to small parameter values, so results are reported in arbitrary units. We defined ODE systems in Simulink (block diagrams showed above) and then solved them using ode15s, a multistep ODE solver for stiff problems.<br />
<br><br />
Parameters have been calibrated considering the values reported in literature and in some other iGEM wiki pages. In some cases values were missing, so we fixed these missing values at 1*10^om, where "om" is the (median) order of magnitude of the parameters having the same meaning (e.g. protein degradation rates).<br />
In other cases we found different values for the same parameter, so we computed the mean value.<br />
<br><br />
An important feature that will have to be studied is systems' basic activity. In other words, can genetic Mux discriminate the output in response to a low input signal and the output in response to a high input signal? Moreover, does Demux have a non negligible baseline activity that turns on the output channel that should be unactive? One of the parameters that plays a very important role in basic activities is the leakage factor ("a", that is the probability that a mRNA is transcribed randomly). This parameter depends on the specific promoter. Future estimation of "a" for all the promoters will be useful to predict "false positive" system outputs. High "a" values give high basic activities, while for a->0 transcriptional regulation becomes "ideal".<br />
<br><br />
At first, we wrote the ODE models with only one "a" parameter, but then we decided to consider different leakage factors for the promoters in the network.<br />
<br><br />
Here we report some simulation examples. The initial states (t=0) have been fixed to 0, but here we are interested into steady state values.<br />
=== '''Mux'''===<br />
We distinguished lux and las system contributions for output GFP.<br />
<br />
'''SIMULATION 1'''<br />
*Tetracycline = 10<br />
*IPTG = 0<br />
*Arabinose = 0<br />
**Tetracycline channel (CH0) is selected and Tetracycline is present, so we expect to find a high GFP(lux) value and a low GFP(las) value.<br />
<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_sim1.png|thumb|600px|left|Mux SIMULATION 1 - tetR, lacI, cI and GFPs]]<br />
|}<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_sim1bis.png|thumb|600px|left|Mux SIMULATION 1 - luxI, luxR, lasI and lasR]]<br />
|}<br />
<br />
*Comments: GFP(las) in low, but not null, because of leakage factor. Posing "a"=0, GFP(las) becomes null. Mux output is the sum of GFP(lux) and GFP(las). If we sum these values at steady state, we have 870.2330 (arbitrary units).<br />
<br />
'''SIMULATION 2'''<br />
*Tetracycline = 10<br />
*IPTG = 0<br />
*Arabinose = 10<br />
**IPTG channel (CH1) is selected, but IPTG is not present, so we expect to find low steady state GFP values for both lux and las systems.<br />
<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_sim2.png|thumb|600px|left|Mux SIMULATION 2 - tetR, lacI, cI and GFPs]]<br />
|}<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_sim2bis.png|thumb|600px|left|Mux SIMULATION 2 - luxI, luxR, lasI and lasR]]<br />
|}<br />
<br />
*Comments: total GFP is low after a transient time. If we sum GFP(lux) and GFP(las) values, we have 14.2292 (arbitrary units), that is much lower than the output total value in SIMULATION 1, confirming that the chosen parameter set gives low leakage.<br />
<br />
=== '''Demux'''===<br />
<br><br />
'''SIMULATION 1'''<br />
*Tetracycline = 0<br />
*IPTG = 0<br />
**IPTG signal (INPUT) is absent, so we expect to find a low RFP and GFP values.<br />
<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_demux_sim1.png|thumb|600px|left|Demux SIMULATION 1 - tetR, lacI, cI, GFP and RFP]]<br />
|}<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_demux_sim1bis.png|thumb|600px|left|Demux SIMULATION 1 - luxI, luxR, lasI and lasR]]<br />
|}<br />
<br />
*Comments: RFP and GFP values are not null, because of leakage. The numeric steady state values are 0.869 for GFP and 2.8753 for RFP, in arbitrary units.<br />
<br />
'''SIMULATION 2'''<br />
*Tetracycline = 0<br />
*IPTG = 10<br />
**IPTG signal (INPUT) is present and Tetracycline signal is not. So, INPUT is transferred into RFP channel (OUT1).<br />
<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_demux_sim2.png|thumb|600px|left|Demux SIMULATION 2 - tetR, lacI, cI, GFP and RFP]]<br />
|}<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_demux_sim2bis.png|thumb|600px|left|Demux SIMULATION 2 - luxI, luxR, lasI and lasR]]<br />
|}<br />
<br />
*Comments: GFP steady state value (0.8709 arbitrary units) is comparable to GFP value in SIMULATION 1. This value is much lower that RFP steady state value (362.11 arbitrary units), confirming that the selected output channel has a significantly higher output value.<br />
<br />
All these simulations will have to be supported with experimental results and parameters will have to be identified in order to characterize our physical devices.<br />
<br />
== '''References''' ==<br />
[1] ''Modelli multiscala per studi di proteomica e genomica funzionale'', S. Cavalcanti, S. Furini, E. Giordano. "Genomica e proteomica computazionale", GNB Book, Publisher: Pàtron, 2007<br />
<br />
[2] ETH Zurich iGEM 2007 Wiki - Modeling basics page</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/ModelingTeam:UNIPV-Pavia/Modeling2008-10-27T15:32:29Z<p>Magni: /* Binding of a ligand to a molecule: Hill equation */</p>
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= '''Mathematical modeling page''' =<br />
In this section we explain two dynamic models that can be used to describe the gene networks in our project. After a brief overview about the motivation of a mathematical model, we will illustrate the general formulas we used, we will show the complete ODE models for Mux and Demux gene networks, and then we will report results of some simulations performed with Matlab and Simulink.<br />
<br><br />
<br><br />
<br />
== '''Why writing a mathematical model?''' ==<br />
The purposes of writing mathematical models for gene networks can be:<br />
*'''Prediction''': a good and well identificated model can be used in simulations to predict real system behavior. In particular we could be interested in system output in response to never seen inputs. In this way, the system can be tested 'in silico', without performing real experiments 'in vitro' or 'in vivo'.<br />
*'''Parameter identification''': we already wrote that it is very important to estimate all the parameters involved in the model, in order to perform realistic simulations. Another goal that can be reached with parameter identification is 'network summarization', in fact estimated parameters can be used as 'behavior indexes' for the network (or a part of it). These indexes can be very useful for synthetic biologists to choose and compare BioBrick standard parts for genetic circuits design, just like electronic engineers choose, for example, a Zener diode, knowing its Zener voltage.<br />
<br><br />
<br><br />
<br />
== '''Equations for gene networks''' ==<br />
In this paragraph, mathematical modeling for gene and protein interactions will be described.<br />
<br><br />
<br />
=== '''Binding of a ligand to a molecule: Hill equation''' ===<br />
The fraction of a molecule saturated by a ligand ( = the probability that the molecule is bound to a ligand) can be expressed as a function of ligand concentration using Hill equation:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_hill.jpg|200px|left]]<br />
|}<br />
*'''Prob:''' the probability that the molecule is bound to the ligand<br />
*'''[L]:''' concentration of the ligand<br />
*'''K50:''' dissociation constant. It is the concentration producing a probability of 0.5<br />
*'''n:''' Hill coefficient. It describes binding cooperativity<br />
<br><br />
<br />
=== '''Regulated transcription''' ===<br />
Transcription of a mRNA molecule can be regulated by transcription factors in active form. These factors can be activators or inhibitors: they respectively increase and decrease the probability that RNA polymerase binds the promoter.<br />
This probability must be function of active transcription factor concentration and can be modeled using Hill equation.<br />
If factor in active form activates transcription, we can write:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_acttranscr.jpg|200px|left]]<br />
|}<br />
*'''Prob:''' probability that the gene is transcripted<br />
*'''[T]:''' concentration of the transcription activator in active form<br />
*'''K50:''' dissociation constant. It is the concentration producing a probability of 0.5<br />
*'''n:''' Hill coefficient (n>0)<br />
<br />
while, if factor in active form inhibits transcription, we are interested in unbound promoter and so we can write:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_inhtranscr.jpg|400px|left]]<br />
|}<br />
*'''Prob:''' probability that the gene is transcripted<br />
*'''[T]:''' concentration of the transcription inhibitor in active form<br />
*'''K50:''' dissociation constant. It is the concentration producing a probability of 0.5<br />
*'''n:''' Hill coefficient (n>0)<br />
<br />
The equations show that:<br />
*In activation formula, if [T]=0 the trascription probability is Prob=0, while the maximum probability, Prob=1, is reached asymptotically for [T]->Inf<br />
*In inhibition formula, if [T]=0 the trascription probability is Prob=1, while the minimum probability, Prob=0, is reached asymptotically for [T]->Inf<br />
<br />
These formulas can be included in a differential equation that can describe the dynamic behavior of a mRNA molecule as a function of the factor that regulates its transcription.<br />
<br><br />
<br />
=== ODE for activated transcription ===<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_ode_acttranscr.jpg|400px|left]]<br />
|}<br />
*'''Vmax:''' maximum transcription rate<br />
*'''[m]:''' concentration of mRNA molecule<br />
*'''[T]:''' concentration of the transcription activator in active form<br />
*'''K50:''' dissociation constant<br />
*'''n:''' Hill coefficient (n>0)<br />
*'''delta:''' mRNA degradation constant<br />
*'''a:''' leakage factor that can modellize promoter basic activity. It is a percentage of Vmax<br />
<br><br />
<br />
=== ODE for inhibited transcription ===<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_ode_inhtranscr.jpg|400px|left]]<br />
|}<br />
*'''Vmax:''' maximum transcription rate<br />
*'''[m]:''' concentration of mRNA molecule<br />
*'''[T]:''' concentration of the transcription inhibitor in active form<br />
*'''K50:''' dissociation constant<br />
*'''n:''' Hill coefficient (n>0)<br />
*'''delta:''' mRNA degradation rate<br />
*'''a:''' leakage factor that can modellize promoter basic activity. It is a percentage of Vmax<br />
<br><br />
<br />
=== ODE for constitutive transcription ===<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_odecontranscr.jpg|180px|left]]<br />
|}<br />
*'''c:''' constant transcription rate<br />
*'''[m]:''' concentration of mRNA molecule<br />
*'''delta:''' mRNA degradation rate<br />
<br><br />
<br />
=== '''Transcription factors in active form''' ===<br />
Formulas described so far are dependent on the concentration of transcription factor in active form. But how can we calculate the amount of transcription factor in active form?<br />
Assuming that the transcription factor can be activated or inhibited by a ''inducer'' factor (that can be an exogenous input), we can use Hill equation again to calculate the concentration of bound and unbound transcription factor as a function of the inducer factor:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_inducers.jpg|490px|left]]<br />
|}<br />
*'''[I]:''' concentration of the inducer<br />
*'''[Tt]:''' total concentration of the transcription factor<br />
*'''[Tu]:''' concentration of unbound transcription factor<br />
*'''[Tb]:''' concentration of bound transcription factor<br />
*'''K50:''' dissociation constant<br />
*'''n:''' Hill coefficient (n>0)<br />
<br />
If active form is bound transcripion factor, we are interested in Tb, while if active form is unbound transcription factor, we are interested in Tu.<br />
<br><br />
<br />
=== '''ODE for protein production''' ===<br />
We have to modellize translation of mRNA molecules. We chose to describe it as a differential equation in which protein production and protein degradation are linear:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_protprod.jpg|200px|left]]<br />
|}<br />
*'''[P]:''' concentration of protein<br />
*'''[m]:''' concentration of mRNA molecule<br />
*'''alpha:''' translation rate<br />
*'''beta''' protein degradation rate<br />
<br><br />
<br />
== '''Our model''' ==<br />
We considered Tetracycline, IPTG, Arabinose and GFP respectively as CH0, CH1, SEL and OUT signals for Mux, while we considered IPTG, Tetracycline, RFP and GFP respectively as IN, SEL, OUT0 and OUT1 signals for Demux. For all these signals, logic 1 corresponds to the presence of the molecule.<br />
<br><br />
According to the equations described so far, every protein and mRNA is a state variable for the model. Mux and Demux models are described in the next paragraphs.<br />
<br><br />
We considered full ODE models, in which transcription and translation processes are separated. In many contexts, anyway, we can write only one differential equation to describe a "regulated protein production", in which trascription process is hidden. In this section we preferred to be general and we considered the transcription dynamic because our aim is to provide a mathematical model for future parameter identification. We also decided to neglect AHL synthesis dynamic, considering luxI protein a "direct" activator for luxR.<br />
<br><br />
In the following paragraphs, the complete ODE systems and the Simulink block diagrams will be reported.<br />
=== '''Mux'''===<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_simulink.jpg|200%|left]]<br />
|}<br />
<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_ODEmux.jpg|200%|left]]<br />
|}<br />
=== '''Demux'''===<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_demux_simulink.jpg|200%|left]]<br />
|}<br />
<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_ODEdemux.jpg|200%|left]]<br />
|}<br />
<br><br />
<br />
== '''Simulations''' ==<br />
The written models have been simulated in Matlab and Simulink, in order to perform a preliminary heuristic study of the system behavior and to search for critical parameters. For simulations, we used dimensionless ODEs to avoid numeric errors due to small parameter values, so results are reported in arbitrary units. We defined ODE systems in Simulink (block diagrams showed above) and then solved them using ode15s, a multistep ODE solver for stiff problems.<br />
<br><br />
Parameters have been calibrated considering the values reported in literature and in some other iGEM wiki pages. In some cases values were missing, so we fixed these missing values at 1*10^om, where "om" is the median order of magnitude of the parameters having the same meaning (e.g. protein degradation rates).<br />
In other cases we found different values for the same parameter, so we computed the mean value.<br />
<br><br />
An important feature that will have to be studied is systems' basic activity. In other words, can genetic Mux discriminate the output in response to a low input signal and the output in response to a high input signal? Moreover, does Demux have a non negligible baseline activity that turns on the output channel that should be unactive? One of the parameters that plays a very important role in basic activities is the leakage factor ("a", that is the probability that a mRNA is transcribed randomly). This parameter depends on the specific promoter. Future estimation of "a" for all the promoters will be useful to predict "false positive" system outputs. High "a" values give high basic activities, while for a->0 transcriptional regulation becomes "ideal".<br />
<br><br />
At first, we wrote the ODE models with only one "a" parameter, but then we decided to consider different leakage factors for the promoters in the network.<br />
<br><br />
Here we report some simulation examples. The initial states (t=0) have been fixed to 0, but here we are interested into steady state values.<br />
=== '''Mux'''===<br />
We distinguished lux and las system contributions for output GFP.<br />
<br />
'''SIMULATION 1'''<br />
*Tetracycline = 10<br />
*IPTG = 0<br />
*Arabinose = 0<br />
**Tetracycline channel (CH0) is selected and Tetracycline is present, so we expect to find a high GFP(lux) value and a low GFP(las) value.<br />
<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_sim1.png|thumb|600px|left|Mux SIMULATION 1 - tetR, lacI, cI and GFPs]]<br />
|}<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_sim1bis.png|thumb|600px|left|Mux SIMULATION 1 - luxI, luxR, lasI and lasR]]<br />
|}<br />
<br />
*Comments: GFP(las) in low, but not null, because of leakage factor. Posing "a"=0, GFP(las) becomes null. Mux output is the sum of GFP(lux) and GFP(las). If we sum these values at steady state, we have 870.2330 (arbitrary units).<br />
<br />
'''SIMULATION 2'''<br />
*Tetracycline = 10<br />
*IPTG = 0<br />
*Arabinose = 10<br />
**IPTG channel (CH1) is selected, but IPTG is not present, so we expect to find low steady state GFP values for both lux and las systems.<br />
<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_sim2.png|thumb|600px|left|Mux SIMULATION 2 - tetR, lacI, cI and GFPs]]<br />
|}<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_sim2bis.png|thumb|600px|left|Mux SIMULATION 2 - luxI, luxR, lasI and lasR]]<br />
|}<br />
<br />
*Comments: total GFP is low after a transient time. If we sum GFP(lux) and GFP(las) values, we have 14.2292 (arbitrary units), that is much lower than the output total value in SIMULATION 1, confirming that the chosen parameter set gives low leakage.<br />
<br />
=== '''Demux'''===<br />
<br><br />
'''SIMULATION 1'''<br />
*Tetracycline = 0<br />
*IPTG = 0<br />
**IPTG signal (INPUT) is absent, so we expect to find a low RFP and GFP values.<br />
<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_demux_sim1.png|thumb|600px|left|Demux SIMULATION 1 - tetR, lacI, cI, GFP and RFP]]<br />
|}<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_demux_sim1bis.png|thumb|600px|left|Demux SIMULATION 1 - luxI, luxR, lasI and lasR]]<br />
|}<br />
<br />
*Comments: RFP and GFP values are not null, because of leakage. The numeric steady state values are 0.869 for GFP and 2.8753 for RFP, in arbitrary units.<br />
<br />
'''SIMULATION 2'''<br />
*Tetracycline = 0<br />
*IPTG = 10<br />
**IPTG signal (INPUT) is present and Tetracycline signal is not. So, INPUT is transferred into RFP channel (OUT1).<br />
<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_demux_sim2.png|thumb|600px|left|Demux SIMULATION 2 - tetR, lacI, cI, GFP and RFP]]<br />
|}<br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_demux_sim2bis.png|thumb|600px|left|Demux SIMULATION 2 - luxI, luxR, lasI and lasR]]<br />
|}<br />
<br />
*Comments: GFP steady state value (0.8709 arbitrary units) is comparable to GFP value in SIMULATION 1. This value is much lower that RFP steady state value (362.11 arbitrary units), confirming that the selected output channel have a significantly higher output value.<br />
<br />
All these simulations will have to be supported with experimental results and parameters will have to be identified in order to characterize our physical devices.<br />
<br />
== '''References''' ==<br />
[1] ''Modelli multiscala per studi di proteomica e genomica funzionale'', S. Cavalcanti, S. Furini, E. Giordano. "Genomica e proteomica computazionale", GNB Book, Publisher: Pàtron, 2007<br />
<br />
[2] ETH Zurich iGEM 2007 Wiki - Modeling basics page</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/ProjectTeam:UNIPV-Pavia/Project2008-10-27T15:28:27Z<p>Magni: /* Functional tests */</p>
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<br />
<br><br />
<br />
== '''Overall project''' ==<br />
<br />
We are trying to mimic Multiplexer (Mux) and Demultiplexer (Demux) logic functions in E. coli.<br />
<br><br />
In the following paragraphs project details will be described from both digital electronic and genetic points of view.<br />
<br><br />
<br><br />
<br />
== '''Electronic Implementation''' ==<br />
<br><br />
=== '''What kind of components are Mux and Demux?''' ===<br />
'''Mux''' is a component which conveys one of the two input channels values into a single output channel. The choice of the input channel is made by a selector.<br />
<br><br />
'''Demux''' is a component which conveys the only input channel value into one of the two output channels. The choice of the output channel is made by a selector.<br />
<br><br />
<br><br />
The following pictures show data flow in Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_dataflow0.png|thumb|300px|left|Data flow in Multiplexer - SELECTOR=0]]<br />
|[[Image:pv_mux_dataflow1.png|thumb|300px|left|Data flow in Multiplexer - SELECTOR=1]]<br />
|-<br />
|[[Image:pv_demux_dataflow0.png|thumb|300px|left|Data flow in Demultiplexer - SELECTOR=0]]<br />
|[[Image:pv_demux_dataflow1.png|thumb|300px|left|Data flow in Demultiplexer - SELECTOR=1]]<br />
|}<br />
<br><br />
<br />
=== '''What kind of signals do we process?''' ===<br />
In this project we consider Boolean logic signals, thus every input/output value can assume only the values 0 and 1. A function that processes Boolean values is called logic function.<br />
<br><br />
Mux and Demux can be considered by now as black boxes which implement a logic function that can process input signals to output signals. Here you can see examples of Boolean data flow in Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_bool.png|thumb|300px|left|Example: Mux Boolean data flow]]<br />
|[[Image:pv_demux_bool.png|thumb|300px|left|Example: Demux Boolean data flow]]<br />
|}<br />
In the following documentation we will see what is inside these black boxes.<br />
<br><br />
<br><br />
=== How can we formalize Mux and Demux logic behavior? ===<br />
Logic functions can be formalized writing a truth table; a truth table is a mathematical table in which every row represents a combination of input values and its respective output values. The table has to be filled with every input combination.<br />
<br><br />
<br><br />
Here you can see Mux and Demux truth tables (output columns are gray):<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_truth.png|thumb|300px|left|Mux truth table]]<br />
|[[Image:pv_demux_truth.png|thumb|300px|left|Demux truth table]]<br />
|}<br />
<br><br />
<br />
=== Building a logic circuit from a truth table ===<br />
Our goal in this section is to project two logic gates networks which behave like Mux and Demux truth tables. A very useful tool to transform a truth table into a logic network is Karnaugh map.<br />
<br><br />
It is possible to read about Karnaugh maps at: [http://en.wikipedia.org/wiki/Karnaugh_map]<br />
<br><br />
<br><br />
Following Karnaugh maps method, we can write these two logic networks for Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux.png|thumb|340px|left|Mux - logic circuit]]<br />
|[[Image:pv_mux_example.png|thumb|340px|left|Mux - Example]]<br />
|-<br />
|[[Image:pv_demux.png|thumb|340px|left|Demux - logic circuit]]<br />
|[[Image:pv_demux_example.png|thumb|340px|left|Demux - Example]]<br />
|}<br />
<br />
<br><br />
<br />
== '''Genetic Implementation''' ==<br />
Our goal is to mimic Mux and Demux logic networks in a biological device, such as E. coli. To perform this, we use protein/DNA and protein/protein interactions to build up biological logic gates.<br />
Mux and Demux logic circuits are composed by three fundamental logic gates, AND, OR, NOT: in the next paragraphs genetic implementation of these logic gates will be provided.<br />
<br><br />
<br><br />
=== AND ===<br />
To mimic an AND gate, we need a biological function, such as a promoter activation, which is directly turned on by the interaction between two upstream genes. In our synthetic devices, we use the luxR/luxI system: luxR can activate Plux promoter only upon 3-oxo-hexanoyl-homoserine lactone (HSL) binding; luxI generates HSL; so, only the contemporary expression of LuxR and luxI proteins can activate the downstream Plux-dependent gene expression. Another AND gate we use is the lasR/lasI system, which works in a very similar way but through another chemical intermediate, N-(3-oxododecanoyl) homoserine lactone (PAI-1).<br />
{|<br />
|[[Image:pv_proj_AND.png|thumb|340px|left|Genetic AND: Plux can be turned on only when the two proteins luxI and luxR are present.]]<br />
|}<br />
=== OR ===<br />
To mimic an OR gate in Mux, we need a biological function which can be activated alternatively by two independent upstream signals or by both. Thus, we combine the outputs of the upstream AND gates to assemble directly an OR reporter function, by simply repeating the reporter gene (GFP) under two different promoters (Plux and Plas). It’s sufficient to activate one of the two promoters (or both) to recover the GFP signal from engineered bacteria.<br />
There should not be an over-expression problem for GFP, in fact, in Mux device, only one promoter can be active, either Plux or Plas. Here we considered GFP output, but OR device can be generalized for every output gene.<br />
{|<br />
|[[Image:pv_proj_OR.png|thumb|340px|left|Genetic OR: it is sufficient that one of the two input promoters is active to obtain GFP expression.]]<br />
|}<br />
=== NOT ===<br />
To mimic a NOT gate, we need an efficient and regulated repressor of a specific downstream promoter: in this case, we choose cI repression on Plambda, which should be specific and, upon cI inactivation, quick and efficient.<br />
{|<br />
|[[Image:pv_proj_NOT.png|thumb|340px|left|Genetic NOT: Plambda can be turned on only when cI protein is not present.]]<br />
|}<br />
<br />
According to what above stated, genetic implementation of Mux and Demux can be obtained connecting these basic logic gates and can be summarized in this way:<br />
<br><br />
<br><br />
<br><br />
<br />
=== Genetic Mux ===<br />
Let PA, PB and PS be three generic promoters that can be ACTIVATED respectively by the three exogenous molecules "A", "B" and "S". A genetic Mux with inputs "A" (CH0) and "B" (CH1), selector "S" and the generic protein GOI as output, can be implemented by the following gene network:<br />
{|align="center"<br />
|[[Image:pv_MUX.png|thumb|420px|left|Genetic Mux]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_MUXint.png|thumb|420px|left|Genetic Mux - interactions]]<br />
|}<br />
We want to supply a device that can be generalized to detect every kind of input and to express every kind of genes as output. So, input promoters and output protein generators are not part of the genetic Mux we want to build up. In this way, a hypothetical user can re-use our device assembling the desired input and output elements.<br />
<br><br />
Note that the output genes are two, but, as explained in "OR" section, if the genes are identical, we can say that there is only one output; in fact, the real output is a protein synthesis and so it is not important which of the two identical genes is expressed.<br />
<br><br />
However, it is possible to assemble two different genes downstream of Plux and Plas, for example two reporters. In this way, debugging process becomes easy, because we can discriminate lux system and las system activities.<br />
<br><br />
Here we report the truth table of this network:<br />
{|align="center"<br />
|[[Image:pv_gmux_truth.png|thumb|420px|left|Genetic Mux - truth table]]<br />
|}<br />
<br><br />
Now we report two examples of genetic Mux behavior, in response to generic inputs.<br />
<br><br />
<br><br />
====Genetic Mux behavior - Example 1====<br />
{|<br />
|[[Image:pv_genmux_example1.png|thumb|420px|left|First example of genetic Mux behavior]]<br />
|}<br />
According to Mux truth table, if "A" is not present and "B" and "S" are present, we expect to have GOI synthesis.<br />
<br><br />
"A" molecule is not present, so PA promoter is not active and luxI gene is not expressed.<br />
<br><br />
"B" molecule is present, so PB promoter is active and lasI gene is expressed.<br />
<br><br />
"S" molecule is present, so PS promoter is active and lasR and cI genes are expressed.<br />
<br><br />
cI protein represses transcription of the gene downstream of Plambda promoter, which is luxR.<br />
<br><br />
lasI protein can synthesize 3-OC12-HSL. This molecule activates transcription factor lasR, which can activate Plas promoter. So, the copy of GOI gene under Plas regulation can be expressed.<br />
<br><br />
The other copy of GOI gene, which is under Plux promoter regulation, is not expressed. In fact Plux is not active, because both luxI and luxR proteins are not present.<br />
<br><br />
Only one of the two GOI genes is expressed, but this is sufficient to synthesize the output protein GOI.<br />
<br><br />
<br><br />
====Genetic Mux behavior - Example 2====<br />
{|<br />
|[[Image:pv_genmux_example2.png|thumb|420px|left|Second example of genetic Mux behavior]]<br />
|}<br />
We expect to have no GOI synthesis in response to this input combination.<br />
<br><br />
"A" molecule is not present, so PA promoter is not active and luxI gene is not expressed.<br />
<br><br />
"B" molecule is present, so PB promoter is active and lasI gene is expressed.<br />
<br><br />
"S" molecule is not present, so PS promoter can't transcribe cI and lasR genes.<br />
<br><br />
cI protein is not present, so Plambda promoter can transcribe luxR gene.<br />
<br><br />
None of the two logic AND systems is active, because lux system lacks of luxI and las system lacks of lasR. So, Plux and Plas promoters are unactive and can't express GOI genes. For this reason, GOI protein is not synthesized.<br />
<br><br />
<br><br />
These two examples show how "S" molecule can select the input to be conveyed into the single output channel: in fact its presence allows lasR expression and represses luxR expression, while "S" absence represses lasR expression and allows luxR expression.<br />
<br><br />
<br><br />
<br />
=== Genetic Demux ===<br />
Let PI and PS be two generic promoters that can be ACTIVATED respectively by the two exogenous molecules "I" and "S". A genetic Demux with input "I", selector "S" and the generic proteins GOI0 and GOI1 as outputs (OUT0 and OUT1 respectively), can be implemented by the following gene network:<br />
{|align="center"<br />
|[[Image:pv_DEMUX.png|thumb|420px|left|Genetic Demux]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_DEMUXint.png|thumb|420px|left|Genetic Demux - interactions]]<br />
|}<br />
As described for genetic Mux, we want to supply a device that can be generalized to detect every kind of inputs and to express every kind of genes as output. So, input promoters and output protein generators are not part of the genetic Demux. In this way, a hypothetical user can re-use our device assembling the desired input and output elements.<br />
<br><br />
Here we report the truth table of this network:<br />
{|align="center"<br />
|[[Image:pv_gdemux_truth.png|thumb|420px|left|Genetic Demux - truth table]]<br />
|}<br />
<br><br />
Now we report two examples of genetic Demux behavior, in response to generic inputs.<br />
<br><br />
<br><br />
====Genetic Demux behavior - Example 1====<br />
{|<br />
|[[Image:pv_gendemux_example1.png|thumb|420px|left|First example of genetic Demux behavior]]<br />
|}<br />
According to Demux truth table, if "I" molecule is present and "S" molecule is absent, we expect to have GOI0 protein synthesis and no GOI1 protein synthesis.<br />
<br><br />
"I" is present, so PI promoter is active and luxI and lasI genes are expressed.<br />
<br><br />
"S" is not present, so PS promoter can't transcribe lasR and cI genes.<br />
<br><br />
cI protein is not present, so Plambda promoter is not inhibited and luxR gene is expressed.<br />
<br><br />
luxI protein can synthesize 3-OC6-HSL lactone. This molecule activates luxR transcription factor which can activate Plux promoter. In this way, GOI0 gene, which is under Plux regulation, is expressed.<br />
<br><br />
On the other hand, lasI protein can synthesize 3-OC12-HSL, but lasR transcription factor is not present and so Plas promoter cannot be activated. In this way, GOI1 gene, which is under Plas regulation, is not expressed.<br />
<br><br />
So, we have GOI0 protein synthesis and no GOI1 protein synthesis.<br />
<br><br />
<br><br />
====Genetic Demux behavior - Example 2====<br />
{|<br />
|[[Image:pv_gendemux_example2.png|thumb|420px|left|Second example of genetic Demux behavior]]<br />
|}<br />
We expect to have GOI1 synthesis and no GOI0 synthesis in response to this input combination.<br />
<br><br />
"I" is present, so PI promoter is active and luxI and lasI genes are expressed.<br />
<br><br />
"S" is present, so PS promoter lasR and cI genes are expressed.<br />
<br><br />
cI protein is present, so Plambda promoter is inhibited and luxR gene is not expressed.<br />
<br><br />
lasI protein can synthesize 3-OC12-HSL lactone. This molecule activates lasR transcription factor which can activate Plas promoter. In this way, GOI1 gene, which is under Plas regulation, is expressed.<br />
<br><br />
On the other hand, luxI protein can synthesize 3-OC6-HSL, but luxR transcription factor is not present and so Plux promoter cannot be activated. In this way, GOI0 gene, which is under Plux regulation, is not expressed.<br />
<br><br />
So, we have GOI1 protein synthesis and no GOI0 protein synthesis.<br />
<br />
<br />
=== A complete genetic Mux===<br />
In this section a complete Mux gene network is described.<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_implementation.png|thumb|420px|left|Example of a complete genetic Mux]]<br />
|[[Image:pv_mux_gn_implementation.png|thumb|420px|left|Example of a complete genetic Mux - Gene network]]<br />
|}<br />
We want to build up a device in which Channel 0, Channel 1 and Selector are respectively sensitive to Tetracycline, IPTG and red light. The presence of each input corresponds to logic 1.<br />
We chose green fluorescence as Mux output: expression of GFP corresponds to logic 1, while absence of fluorescence corresponds to logic 0.<br />
<br><br />
Tetracycline and IPTG can be considered as "A" and "B" molecules, introduced in "Genetic Mux" section, because both Tetracycline and IPTG are indirect ACTIVATORS of Ptet and Plac promoters respectively.<br />
On the other hand, red light is quite different from "S" molecule, because red light is an indirect REPRESSOR of Pomp promoter and not an activator as required by the original schema. For this reason, if we want a device in which the presence of Tetracycline, IPTG and red light correspond to logic 1, red light input should be ''inverted''. The simplest way to do this, is to cross-exchange the two input channels. So, Tetracycline sensor (CH0) has to be assembled to las system and IPTG sensor (CH1) has to be assembled to lux system.<br />
<br />
<br />
====Complete genetic Mux - Example====<br />
{|<br />
|[[Image:pv_mux_example1.png|thumb|420px|left|Example of a complete genetic Mux behavior]]<br />
|}<br />
According to Mux truth table, if SEL=0, CH0=1, CH1=0, output channel must be logic 1.<br />
Red light is not present, so it can't dephosphorylate cph8-ho1-pcyA complex, which is constitutively expressed. cph8-ho1-pcyA complex activates endogenous ompR, which can activate Pomp promoter, so cI and lasR genes are transcribed.<br />
cI protein binds Plambda promoter and so luxR expression is inhibited.<br />
TetR is a repressor for Ptet promoter, but Tetracycline is present, so it can bind tetR protein, which is constitutively expressed, and can activate transcription of downstream gene: lasI.<br />
Simultaneous expression of lasI and lasR can activate GFP, which is under Plas promoter.<br />
IPTG is not present, so lacI protein binds Plac promoter and represses luxI transcription.<br />
<br />
<br />
=== A complete genetic Demux===<br />
In this section a complete Demux gene network is described.<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_demux_implementation.png|thumb|420px|left|Example of a complete genetic Demux]]<br />
|[[Image:pv_demux_gn_implementation.png|thumb|420px|left|Example of a complete genetic Demux - Gene network]]<br />
|}<br />
<br />
We want to build up a device in which Input and Selector are respectively IPTG and Tetracycline.<br><br />
In Demux we have two output channels: red fluorescence corresponds to logic 1 at Channel 0, while green fluorescence corresponds to logic 1 at Channel 1.<br><br />
Absence of reporters expression corresponds to logic 0 at Channel 0 and Channel 1.<br><br />
There isn't any input combination that corresponds to logic 1 at Channel 0 and Channel 1 together.<br />
<br />
====Complete genetic Demux - Example====<br />
{|<br />
|[[Image:pv_demux_example1.png|thumb|420px|left|Example of a complete genetic Demux behavior]]<br />
|}<br />
According to Demux truth table, if IN=1 and SEL=1, output channel 0 is logic 0 and output channel 1 is logic 1.<br><br />
IPTG is present, so Plac promoter is active because IPTG binds lacI protein. This allows lasI and luxI transcription.<br><br />
Tetracycline is present, so Ptet promoter is active because Tetracycline binds tetR protein. This allows cI and lasR transcription.<br><br />
cI protein binds Plambda promoter and so luxR expression is inhibited.<br><br />
Simultaneous expression of lasI and lasR can activate GFP, which is under Plas promoter.<br><br />
There is no simultaneous expression of luxI and luxR, so RFP (which is under Plux regulation) cannot be expressed.<br />
<br />
== Final devices==<br />
BioBrick standard parts for genetic Mux and Demux are summarized in the following schemas:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_final.png|thumb|420px|left|Three standard parts for mux]]<br />
|[[Image:pv_demux_final.png|thumb|450px|left|Two standard parts for demux]]<br />
|}<br />
<br />
A hypothetical user of Mux or Demux has to ligate our standard parts with desired inputs and outputs, as shown in the pictures above.<br />
<br><br />
The structure of our devices show that Mux and Demux systems both conform to the PoPS device boundary standard.<br />
<br><br />
<br />
==Applications==<br />
Mux and Demux are two fundamental devices in electronics. They are used in several applications, for example in communication devices, in Arithmetic Logic Units (ALUs), or, in general, in applications that involve channel sharing.<br />
<br><br />
Analogously, they could play a crucial role in the building of complex genetic circuits. In fact, both of them can be used as controlled genetic switches.<br />
<br><br />
Because our devices had been designed to be general, their application field is very wide. For example, genetic Mux can be used to integrate signals from the environment in a two-inputs and one-output biosensor; once detected, the selector controls which of the two inputs must be transferred in output. In this way, two sensing devices can be integrated in only one circuit that can compute multiplexing logic function.<br />
On the other hand, genetic Demux can be used for controlled protein productions, where the choice of the protein to produce is made by the selector. In this case, we can imagine an industrial process where two consequent enzyme reactions are necessary to build a final product. Using a Demux, we can consider the two enzymes as system outputs and then we can easily switch on and off their production modulating selection signal.<br />
<br><br />
Selector in Mux and Demux can be set manually, but also controlled automatically. In fact, it is possible to use feedback control to pilot the selector. For example, in Demux it is possible to switch the enzyme production when a specific condition (for example a pH threshold) is reached.<br />
<br><br />
Switching activity in these two devices is a very powerful tool to manage multi-input and multi-output systems.<br />
<br />
== Experiments and results ==<br />
=== Assemblies ===<br />
We successfully amplified the following BioBrick standard parts from Spring 2008 DNA Distribution:<br />
{|align="center"<br />
|[[Image:pv_resusp.png|thumb|600px|left|Successful amplifications]]<br />
|}<br />
<br><br />
while we couldn't amplify correctly the following parts:<br />
{|align="center"<br />
|[[Image:pv_notresusp.png|thumb|600px|left|Unsuccessful amplifications]]<br />
|}<br />
<br><br />
To build up our designed devices, we followed and completed this assembly tree schema:<br />
{|align="center"<br />
|[[Image:pv_assemblyschema.png|thumb|600px|left|Assembly tree schema]]<br />
|}<br />
<br />
=== Functional tests ===<br />
We performed these boolean (on/off) fluorescence tests:<br />
<br><br />
<hr><br />
*'''TEST 1'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test1.png|thumb|250px|left|GFP protein generator under Plambda]]<br />
|}<br />
'''Description:''' we assembled an available GFP protein generator (E0240) under Plambda promoter, keeping pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' cI protein was not present, so Plambda should work like a constitutive promoter. A reporter gene downstream of this promoter allows us to validate Plambda costitutive activity. That can be very useful to validate our NOT logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of R0051 and E0240, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test1.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that GFP was expressed in positive transformants, so Plambda activity was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 2'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test2.png|thumb|250px|left|GFP protein generator under Ptet]]<br />
|}<br />
'''Description:''' we assembled E0240 under Ptet promoter, keeping pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' tetR protein was not present, so Ptet should work like a constitutive promoter. A reporter gene downstream of this promoter allows us to validate Ptet costitutive activity. Ptet is useful for specific input building.<br />
<br><br />
'''Methods:''' after ligation reaction of R0040 and E0240, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test2.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that GFP was expressed in positive transformants, so Ptet activity was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 3'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test3.png|thumb|250px|left|RFP protein generator under constitutive promoter]]<br />
|}<br />
'''Description:''' we didn't perform any assembly for this experiment, because the promoter we wanted to test was contained into BBa_J61002 vector, which places a RFP protein generator between SpeI and PstI restriction sites.<br />
<br><br />
'''Motivation:''' J23100, which we call Pcon, should have a strong constitutive activity. A reporter gene downstream of this promoter allows us to validate this activity. Pcon is useful to build our inputs because some sensors like IPTG and Tetracycline sensors need the constitutive production of specific proteins, in this case lacI and tetR respectively.<br />
<br><br />
'''Methods''' we transformed J23100 using Invitrogen TOP10 and plated transformed bacteria. We expected to observe red fluorescence (using transluminator) in all the colonies. We also picked up a colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through TRITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' all the grown colonies glowed under UV rays and red fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test3.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that RFP was expressed in transformed bacteria, so Pcon functionality was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 4'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test4.png|thumb|250px|left|Our GFP protein generator (K081012) under a constitutive promoter]]<br />
|}<br />
'''Description:''' we assembled a constitutive promoter family member (J23100) upstream of K081012, a GFP protein generator we built. We decided to excide J23100 from its plasmid (E-S) and to open K081012 plasmid (pSB1AK3) (E-X).<br />
<br><br />
'''Motivation:''' we already validated qualitatively J23100 constitutive activity, so this promoter can be used to test the GFP protein generator we built. We wanted to check if K081012 actually generates GFP.<br />
<br><br />
'''Methods''' after ligation reaction of J23100 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays (see figure) and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test4bis.jpg|thumb|400px|left|TOP10 colonies under UV]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test4.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that our GFP protein generator actually generates GFP.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 5'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test5.png|thumb|250px|left|Our RFP protein generator (K081014) under a constitutive promoter]]<br />
|}<br />
'''Description:''' we assembled a constitutive promoter family member (J23100) upstream of K081014, a RFP protein generator we built. We decided to excide J23100 from its plasmid (E-S) and to open K081014 plasmid (pSB1AK3) (E-X).<br />
<br><br />
'''Motivation:''' we already validated qualitatively J23100 constitutive activity, so this promoter can be used to test the RFP protein generator we built. We wanted to check if K081014 actually generates RFP.<br />
<br><br />
'''Methods''' after ligation reaction of J23100 and K081014, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through TRITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays (see figure) and red fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test5bis.jpg|thumb|400px|left|TOP10 colonies under UV]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test5.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that our RFP protein generator actually generates RFP.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 6'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test6.png|thumb|350px|left|GFP protein generator under Plux]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081000. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we didn't assemble any input to K081000 device, so we could study only the promoter basic activity. We expected to find a weak basic activity. Plux is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of K081000 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we performed a screening on 5 colonies through colony PCR to search for correctly ligated plasmid. We infected 9 ml of LB + Amp with the chosen colony and incubated the culture at 37°C, 220 rpm for 15 hours. Then we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test6.png|thumb|400px|left|No fluorescent TOP10 cells can be seen at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' the picture above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria. Increasing exposition time, green fluorescence could be observed (picture not reported). So, we can say that GFP was expressed very weakly. This experiment confirmed that Plux promoter has a weak activity without luxI and luxR proteins.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 7'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test7.png|thumb|350px|left|GFP protein generator under Plas]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plas promoter that was contained into K081001. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we didn't assemble any input to K081001 device, so we could study only the promoter basic activity. We expected to find a weak basic activity. Plas is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of K081001 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we performed a screening on 5 colonies through colony PCR to search for correctly ligated plasmid. We infected 9 ml of LB + Amp with the chosen colony and incubated the culture at 37°C, 220 rpm for 15 hours. Then we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test7.png|thumb|400px|left|No fluorescent TOP10 cells can be seen at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' the picture above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria. Increasing exposition time, green fluorescence could be observed (picture not reported). So, we can say that GFP was expressed very weakly. This experiment confirmed that Plas promoter has a weak activity without lasI and lasR proteins.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 8'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test8.png|thumb|600px|left|GFP protein generator under Plux and constitutive expression of luxI and luxR]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081000. We also assembled K081011 upstream of K081000. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' Plux promoter activity was studied in response of a constitutive expression of luxI and luxR. Plambda promoter without cI repressor guaranteed the constitutive expression of these genes. We expected to find a strong activity because Plux is turned on. lux system is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' we ligated K081012 under K081000, transformed ligation, plated transformed bacteria, performed PCR screening on some colonies and extracted correctly ligated plasmids. We repeated these steps to assemble K081011 upstream of K081000-K081012, but we didn't perform PCR screening. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test8.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that Plux promoter can be activated by the contemporary presence of luxI and luxR. This is a crucial result for our AND logic gate.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 9'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test9.png|thumb|500px|left|GFP protein generator under Plux and constitutive expression of luxR]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081022. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' Plux promoter activity was studied in response to a constitutive expression of luxR. Plambda promoter without cI repressor guaranteed the constitutive expression of this gene. We expected to find a weak activity because luxR transcription factor is not active and so Plux cannot be turned on. We also expected to find a strong activity if we induce luxR activation using 3OC6-HSL (this is equivalent to TEST 8 conditions, because 3OC6-HSL is synthesized by luxI). lux system is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' we ligated K081012 downstream of K081022, transformed ligation, plated transformed bacteria and screened three colonies to insulate a colony containing correctly ligated plasmids. We inoculated the positive colony into 9 ml of LB + Amp and incubated the culture at 37°C, 220 rpm for 15 hours. Then we diluted 1:10 the culture in two falcon tubes (5 ml cultures). One of these cultures was induced with 3OC6-HSL 1 uM. We let the two cultures grow for 2 hours and then we watched 50 ul at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture.<br />
{|align="center"<br />
|[[Image:pv_fig_test9.png|thumb|400px|left|Non induced culture: TOP10 cells cannot be seen at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test9bis.png|thumb|400px|left|Induced culture: fluorescent TOP10 cells at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test9superexp.png|thumb|400px|left|Same frames as above, but superexposed: non induced (left) and induced (right) cultures]]<br />
|}<br />
<br><br />
'''Comments:''' the pictures above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria for the non induced culture, while we can see green fluorescent TOP10 in the induced culture. As we wrote for TEST 6 and TEST 7, increasing exposition time green fluorescence could be observed even for the non induced culture (last picture), confirming the weak activity of Plux promoter in response of unactive luxR. This experiment confirmed that Plux promoter has a weak activity without luxI (or 3OC6-HSL) and luxR protein is not sufficient to induce a strong transcription. Adding 3OC6-HSL, luxR becomes active and so Plux is turned on.<br />
<br><br />
NOTE: K081022 has a point mutation in position 349 of C0062 coding sequence. This mutation changes the aminoacid, but luxR seems to work as expected.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 10'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test10.png|thumb|600px|left|RFP protein generator under Plux and constitutive expression of luxR]]<br />
|}<br />
'''Description:''' this test is equivalent to TEST 9: we assembled K081014 (our RFP) under the Plux promoter that was contained into K081004. We kept pSB1AK3 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we wanted to repeat TEST 9 experiment using a longer construct and a different reporter gene.<br />
<br><br />
'''Methods:''' the same as TEST 9, but using K081004 instead of K081022 and using RFP (K081014) instead of GFP (K081012).<br />
<br><br />
'''Results:''' red fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture.<br />
{|align="center"<br />
|[[Image:pv_fig_test10.png|thumb|400px|left|Non induced culture: TOP10 cells cannot be seen at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test10bis.png|thumb|400px|left|Induced culture: fluorescent TOP10 cells at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test10superexp.png|thumb|400px|left|Same frames as above, but superexposed: non induced (left) and induced (right) cultures]]<br />
|}<br />
<br><br />
'''Comments:''' the same as TEST 9.<br />
<br><br />
NOTE: C0062 has the same mutation described in TEST 9. We also performed this test with an old version of K081004 carrying another point mutation (C->T at position 704 of C0062 coding sequence) that changed an aminoacid. Even in this case K081004 seemed to work as expected (results not shown).<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 11'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test11.png|thumb|600px|left|Terminator efficiency test]]<br />
|}<br />
'''Description:''' we assembled K081014 (our RFP) under the artificial 39 bp terminator (B1006) that is at the end of K081022-K081012 (composite part used for TEST 9). We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we wanted to test qualitatively if our terminator actually stops transcription.<br />
<br><br />
'''Methods:''' we assembled K081014 downstream of K081022, transformed ligation, plated transformed bacteria and insulated a colony containing correctly ligated plasmid. We inoculated the colony into 9 ml of LB + Amp and incubated the culture at 37°C, 220 rpm for 15 hours. Then we diluted 1:10 the culture in two falcon tubes (5 ml cultures). One of these cultures was induced with 3OC6-HSL 1 uM. We let the two cultures grow for 2 hours and then we watched 50 ul at microscope through FITC channel (positive control), TRITC channel and DAPI channel (negative control).<br />
<br><br />
'''Results:''' soon<br />
<br><br />
'''Comments:''' soon<br />
<br><br />
<br><br />
<hr><br />
<br><br />
We also performed these quantitative fluorescence tests:<br />
<hr><br />
*'''TEST A'''<br />
<br><br />
'''Description:''' this test is an extension of TEST 9. We induced six cultures with 3OC6-HSL at different concentrations to study quantitatively HSL-GFP static transfer function.<br />
<br><br />
'''Motivation:''' we want to know cutoff point of this device.<br />
<br><br />
'''Methods:''' the same as TEST 9, but we diluted the overnight 9 ml culture 1:10 in six falcon tubes (5 ml cultures). We induced the six cultures with 3OC6-HSL 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM and 1 uM respectively. We incubated the cultures at 37°C, 220 rpm for 2 hours and then we watched 50 ul at microscope through FITC channel and DAPI channel for negative control. We prepared two 50 ul glasses for each culture. We acquired three frames at 2.5 s (maximum exposition time) and 10 ms exposition time for every sample. We used ImageJ software to count automatically the number of cells for every frame. Then we computed n10/n2.5 (where n is the number of cells) to calculate the percentage of cells that glowed in the 10 ms acquisition, assuming that in the 2.5 s acquisition we can see the total number of cells. For each HSL concentration, we calculated the mean value of the 6 statistics (3 frames for each of the 2 glasses).<br />
<br><br />
'''Results:'''<br />
{|align="center"<br />
|[[Image:pv_fig_test1q.png|thumb|600px|left|GFP (arbitrary units) vs 3OC6-HSL concentration: 1=0nM, 2=0.1nM, 3=1nM, 4=10nM, 5=100nM, 6=1uM]]<br />
|}<br />
<br><br />
'''Comments:''' we computed the statistic n10/n2.5 because we know that when fluorescence is weak, cells cannot be seen at low exposition times. So, we calculate the ratio between the cells we can see at a low exposition time and the total number of cells in the frame. We chose 10 ms because the previous experiments with GFP had been performed using this parameter. We know that this is not an exact statistic, in fact we have to consider the count errors of ImageJ software, especially when cells are superimposed. Further quantitative experiments will have to be performed using standard measurement, in order to characterize parts.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST B'''<br />
<br><br />
'''Description:''' this test is an extension of TEST 10. We induced seven cultures with 3OC6-HSL at different concentrations to study quantitatively HSL-RFP static transfer function.<br />
<br><br />
'''Motivation:''' we want to know cutoff point of this device.<br />
<br><br />
'''Methods:''' the same as TEST 10, but we diluted the overnight 9 ml culture 1:10 in seven falcon tubes (5 ml cultures). We induced the seven cultures with 3OC6-HSL 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 uM and 10 uM respectively. We incubated the cultures at 37°C, 220 rpm for 2 hours and then we watched 50 ul at microscope through TRITC channel and DAPI channel for negative control. We prepared two 50 ul glasses for each culture. We acquired three frames at 2.5 s (maximum exposition time) and 90 ms exposition time for every sample. We used ImageJ software to count automatically the number of cells for every acquisition. Then we computed n90/n2.5 (where n is the number of cells) to calculate the percentage of cells that glowed in the 90 ms acquisition, assuming that in the 2.5 s acquisition we can see the total number of cells. For each HSL concentration, we calculated the mean value of the 6 statistics (3 frames for each of the 2 glasses).<br />
<br><br />
'''Results:'''<br />
{|align="center"<br />
|[[Image:pv_fig_test2q.png|thumb|600px|left|RFP (arbitrary units) vs 3OC6-HSL concentration: 1=0nM, 2=0.1nM, 3=1nM, 4=10nM, 5=100nM, 6=1uM, 7=10uM]]<br />
|}<br />
<br><br />
'''Comments:''' we computed the statistic n90/n2.5 because we know that when fluorescence is weak, cells cannot be seen at low exposition times. So, we calculate the ratio between the cells we can see at a low exposition time and the total number of cells in the frame. We chose 90 ms because the previous experiments with RFP had been performed using this parameter. We know that this is not an exact statistic, in fact we have to consider the count errors of ImageJ software, especially when cells are superimposed. As we wrote for TEST 9, further quantitative experiments will have to be performed using standard measurement, in order to characterize parts.<br />
<br><br />
<hr><br />
<br />
=== Conclusions ===<br />
All the experiments we performed were consistent with our expectations.<br />
<br><br />
Considering the elementary logic gates of our final devices (AND, OR, NOT), we validated some truth table rows:<br />
{|align="center"<br />
|[[Image:pv_summary.png|thumb|600px|left|Biological truth tables]]<br />
|}<br />
<br />
In particular:<br />
*'''TEST 1 validated the first row of NOT logic gate.'''<br />
*'''TEST 6 validated the first row of AND (lux) logic gate.'''<br />
*'''TEST 7 validated the first row of AND (las) logic gate.'''<br />
*'''TEST 8, TEST 9 (with induction) and TEST 10 (with induction) validated the fourth row of AND (lux) logic gate.'''<br />
*'''TEST 9 (without induction) and TEST 10 (without induction) validated the third row of AND (lux) logic gate.'''</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/ProjectTeam:UNIPV-Pavia/Project2008-10-27T15:12:16Z<p>Magni: /* Applications */</p>
<hr />
<div>{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
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|-<br />
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|}<br />
<br />
<br><br />
<br />
== '''Overall project''' ==<br />
<br />
We are trying to mimic Multiplexer (Mux) and Demultiplexer (Demux) logic functions in E. coli.<br />
<br><br />
In the following paragraphs project details will be described from both digital electronic and genetic points of view.<br />
<br><br />
<br><br />
<br />
== '''Electronic Implementation''' ==<br />
<br><br />
=== '''What kind of components are Mux and Demux?''' ===<br />
'''Mux''' is a component which conveys one of the two input channels values into a single output channel. The choice of the input channel is made by a selector.<br />
<br><br />
'''Demux''' is a component which conveys the only input channel value into one of the two output channels. The choice of the output channel is made by a selector.<br />
<br><br />
<br><br />
The following pictures show data flow in Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_dataflow0.png|thumb|300px|left|Data flow in Multiplexer - SELECTOR=0]]<br />
|[[Image:pv_mux_dataflow1.png|thumb|300px|left|Data flow in Multiplexer - SELECTOR=1]]<br />
|-<br />
|[[Image:pv_demux_dataflow0.png|thumb|300px|left|Data flow in Demultiplexer - SELECTOR=0]]<br />
|[[Image:pv_demux_dataflow1.png|thumb|300px|left|Data flow in Demultiplexer - SELECTOR=1]]<br />
|}<br />
<br><br />
<br />
=== '''What kind of signals do we process?''' ===<br />
In this project we consider Boolean logic signals, thus every input/output value can assume only the values 0 and 1. A function that processes Boolean values is called logic function.<br />
<br><br />
Mux and Demux can be considered by now as black boxes which implement a logic function that can process input signals to output signals. Here you can see examples of Boolean data flow in Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_bool.png|thumb|300px|left|Example: Mux Boolean data flow]]<br />
|[[Image:pv_demux_bool.png|thumb|300px|left|Example: Demux Boolean data flow]]<br />
|}<br />
In the following documentation we will see what is inside these black boxes.<br />
<br><br />
<br><br />
=== How can we formalize Mux and Demux logic behavior? ===<br />
Logic functions can be formalized writing a truth table; a truth table is a mathematical table in which every row represents a combination of input values and its respective output values. The table has to be filled with every input combination.<br />
<br><br />
<br><br />
Here you can see Mux and Demux truth tables (output columns are gray):<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_truth.png|thumb|300px|left|Mux truth table]]<br />
|[[Image:pv_demux_truth.png|thumb|300px|left|Demux truth table]]<br />
|}<br />
<br><br />
<br />
=== Building a logic circuit from a truth table ===<br />
Our goal in this section is to project two logic gates networks which behave like Mux and Demux truth tables. A very useful tool to transform a truth table into a logic network is Karnaugh map.<br />
<br><br />
It is possible to read about Karnaugh maps at: [http://en.wikipedia.org/wiki/Karnaugh_map]<br />
<br><br />
<br><br />
Following Karnaugh maps method, we can write these two logic networks for Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux.png|thumb|340px|left|Mux - logic circuit]]<br />
|[[Image:pv_mux_example.png|thumb|340px|left|Mux - Example]]<br />
|-<br />
|[[Image:pv_demux.png|thumb|340px|left|Demux - logic circuit]]<br />
|[[Image:pv_demux_example.png|thumb|340px|left|Demux - Example]]<br />
|}<br />
<br />
<br><br />
<br />
== '''Genetic Implementation''' ==<br />
Our goal is to mimic Mux and Demux logic networks in a biological device, such as E. coli. To perform this, we use protein/DNA and protein/protein interactions to build up biological logic gates.<br />
Mux and Demux logic circuits are composed by three fundamental logic gates, AND, OR, NOT: in the next paragraphs genetic implementation of these logic gates will be provided.<br />
<br><br />
<br><br />
=== AND ===<br />
To mimic an AND gate, we need a biological function, such as a promoter activation, which is directly turned on by the interaction between two upstream genes. In our synthetic devices, we use the luxR/luxI system: luxR can activate Plux promoter only upon 3-oxo-hexanoyl-homoserine lactone (HSL) binding; luxI generates HSL; so, only the contemporary expression of LuxR and luxI proteins can activate the downstream Plux-dependent gene expression. Another AND gate we use is the lasR/lasI system, which works in a very similar way but through another chemical intermediate, N-(3-oxododecanoyl) homoserine lactone (PAI-1).<br />
{|<br />
|[[Image:pv_proj_AND.png|thumb|340px|left|Genetic AND: Plux can be turned on only when the two proteins luxI and luxR are present.]]<br />
|}<br />
=== OR ===<br />
To mimic an OR gate in Mux, we need a biological function which can be activated alternatively by two independent upstream signals or by both. Thus, we combine the outputs of the upstream AND gates to assemble directly an OR reporter function, by simply repeating the reporter gene (GFP) under two different promoters (Plux and Plas). It’s sufficient to activate one of the two promoters (or both) to recover the GFP signal from engineered bacteria.<br />
There should not be an over-expression problem for GFP, in fact, in Mux device, only one promoter can be active, either Plux or Plas. Here we considered GFP output, but OR device can be generalized for every output gene.<br />
{|<br />
|[[Image:pv_proj_OR.png|thumb|340px|left|Genetic OR: it is sufficient that one of the two input promoters is active to obtain GFP expression.]]<br />
|}<br />
=== NOT ===<br />
To mimic a NOT gate, we need an efficient and regulated repressor of a specific downstream promoter: in this case, we choose cI repression on Plambda, which should be specific and, upon cI inactivation, quick and efficient.<br />
{|<br />
|[[Image:pv_proj_NOT.png|thumb|340px|left|Genetic NOT: Plambda can be turned on only when cI protein is not present.]]<br />
|}<br />
<br />
According to what above stated, genetic implementation of Mux and Demux can be obtained connecting these basic logic gates and can be summarized in this way:<br />
<br><br />
<br><br />
<br><br />
<br />
=== Genetic Mux ===<br />
Let PA, PB and PS be three generic promoters that can be ACTIVATED respectively by the three exogenous molecules "A", "B" and "S". A genetic Mux with inputs "A" (CH0) and "B" (CH1), selector "S" and the generic protein GOI as output, can be implemented by the following gene network:<br />
{|align="center"<br />
|[[Image:pv_MUX.png|thumb|420px|left|Genetic Mux]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_MUXint.png|thumb|420px|left|Genetic Mux - interactions]]<br />
|}<br />
We want to supply a device that can be generalized to detect every kind of input and to express every kind of genes as output. So, input promoters and output protein generators are not part of the genetic Mux we want to build up. In this way, a hypothetical user can re-use our device assembling the desired input and output elements.<br />
<br><br />
Note that the output genes are two, but, as explained in "OR" section, if the genes are identical, we can say that there is only one output; in fact, the real output is a protein synthesis and so it is not important which of the two identical genes is expressed.<br />
<br><br />
However, it is possible to assemble two different genes downstream of Plux and Plas, for example two reporters. In this way, debugging process becomes easy, because we can discriminate lux system and las system activities.<br />
<br><br />
Here we report the truth table of this network:<br />
{|align="center"<br />
|[[Image:pv_gmux_truth.png|thumb|420px|left|Genetic Mux - truth table]]<br />
|}<br />
<br><br />
Now we report two examples of genetic Mux behavior, in response to generic inputs.<br />
<br><br />
<br><br />
====Genetic Mux behavior - Example 1====<br />
{|<br />
|[[Image:pv_genmux_example1.png|thumb|420px|left|First example of genetic Mux behavior]]<br />
|}<br />
According to Mux truth table, if "A" is not present and "B" and "S" are present, we expect to have GOI synthesis.<br />
<br><br />
"A" molecule is not present, so PA promoter is not active and luxI gene is not expressed.<br />
<br><br />
"B" molecule is present, so PB promoter is active and lasI gene is expressed.<br />
<br><br />
"S" molecule is present, so PS promoter is active and lasR and cI genes are expressed.<br />
<br><br />
cI protein represses transcription of the gene downstream of Plambda promoter, which is luxR.<br />
<br><br />
lasI protein can synthesize 3-OC12-HSL. This molecule activates transcription factor lasR, which can activate Plas promoter. So, the copy of GOI gene under Plas regulation can be expressed.<br />
<br><br />
The other copy of GOI gene, which is under Plux promoter regulation, is not expressed. In fact Plux is not active, because both luxI and luxR proteins are not present.<br />
<br><br />
Only one of the two GOI genes is expressed, but this is sufficient to synthesize the output protein GOI.<br />
<br><br />
<br><br />
====Genetic Mux behavior - Example 2====<br />
{|<br />
|[[Image:pv_genmux_example2.png|thumb|420px|left|Second example of genetic Mux behavior]]<br />
|}<br />
We expect to have no GOI synthesis in response to this input combination.<br />
<br><br />
"A" molecule is not present, so PA promoter is not active and luxI gene is not expressed.<br />
<br><br />
"B" molecule is present, so PB promoter is active and lasI gene is expressed.<br />
<br><br />
"S" molecule is not present, so PS promoter can't transcribe cI and lasR genes.<br />
<br><br />
cI protein is not present, so Plambda promoter can transcribe luxR gene.<br />
<br><br />
None of the two logic AND systems is active, because lux system lacks of luxI and las system lacks of lasR. So, Plux and Plas promoters are unactive and can't express GOI genes. For this reason, GOI protein is not synthesized.<br />
<br><br />
<br><br />
These two examples show how "S" molecule can select the input to be conveyed into the single output channel: in fact its presence allows lasR expression and represses luxR expression, while "S" absence represses lasR expression and allows luxR expression.<br />
<br><br />
<br><br />
<br />
=== Genetic Demux ===<br />
Let PI and PS be two generic promoters that can be ACTIVATED respectively by the two exogenous molecules "I" and "S". A genetic Demux with input "I", selector "S" and the generic proteins GOI0 and GOI1 as outputs (OUT0 and OUT1 respectively), can be implemented by the following gene network:<br />
{|align="center"<br />
|[[Image:pv_DEMUX.png|thumb|420px|left|Genetic Demux]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_DEMUXint.png|thumb|420px|left|Genetic Demux - interactions]]<br />
|}<br />
As described for genetic Mux, we want to supply a device that can be generalized to detect every kind of inputs and to express every kind of genes as output. So, input promoters and output protein generators are not part of the genetic Demux. In this way, a hypothetical user can re-use our device assembling the desired input and output elements.<br />
<br><br />
Here we report the truth table of this network:<br />
{|align="center"<br />
|[[Image:pv_gdemux_truth.png|thumb|420px|left|Genetic Demux - truth table]]<br />
|}<br />
<br><br />
Now we report two examples of genetic Demux behavior, in response to generic inputs.<br />
<br><br />
<br><br />
====Genetic Demux behavior - Example 1====<br />
{|<br />
|[[Image:pv_gendemux_example1.png|thumb|420px|left|First example of genetic Demux behavior]]<br />
|}<br />
According to Demux truth table, if "I" molecule is present and "S" molecule is absent, we expect to have GOI0 protein synthesis and no GOI1 protein synthesis.<br />
<br><br />
"I" is present, so PI promoter is active and luxI and lasI genes are expressed.<br />
<br><br />
"S" is not present, so PS promoter can't transcribe lasR and cI genes.<br />
<br><br />
cI protein is not present, so Plambda promoter is not inhibited and luxR gene is expressed.<br />
<br><br />
luxI protein can synthesize 3-OC6-HSL lactone. This molecule activates luxR transcription factor which can activate Plux promoter. In this way, GOI0 gene, which is under Plux regulation, is expressed.<br />
<br><br />
On the other hand, lasI protein can synthesize 3-OC12-HSL, but lasR transcription factor is not present and so Plas promoter cannot be activated. In this way, GOI1 gene, which is under Plas regulation, is not expressed.<br />
<br><br />
So, we have GOI0 protein synthesis and no GOI1 protein synthesis.<br />
<br><br />
<br><br />
====Genetic Demux behavior - Example 2====<br />
{|<br />
|[[Image:pv_gendemux_example2.png|thumb|420px|left|Second example of genetic Demux behavior]]<br />
|}<br />
We expect to have GOI1 synthesis and no GOI0 synthesis in response to this input combination.<br />
<br><br />
"I" is present, so PI promoter is active and luxI and lasI genes are expressed.<br />
<br><br />
"S" is present, so PS promoter lasR and cI genes are expressed.<br />
<br><br />
cI protein is present, so Plambda promoter is inhibited and luxR gene is not expressed.<br />
<br><br />
lasI protein can synthesize 3-OC12-HSL lactone. This molecule activates lasR transcription factor which can activate Plas promoter. In this way, GOI1 gene, which is under Plas regulation, is expressed.<br />
<br><br />
On the other hand, luxI protein can synthesize 3-OC6-HSL, but luxR transcription factor is not present and so Plux promoter cannot be activated. In this way, GOI0 gene, which is under Plux regulation, is not expressed.<br />
<br><br />
So, we have GOI1 protein synthesis and no GOI0 protein synthesis.<br />
<br />
<br />
=== A complete genetic Mux===<br />
In this section a complete Mux gene network is described.<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_implementation.png|thumb|420px|left|Example of a complete genetic Mux]]<br />
|[[Image:pv_mux_gn_implementation.png|thumb|420px|left|Example of a complete genetic Mux - Gene network]]<br />
|}<br />
We want to build up a device in which Channel 0, Channel 1 and Selector are respectively sensitive to Tetracycline, IPTG and red light. The presence of each input corresponds to logic 1.<br />
We chose green fluorescence as Mux output: expression of GFP corresponds to logic 1, while absence of fluorescence corresponds to logic 0.<br />
<br><br />
Tetracycline and IPTG can be considered as "A" and "B" molecules, introduced in "Genetic Mux" section, because both Tetracycline and IPTG are indirect ACTIVATORS of Ptet and Plac promoters respectively.<br />
On the other hand, red light is quite different from "S" molecule, because red light is an indirect REPRESSOR of Pomp promoter and not an activator as required by the original schema. For this reason, if we want a device in which the presence of Tetracycline, IPTG and red light correspond to logic 1, red light input should be ''inverted''. The simplest way to do this, is to cross-exchange the two input channels. So, Tetracycline sensor (CH0) has to be assembled to las system and IPTG sensor (CH1) has to be assembled to lux system.<br />
<br />
<br />
====Complete genetic Mux - Example====<br />
{|<br />
|[[Image:pv_mux_example1.png|thumb|420px|left|Example of a complete genetic Mux behavior]]<br />
|}<br />
According to Mux truth table, if SEL=0, CH0=1, CH1=0, output channel must be logic 1.<br />
Red light is not present, so it can't dephosphorylate cph8-ho1-pcyA complex, which is constitutively expressed. cph8-ho1-pcyA complex activates endogenous ompR, which can activate Pomp promoter, so cI and lasR genes are transcribed.<br />
cI protein binds Plambda promoter and so luxR expression is inhibited.<br />
TetR is a repressor for Ptet promoter, but Tetracycline is present, so it can bind tetR protein, which is constitutively expressed, and can activate transcription of downstream gene: lasI.<br />
Simultaneous expression of lasI and lasR can activate GFP, which is under Plas promoter.<br />
IPTG is not present, so lacI protein binds Plac promoter and represses luxI transcription.<br />
<br />
<br />
=== A complete genetic Demux===<br />
In this section a complete Demux gene network is described.<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_demux_implementation.png|thumb|420px|left|Example of a complete genetic Demux]]<br />
|[[Image:pv_demux_gn_implementation.png|thumb|420px|left|Example of a complete genetic Demux - Gene network]]<br />
|}<br />
<br />
We want to build up a device in which Input and Selector are respectively IPTG and Tetracycline.<br><br />
In Demux we have two output channels: red fluorescence corresponds to logic 1 at Channel 0, while green fluorescence corresponds to logic 1 at Channel 1.<br><br />
Absence of reporters expression corresponds to logic 0 at Channel 0 and Channel 1.<br><br />
There isn't any input combination that corresponds to logic 1 at Channel 0 and Channel 1 together.<br />
<br />
====Complete genetic Demux - Example====<br />
{|<br />
|[[Image:pv_demux_example1.png|thumb|420px|left|Example of a complete genetic Demux behavior]]<br />
|}<br />
According to Demux truth table, if IN=1 and SEL=1, output channel 0 is logic 0 and output channel 1 is logic 1.<br><br />
IPTG is present, so Plac promoter is active because IPTG binds lacI protein. This allows lasI and luxI transcription.<br><br />
Tetracycline is present, so Ptet promoter is active because Tetracycline binds tetR protein. This allows cI and lasR transcription.<br><br />
cI protein binds Plambda promoter and so luxR expression is inhibited.<br><br />
Simultaneous expression of lasI and lasR can activate GFP, which is under Plas promoter.<br><br />
There is no simultaneous expression of luxI and luxR, so RFP (which is under Plux regulation) cannot be expressed.<br />
<br />
== Final devices==<br />
BioBrick standard parts for genetic Mux and Demux are summarized in the following schemas:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_final.png|thumb|420px|left|Three standard parts for mux]]<br />
|[[Image:pv_demux_final.png|thumb|450px|left|Two standard parts for demux]]<br />
|}<br />
<br />
A hypothetical user of Mux or Demux has to ligate our standard parts with desired inputs and outputs, as shown in the pictures above.<br />
<br><br />
The structure of our devices show that Mux and Demux systems both conform to the PoPS device boundary standard.<br />
<br><br />
<br />
==Applications==<br />
Mux and Demux are two fundamental devices in electronics. They are used in several applications, for example in communication devices, in Arithmetic Logic Units (ALUs), or, in general, in applications that involve channel sharing.<br />
<br><br />
Analogously, they could play a crucial role in the building of complex genetic circuits. In fact, both of them can be used as controlled genetic switches.<br />
<br><br />
Because our devices had been designed to be general, their application field is very wide. For example, genetic Mux can be used to integrate signals from the environment in a two-inputs and one-output biosensor; once detected, the selector controls which of the two inputs must be transferred in output. In this way, two sensing devices can be integrated in only one circuit that can compute multiplexing logic function.<br />
On the other hand, genetic Demux can be used for controlled protein productions, where the choice of the protein to produce is made by the selector. In this case, we can imagine an industrial process where two consequent enzyme reactions are necessary to build a final product. Using a Demux, we can consider the two enzymes as system outputs and then we can easily switch on and off their production modulating selection signal.<br />
<br><br />
Selector in Mux and Demux can be set manually, but also controlled automatically. In fact, it is possible to use feedback control to pilot the selector. For example, in Demux it is possible to switch the enzyme production when a specific condition (for example a pH threshold) is reached.<br />
<br><br />
Switching activity in these two devices is a very powerful tool to manage multi-input and multi-output systems.<br />
<br />
== Experiments and results ==<br />
=== Assemblies ===<br />
We successfully amplified the following BioBrick standard parts from Spring 2008 DNA Distribution:<br />
{|align="center"<br />
|[[Image:pv_resusp.png|thumb|600px|left|Successful amplifications]]<br />
|}<br />
<br><br />
while we couldn't amplify correctly the following parts:<br />
{|align="center"<br />
|[[Image:pv_notresusp.png|thumb|600px|left|Unsuccessful amplifications]]<br />
|}<br />
<br><br />
To build up our designed devices, we followed and completed this assembly tree schema:<br />
{|align="center"<br />
|[[Image:pv_assemblyschema.png|thumb|600px|left|Assembly tree schema]]<br />
|}<br />
<br />
=== Functional tests ===<br />
We performed these boolean (on/off) fluorescence tests:<br />
<br><br />
<hr><br />
*'''TEST 1'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test1.png|thumb|250px|left|GFP protein generator under Plambda]]<br />
|}<br />
'''Description:''' we assembled an available GFP protein generator (E0240) under Plambda promoter, keeping pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' cI protein was not present, so Plambda should work like a constitutive promoter. A reporter gene downstream of this promoter allows us to validate Plambda costitutive activity. That can be very useful to validate our NOT logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of R0051 and E0240, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test1.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that GFP was expressed in positive transformants, so Plambda activity was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 2'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test2.png|thumb|250px|left|GFP protein generator under Ptet]]<br />
|}<br />
'''Description:''' we assembled E0240 under Ptet promoter, keeping pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' tetR protein was not present, so Ptet should work like a constitutive promoter. A reporter gene downstream of this promoter allows us to validate Ptet costitutive activity. Ptet is useful for specific input building.<br />
<br><br />
'''Methods:''' after ligation reaction of R0040 and E0240, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test2.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that GFP was expressed in positive transformants, so Ptet activity was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 3'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test3.png|thumb|250px|left|RFP protein generator under constitutive promoter]]<br />
|}<br />
'''Description:''' we didn't perform any assembly for this experiment, because the promoter we wanted to test was contained into BBa_J61002 vector, which places a RFP protein generator between SpeI and PstI restriction sites.<br />
<br><br />
'''Motivation:''' J23100, which we call Pcon, should have a strong constitutive activity. A reporter gene downstream of this promoter allows us to validate this activity. Pcon is useful to build our inputs because some sensors like IPTG and Tetracycline sensors need the constitutive production of specific proteins, in this case lacI and tetR respectively.<br />
<br><br />
'''Methods''' we transformed J23100 using Invitrogen TOP10 and plated transformed bacteria. We expected to observe red fluorescence (using transluminator) in all the colonies. We also picked up a colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through TRITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' all the grown colonies glowed under UV rays and red fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test3.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that RFP was expressed in transformed bacteria, so Pcon functionality was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 4'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test4.png|thumb|250px|left|Our GFP protein generator (K081012) under a constitutive promoter]]<br />
|}<br />
'''Description:''' we assembled a constitutive promoter family member (J23100) upstream of K081012, a GFP protein generator we built. We decided to excide J23100 from its plasmid (E-S) and to open K081012 plasmid (pSB1AK3) (E-X).<br />
<br><br />
'''Motivation:''' we already validated qualitatively J23100 constitutive activity, so this promoter can be used to test the GFP protein generator we built. We wanted to check if K081012 actually generates GFP.<br />
<br><br />
'''Methods''' after ligation reaction of J23100 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays (see figure) and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test4bis.jpg|thumb|400px|left|TOP10 colonies under UV]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test4.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that our GFP protein generator actually generates GFP.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 5'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test5.png|thumb|250px|left|Our RFP protein generator (K081014) under a constitutive promoter]]<br />
|}<br />
'''Description:''' we assembled a constitutive promoter family member (J23100) upstream of K081014, a RFP protein generator we built. We decided to excide J23100 from its plasmid (E-S) and to open K081014 plasmid (pSB1AK3) (E-X).<br />
<br><br />
'''Motivation:''' we already validated qualitatively J23100 constitutive activity, so this promoter can be used to test the RFP protein generator we built. We wanted to check if K081014 actually generates RFP.<br />
<br><br />
'''Methods''' after ligation reaction of J23100 and K081014, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through TRITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays (see figure) and red fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test5bis.jpg|thumb|400px|left|TOP10 colonies under UV]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test5.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that our RFP protein generator actually generates RFP.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 6'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test6.png|thumb|350px|left|GFP protein generator under Plux]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081000. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we didn't assemble any input to K081000 device, so we could study only the promoter basic activity. We expected to find a weak basic activity. Plux is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of K081000 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we performed a screening on 5 colonies through colony PCR to search for correctly ligated plasmid. We infected 9 ml of LB + Amp with the chosen colony and incubated the culture at 37°C, 220 rpm for 15 hours. Then we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test6.png|thumb|400px|left|No fluorescent TOP10 cells can be seen at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' the picture above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria. Increasing exposition time, green fluorescence could be observed (picture not reported). So, we can say that GFP was expressed very weakly. This experiment confirmed that Plux promoter has a weak activity without luxI and luxR proteins.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 7'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test7.png|thumb|350px|left|GFP protein generator under Plas]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plas promoter that was contained into K081001. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we didn't assemble any input to K081001 device, so we could study only the promoter basic activity. We expected to find a weak basic activity. Plas is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of K081001 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we performed a screening on 5 colonies through colony PCR to search for correctly ligated plasmid. We infected 9 ml of LB + Amp with the chosen colony and incubated the culture at 37°C, 220 rpm for 15 hours. Then we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test7.png|thumb|400px|left|No fluorescent TOP10 cells can be seen at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' the picture above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria. Increasing exposition time, green fluorescence could be observed (picture not reported). So, we can say that GFP was expressed very weakly. This experiment confirmed that Plas promoter has a weak activity without lasI and lasR proteins.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 8'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test8.png|thumb|600px|left|GFP protein generator under Plux and constitutive expression of luxI and luxR]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081000. We also assembled K081011 upstream of K081000. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' Plux promoter activity was studied in response of a constitutive expression of luxI and luxR. Plambda promoter without cI repressor guaranteed the constitutive expression of these genes. We expected to find a strong activity because Plux is turned on. lux system is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' we ligated K081012 under K081000, transformed ligation, plated transformed bacteria, performed PCR screening on some colonies and extracted correctly ligated plasmids. We repeated these steps to assemble K081011 upstream of K081000-K081012, but we didn't perform PCR screening. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test8.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that Plux promoter can be activated by the contemporary presence of luxI and luxR. This is a crucial result for our AND logic gate.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 9'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test9.png|thumb|500px|left|GFP protein generator under Plux and constitutive expression of luxR]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081022. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' Plux promoter activity was studied in response to a constitutive expression of luxR. Plambda promoter without cI repressor guaranteed the constitutive expression of this gene. We expected to find a weak activity because luxR transcription factor is not active and so Plux cannot be turned on. We also expected to find a strong activity if we induce luxR activation using 3OC6-HSL (this is equivalent to TEST 8 conditions, because 3OC6-HSL is synthesized by luxI). lux system is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' we ligated K081012 downstream of K081022, transformed ligation, plated transformed bacteria and screened three colonies to insulate a colony containing correctly ligated plasmids. We inoculated the positive colony into 9 ml of LB + Amp and incubated the culture at 37°C, 220 rpm for 15 hours. Then we diluted 1:10 the culture in two falcon tubes (5 ml cultures). One of these cultures was induced with 3OC6-HSL 1 uM. We let the two cultures grow for 2 hours and then we watched 50 ul at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture.<br />
{|align="center"<br />
|[[Image:pv_fig_test9.png|thumb|400px|left|Non induced culture: TOP10 cells cannot be seen at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test9bis.png|thumb|400px|left|Induced culture: fluorescent TOP10 cells at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test9superexp.png|thumb|400px|left|Same frames as above, but superexposed: non induced (left) and induced (right) cultures]]<br />
|}<br />
<br><br />
'''Comments:''' the pictures above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria for the non induced culture, while we can see green fluorescent TOP10 in the induced culture. As we wrote for TEST 6 and TEST 7, increasing exposition time green fluorescence could be observed even for the non induced culture (last picture), confirming the weak activity of Plux promoter in response of unactive luxR. This experiment confirmed that Plux promoter has a weak activity without luxI (or 3OC6-HSL) and luxR protein is not sufficient to induce a strong transcription. Adding 3OC6-HSL, luxR becomes active and so Plux is turned on.<br />
<br><br />
NOTE: K081022 has a point mutation in position 349 of C0062 coding sequence. This mutation changes the aminoacid, but luxR seems to work as expected.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 10'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test10.png|thumb|600px|left|RFP protein generator under Plux and constitutive expression of luxR]]<br />
|}<br />
'''Description:''' this test is equivalent to TEST 9: we assembled K081014 (our RFP) under the Plux promoter that was contained into K081004. We kept pSB1AK3 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we wanted to repeat TEST 9 experiment using a longer construct and a different reporter gene.<br />
<br><br />
'''Methods:''' the same as TEST 9, but using K081004 instead of K081022 and using RFP (K081014) instead of GFP (K081012).<br />
<br><br />
'''Results:''' red fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture.<br />
{|align="center"<br />
|[[Image:pv_fig_test10.png|thumb|400px|left|Non induced culture: TOP10 cells cannot be seen at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test10bis.png|thumb|400px|left|Induced culture: fluorescent TOP10 cells at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test10superexp.png|thumb|400px|left|Same frames as above, but superexposed: non induced (left) and induced (right) cultures]]<br />
|}<br />
<br><br />
'''Comments:''' the same as TEST 9.<br />
<br><br />
NOTE: C0062 has the same mutation described in TEST 9. We also performed this test with an old version of K081004 carrying another point mutation (C->T at position 704 of C0062 coding sequence) that changed an aminoacid. Even in this case K081004 seemed to work as expected (results not shown).<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 11'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test11.png|thumb|600px|left|Terminator efficiency test]]<br />
|}<br />
'''Description:''' we assembled K081014 (our RFP) under the artificial 39 bp terminator (B1006) that is at the end of K081022-K081012 (composite part used for TEST 9). We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we wanted to test qualitatively if our terminator actually stops transcription.<br />
<br><br />
'''Methods:''' we assembled K081014 downstream of K081022, transformed ligation, plated transformed bacteria and insulated a colony containing correctly ligated plasmid. We inoculated the colony into 9 ml of LB + Amp and incubated the culture at 37°C, 220 rpm for 15 hours. Then we diluted 1:10 the culture in two falcon tubes (5 ml cultures). One of these cultures was induced with 3OC6-HSL 1 uM. We let the two cultures grow for 2 hours and then we watched 50 ul at microscope through FITC channel (positive control), TRITC channel and DAPI channel (negative control).<br />
<br><br />
'''Results:''' soon<br />
<br><br />
'''Comments:''' soon<br />
<br><br />
<br><br />
<hr><br />
<br><br />
We also performed these quantitative fluorescence tests:<br />
<hr><br />
*'''TEST 1'''<br />
<br><br />
'''Description:''' this test is an extension of TEST 9. We induced six cultures with 3OC6-HSL at different concentrations to study quantitatively HSL-GFP static transfer function.<br />
<br><br />
'''Motivation:''' we want to know cutoff point of this device.<br />
<br><br />
'''Methods:''' the same as TEST 9, but we diluted the overnight 9 ml culture 1:10 in six falcon tubes (5 ml cultures). We induced the six cultures with 3OC6-HSL 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM and 1 uM respectively. We incubated the cultures at 37°C, 220 rpm for 2 hours and then we watched 50 ul at microscope through FITC channel and DAPI channel for negative control. We prepared two 50 ul glasses for each culture. We acquired three frames at 2.5 s (maximum exposition time) and 10 ms exposition time for every sample. We used ImageJ software to count automatically the number of cells for every frame. Then we computed n10/n2.5 (where n is the number of cells) to calculate the percentage of cells that glowed in the 10 ms acquisition, assuming that in the 2.5 s acquisition we can see the total number of cells. For each HSL concentration, we calculated the mean value of the 6 statistics (3 frames for each of the 2 glasses).<br />
<br><br />
'''Results:'''<br />
{|align="center"<br />
|[[Image:pv_fig_test1q.png|thumb|600px|left|GFP (arbitrary units) vs 3OC6-HSL concentration: 1=0nM, 2=0.1nM, 3=1nM, 4=10nM, 5=100nM, 6=1uM]]<br />
|}<br />
<br><br />
'''Comments:''' we computed the statistic n10/n2.5 because we know that when fluorescence is weak, cells cannot be seen at low exposition times. So, we calculate the ratio between the cells we can see at a low exposition time and the total number of cells in the frame. We chose 10 ms because the previous experiments with GFP had been performed using this parameter. We know that this is not an exact statistic, in fact we have to consider the count errors of ImageJ software, especially when cells are superimposed. Further quantitative experiments will have to be performed using standard measurement, in order to characterize parts.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 2'''<br />
<br><br />
'''Description:''' this test is an extension of TEST 10. We induced seven cultures with 3OC6-HSL at different concentrations to study quantitatively HSL-RFP static transfer function.<br />
<br><br />
'''Motivation:''' we want to know cutoff point of this device.<br />
<br><br />
'''Methods:''' the same as TEST 10, but we diluted the overnight 9 ml culture 1:10 in seven falcon tubes (5 ml cultures). We induced the seven cultures with 3OC6-HSL 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 uM and 10 uM respectively. We incubated the cultures at 37°C, 220 rpm for 2 hours and then we watched 50 ul at microscope through TRITC channel and DAPI channel for negative control. We prepared two 50 ul glasses for each culture. We acquired three frames at 2.5 s (maximum exposition time) and 90 ms exposition time for every sample. We used ImageJ software to count automatically the number of cells for every acquisition. Then we computed n90/n2.5 (where n is the number of cells) to calculate the percentage of cells that glowed in the 90 ms acquisition, assuming that in the 2.5 s acquisition we can see the total number of cells. For each HSL concentration, we calculated the mean value of the 6 statistics (3 frames for each of the 2 glasses).<br />
<br><br />
'''Results:'''<br />
{|align="center"<br />
|[[Image:pv_fig_test2q.png|thumb|600px|left|RFP (arbitrary units) vs 3OC6-HSL concentration: 1=0nM, 2=0.1nM, 3=1nM, 4=10nM, 5=100nM, 6=1uM, 7=10uM]]<br />
|}<br />
<br><br />
'''Comments:''' we computed the statistic n90/n2.5 because we know that when fluorescence is weak, cells cannot be seen at low exposition times. So, we calculate the ratio between the cells we can see at a low exposition time and the total number of cells in the frame. We chose 90 ms because the previous experiments with RFP had been performed using this parameter. We know that this is not an exact statistic, in fact we have to consider the count errors of ImageJ software, especially when cells are superimposed. As we wrote for TEST 9, further quantitative experiments will have to be performed using standard measurement, in order to characterize parts.<br />
<br><br />
<hr><br />
<br />
=== Conclusions ===<br />
All the experiments we performed were consistent with our expectations.<br />
<br><br />
Considering the elementary logic gates of our final devices (AND, OR, NOT), we validated some truth table rows:<br />
{|align="center"<br />
|[[Image:pv_summary.png|thumb|600px|left|Biological truth tables]]<br />
|}<br />
<br />
In particular:<br />
*'''TEST 1 validated the first row of NOT logic gate.'''<br />
*'''TEST 6 validated the first row of AND (lux) logic gate.'''<br />
*'''TEST 7 validated the first row of AND (las) logic gate.'''<br />
*'''TEST 8, TEST 9 (with induction) and TEST 10 (with induction) validated the fourth row of AND (lux) logic gate.'''<br />
*'''TEST 9 (without induction) and TEST 10 (without induction) validated the third row of AND (lux) logic gate.'''</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/ProjectTeam:UNIPV-Pavia/Project2008-10-27T14:36:08Z<p>Magni: /* Applications */</p>
<hr />
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|}<br />
<br />
<br><br />
<br />
== '''Overall project''' ==<br />
<br />
We are trying to mimic Multiplexer (Mux) and Demultiplexer (Demux) logic functions in E. coli.<br />
<br><br />
In the following paragraphs project details will be described from both digital electronic and genetic points of view.<br />
<br><br />
<br><br />
<br />
== '''Electronic Implementation''' ==<br />
<br><br />
=== '''What kind of components are Mux and Demux?''' ===<br />
'''Mux''' is a component which conveys one of the two input channels values into a single output channel. The choice of the input channel is made by a selector.<br />
<br><br />
'''Demux''' is a component which conveys the only input channel value into one of the two output channels. The choice of the output channel is made by a selector.<br />
<br><br />
<br><br />
The following pictures show data flow in Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_dataflow0.png|thumb|300px|left|Data flow in Multiplexer - SELECTOR=0]]<br />
|[[Image:pv_mux_dataflow1.png|thumb|300px|left|Data flow in Multiplexer - SELECTOR=1]]<br />
|-<br />
|[[Image:pv_demux_dataflow0.png|thumb|300px|left|Data flow in Demultiplexer - SELECTOR=0]]<br />
|[[Image:pv_demux_dataflow1.png|thumb|300px|left|Data flow in Demultiplexer - SELECTOR=1]]<br />
|}<br />
<br><br />
<br />
=== '''What kind of signals do we process?''' ===<br />
In this project we consider Boolean logic signals, thus every input/output value can assume only the values 0 and 1. A function that processes Boolean values is called logic function.<br />
<br><br />
Mux and Demux can be considered by now as black boxes which implement a logic function that can process input signals to output signals. Here you can see examples of Boolean data flow in Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_bool.png|thumb|300px|left|Example: Mux Boolean data flow]]<br />
|[[Image:pv_demux_bool.png|thumb|300px|left|Example: Demux Boolean data flow]]<br />
|}<br />
In the following documentation we will see what is inside these black boxes.<br />
<br><br />
<br><br />
=== How can we formalize Mux and Demux logic behavior? ===<br />
Logic functions can be formalized writing a truth table; a truth table is a mathematical table in which every row represents a combination of input values and its respective output values. The table has to be filled with every input combination.<br />
<br><br />
<br><br />
Here you can see Mux and Demux truth tables (output columns are gray):<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_truth.png|thumb|300px|left|Mux truth table]]<br />
|[[Image:pv_demux_truth.png|thumb|300px|left|Demux truth table]]<br />
|}<br />
<br><br />
<br />
=== Building a logic circuit from a truth table ===<br />
Our goal in this section is to project two logic gates networks which behave like Mux and Demux truth tables. A very useful tool to transform a truth table into a logic network is Karnaugh map.<br />
<br><br />
It is possible to read about Karnaugh maps at: [http://en.wikipedia.org/wiki/Karnaugh_map]<br />
<br><br />
<br><br />
Following Karnaugh maps method, we can write these two logic networks for Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux.png|thumb|340px|left|Mux - logic circuit]]<br />
|[[Image:pv_mux_example.png|thumb|340px|left|Mux - Example]]<br />
|-<br />
|[[Image:pv_demux.png|thumb|340px|left|Demux - logic circuit]]<br />
|[[Image:pv_demux_example.png|thumb|340px|left|Demux - Example]]<br />
|}<br />
<br />
<br><br />
<br />
== '''Genetic Implementation''' ==<br />
Our goal is to mimic Mux and Demux logic networks in a biological device, such as E. coli. To perform this, we use protein/DNA and protein/protein interactions to build up biological logic gates.<br />
Mux and Demux logic circuits are composed by three fundamental logic gates, AND, OR, NOT: in the next paragraphs genetic implementation of these logic gates will be provided.<br />
<br><br />
<br><br />
=== AND ===<br />
To mimic an AND gate, we need a biological function, such as a promoter activation, which is directly turned on by the interaction between two upstream genes. In our synthetic devices, we use the luxR/luxI system: luxR can activate Plux promoter only upon 3-oxo-hexanoyl-homoserine lactone (HSL) binding; luxI generates HSL; so, only the contemporary expression of LuxR and luxI proteins can activate the downstream Plux-dependent gene expression. Another AND gate we use is the lasR/lasI system, which works in a very similar way but through another chemical intermediate, N-(3-oxododecanoyl) homoserine lactone (PAI-1).<br />
{|<br />
|[[Image:pv_proj_AND.png|thumb|340px|left|Genetic AND: Plux can be turned on only when the two proteins luxI and luxR are present.]]<br />
|}<br />
=== OR ===<br />
To mimic an OR gate in Mux, we need a biological function which can be activated alternatively by two independent upstream signals or by both. Thus, we combine the outputs of the upstream AND gates to assemble directly an OR reporter function, by simply repeating the reporter gene (GFP) under two different promoters (Plux and Plas). It’s sufficient to activate one of the two promoters (or both) to recover the GFP signal from engineered bacteria.<br />
There should not be an over-expression problem for GFP, in fact, in Mux device, only one promoter can be active, either Plux or Plas. Here we considered GFP output, but OR device can be generalized for every output gene.<br />
{|<br />
|[[Image:pv_proj_OR.png|thumb|340px|left|Genetic OR: it is sufficient that one of the two input promoters is active to obtain GFP expression.]]<br />
|}<br />
=== NOT ===<br />
To mimic a NOT gate, we need an efficient and regulated repressor of a specific downstream promoter: in this case, we choose cI repression on Plambda, which should be specific and, upon cI inactivation, quick and efficient.<br />
{|<br />
|[[Image:pv_proj_NOT.png|thumb|340px|left|Genetic NOT: Plambda can be turned on only when cI protein is not present.]]<br />
|}<br />
<br />
According to what above stated, genetic implementation of Mux and Demux can be obtained connecting these basic logic gates and can be summarized in this way:<br />
<br><br />
<br><br />
<br><br />
<br />
=== Genetic Mux ===<br />
Let PA, PB and PS be three generic promoters that can be ACTIVATED respectively by the three exogenous molecules "A", "B" and "S". A genetic Mux with inputs "A" (CH0) and "B" (CH1), selector "S" and the generic protein GOI as output, can be implemented by the following gene network:<br />
{|align="center"<br />
|[[Image:pv_MUX.png|thumb|420px|left|Genetic Mux]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_MUXint.png|thumb|420px|left|Genetic Mux - interactions]]<br />
|}<br />
We want to supply a device that can be generalized to detect every kind of input and to express every kind of genes as output. So, input promoters and output protein generators are not part of the genetic Mux we want to build up. In this way, a hypothetical user can re-use our device assembling the desired input and output elements.<br />
<br><br />
Note that the output genes are two, but, as explained in "OR" section, if the genes are identical, we can say that there is only one output; in fact, the real output is a protein synthesis and so it is not important which of the two identical genes is expressed.<br />
<br><br />
However, it is possible to assemble two different genes downstream of Plux and Plas, for example two reporters. In this way, debugging process becomes easy, because we can discriminate lux system and las system activities.<br />
<br><br />
Here we report the truth table of this network:<br />
{|align="center"<br />
|[[Image:pv_gmux_truth.png|thumb|420px|left|Genetic Mux - truth table]]<br />
|}<br />
<br><br />
Now we report two examples of genetic Mux behavior, in response to generic inputs.<br />
<br><br />
<br><br />
====Genetic Mux behavior - Example 1====<br />
{|<br />
|[[Image:pv_genmux_example1.png|thumb|420px|left|First example of genetic Mux behavior]]<br />
|}<br />
According to Mux truth table, if "A" is not present and "B" and "S" are present, we expect to have GOI synthesis.<br />
<br><br />
"A" molecule is not present, so PA promoter is not active and luxI gene is not expressed.<br />
<br><br />
"B" molecule is present, so PB promoter is active and lasI gene is expressed.<br />
<br><br />
"S" molecule is present, so PS promoter is active and lasR and cI genes are expressed.<br />
<br><br />
cI protein represses transcription of the gene downstream of Plambda promoter, which is luxR.<br />
<br><br />
lasI protein can synthesize 3-OC12-HSL. This molecule activates transcription factor lasR, which can activate Plas promoter. So, the copy of GOI gene under Plas regulation can be expressed.<br />
<br><br />
The other copy of GOI gene, which is under Plux promoter regulation, is not expressed. In fact Plux is not active, because both luxI and luxR proteins are not present.<br />
<br><br />
Only one of the two GOI genes is expressed, but this is sufficient to synthesize the output protein GOI.<br />
<br><br />
<br><br />
====Genetic Mux behavior - Example 2====<br />
{|<br />
|[[Image:pv_genmux_example2.png|thumb|420px|left|Second example of genetic Mux behavior]]<br />
|}<br />
We expect to have no GOI synthesis in response to this input combination.<br />
<br><br />
"A" molecule is not present, so PA promoter is not active and luxI gene is not expressed.<br />
<br><br />
"B" molecule is present, so PB promoter is active and lasI gene is expressed.<br />
<br><br />
"S" molecule is not present, so PS promoter can't transcribe cI and lasR genes.<br />
<br><br />
cI protein is not present, so Plambda promoter can transcribe luxR gene.<br />
<br><br />
None of the two logic AND systems is active, because lux system lacks of luxI and las system lacks of lasR. So, Plux and Plas promoters are unactive and can't express GOI genes. For this reason, GOI protein is not synthesized.<br />
<br><br />
<br><br />
These two examples show how "S" molecule can select the input to be conveyed into the single output channel: in fact its presence allows lasR expression and represses luxR expression, while "S" absence represses lasR expression and allows luxR expression.<br />
<br><br />
<br><br />
<br />
=== Genetic Demux ===<br />
Let PI and PS be two generic promoters that can be ACTIVATED respectively by the two exogenous molecules "I" and "S". A genetic Demux with input "I", selector "S" and the generic proteins GOI0 and GOI1 as outputs (OUT0 and OUT1 respectively), can be implemented by the following gene network:<br />
{|align="center"<br />
|[[Image:pv_DEMUX.png|thumb|420px|left|Genetic Demux]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_DEMUXint.png|thumb|420px|left|Genetic Demux - interactions]]<br />
|}<br />
As described for genetic Mux, we want to supply a device that can be generalized to detect every kind of inputs and to express every kind of genes as output. So, input promoters and output protein generators are not part of the genetic Demux. In this way, a hypothetical user can re-use our device assembling the desired input and output elements.<br />
<br><br />
Here we report the truth table of this network:<br />
{|align="center"<br />
|[[Image:pv_gdemux_truth.png|thumb|420px|left|Genetic Demux - truth table]]<br />
|}<br />
<br><br />
Now we report two examples of genetic Demux behavior, in response to generic inputs.<br />
<br><br />
<br><br />
====Genetic Demux behavior - Example 1====<br />
{|<br />
|[[Image:pv_gendemux_example1.png|thumb|420px|left|First example of genetic Demux behavior]]<br />
|}<br />
According to Demux truth table, if "I" molecule is present and "S" molecule is absent, we expect to have GOI0 protein synthesis and no GOI1 protein synthesis.<br />
<br><br />
"I" is present, so PI promoter is active and luxI and lasI genes are expressed.<br />
<br><br />
"S" is not present, so PS promoter can't transcribe lasR and cI genes.<br />
<br><br />
cI protein is not present, so Plambda promoter is not inhibited and luxR gene is expressed.<br />
<br><br />
luxI protein can synthesize 3-OC6-HSL lactone. This molecule activates luxR transcription factor which can activate Plux promoter. In this way, GOI0 gene, which is under Plux regulation, is expressed.<br />
<br><br />
On the other hand, lasI protein can synthesize 3-OC12-HSL, but lasR transcription factor is not present and so Plas promoter cannot be activated. In this way, GOI1 gene, which is under Plas regulation, is not expressed.<br />
<br><br />
So, we have GOI0 protein synthesis and no GOI1 protein synthesis.<br />
<br><br />
<br><br />
====Genetic Demux behavior - Example 2====<br />
{|<br />
|[[Image:pv_gendemux_example2.png|thumb|420px|left|Second example of genetic Demux behavior]]<br />
|}<br />
We expect to have GOI1 synthesis and no GOI0 synthesis in response to this input combination.<br />
<br><br />
"I" is present, so PI promoter is active and luxI and lasI genes are expressed.<br />
<br><br />
"S" is present, so PS promoter lasR and cI genes are expressed.<br />
<br><br />
cI protein is present, so Plambda promoter is inhibited and luxR gene is not expressed.<br />
<br><br />
lasI protein can synthesize 3-OC12-HSL lactone. This molecule activates lasR transcription factor which can activate Plas promoter. In this way, GOI1 gene, which is under Plas regulation, is expressed.<br />
<br><br />
On the other hand, luxI protein can synthesize 3-OC6-HSL, but luxR transcription factor is not present and so Plux promoter cannot be activated. In this way, GOI0 gene, which is under Plux regulation, is not expressed.<br />
<br><br />
So, we have GOI1 protein synthesis and no GOI0 protein synthesis.<br />
<br />
<br />
=== A complete genetic Mux===<br />
In this section a complete Mux gene network is described.<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_implementation.png|thumb|420px|left|Example of a complete genetic Mux]]<br />
|[[Image:pv_mux_gn_implementation.png|thumb|420px|left|Example of a complete genetic Mux - Gene network]]<br />
|}<br />
We want to build up a device in which Channel 0, Channel 1 and Selector are respectively sensitive to Tetracycline, IPTG and red light. The presence of each input corresponds to logic 1.<br />
We chose green fluorescence as Mux output: expression of GFP corresponds to logic 1, while absence of fluorescence corresponds to logic 0.<br />
<br><br />
Tetracycline and IPTG can be considered as "A" and "B" molecules, introduced in "Genetic Mux" section, because both Tetracycline and IPTG are indirect ACTIVATORS of Ptet and Plac promoters respectively.<br />
On the other hand, red light is quite different from "S" molecule, because red light is an indirect REPRESSOR of Pomp promoter and not an activator as required by the original schema. For this reason, if we want a device in which the presence of Tetracycline, IPTG and red light correspond to logic 1, red light input should be ''inverted''. The simplest way to do this, is to cross-exchange the two input channels. So, Tetracycline sensor (CH0) has to be assembled to las system and IPTG sensor (CH1) has to be assembled to lux system.<br />
<br />
<br />
====Complete genetic Mux - Example====<br />
{|<br />
|[[Image:pv_mux_example1.png|thumb|420px|left|Example of a complete genetic Mux behavior]]<br />
|}<br />
According to Mux truth table, if SEL=0, CH0=1, CH1=0, output channel must be logic 1.<br />
Red light is not present, so it can't dephosphorylate cph8-ho1-pcyA complex, which is constitutively expressed. cph8-ho1-pcyA complex activates endogenous ompR, which can activate Pomp promoter, so cI and lasR genes are transcribed.<br />
cI protein binds Plambda promoter and so luxR expression is inhibited.<br />
TetR is a repressor for Ptet promoter, but Tetracycline is present, so it can bind tetR protein, which is constitutively expressed, and can activate transcription of downstream gene: lasI.<br />
Simultaneous expression of lasI and lasR can activate GFP, which is under Plas promoter.<br />
IPTG is not present, so lacI protein binds Plac promoter and represses luxI transcription.<br />
<br />
<br />
=== A complete genetic Demux===<br />
In this section a complete Demux gene network is described.<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_demux_implementation.png|thumb|420px|left|Example of a complete genetic Demux]]<br />
|[[Image:pv_demux_gn_implementation.png|thumb|420px|left|Example of a complete genetic Demux - Gene network]]<br />
|}<br />
<br />
We want to build up a device in which Input and Selector are respectively IPTG and Tetracycline.<br><br />
In Demux we have two output channels: red fluorescence corresponds to logic 1 at Channel 0, while green fluorescence corresponds to logic 1 at Channel 1.<br><br />
Absence of reporters expression corresponds to logic 0 at Channel 0 and Channel 1.<br><br />
There isn't any input combination that corresponds to logic 1 at Channel 0 and Channel 1 together.<br />
<br />
====Complete genetic Demux - Example====<br />
{|<br />
|[[Image:pv_demux_example1.png|thumb|420px|left|Example of a complete genetic Demux behavior]]<br />
|}<br />
According to Demux truth table, if IN=1 and SEL=1, output channel 0 is logic 0 and output channel 1 is logic 1.<br><br />
IPTG is present, so Plac promoter is active because IPTG binds lacI protein. This allows lasI and luxI transcription.<br><br />
Tetracycline is present, so Ptet promoter is active because Tetracycline binds tetR protein. This allows cI and lasR transcription.<br><br />
cI protein binds Plambda promoter and so luxR expression is inhibited.<br><br />
Simultaneous expression of lasI and lasR can activate GFP, which is under Plas promoter.<br><br />
There is no simultaneous expression of luxI and luxR, so RFP (which is under Plux regulation) cannot be expressed.<br />
<br />
== Final devices==<br />
BioBrick standard parts for genetic Mux and Demux are summarized in the following schemas:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_final.png|thumb|420px|left|Three standard parts for mux]]<br />
|[[Image:pv_demux_final.png|thumb|450px|left|Two standard parts for demux]]<br />
|}<br />
<br />
A hypothetical user of Mux or Demux has to ligate our standard parts with desired inputs and outputs, as shown in the pictures above.<br />
<br><br />
The structure of our devices show that Mux and Demux systems both conform to the PoPS device boundary standard.<br />
<br><br />
<br />
==Applications==<br />
Mux and Demux are two fundamental devices in electronics. They are used in several applications, for example in communication devices, in Arithmetic Logic Units (ALUs), or, in general, in applications that involve channel sharing.<br />
<br><br />
Analogously, they could play a crucial role in the building of complex genetic circuits. In fact, both of them can be used as controlled genetic switches.<br />
<br><br />
Because our devices had been designed to be general, their application field is very wide. For example, genetic Mux can be used to integrate signals from the environment in a two-inputs and one-output biosensor; once detected, the selector controls which of the two inputs must be transferred in output. In this way, two sensing devices can be integrated in only one circuit that can compute multiplexing logic function.<br />
On the other hand, genetic Demux can be used for controlled protein productions, where the choice of the protein to produce is made by the selector. In this case, we can imagine an industrial process where two consequent enzyme reactions are necessary to build a final product. Using a Demux, we can consider the two enzymes as system outputs and then we can easily switch on and off their production modulating selection signal.<br />
<br><br />
Selector in Mux and Demux can be controlled manually, but also automatically. In fact, it is possible to use feedback control to pilot the selector in order, according with Demux example, to switch enzyme production when a specific condition (for example a pH threshold) is reached.<br />
<br><br />
Switching activity in these two devices is a very powerful tool to manage multi-input and multi-output systems.<br />
<br />
== Experiments and results ==<br />
=== Assemblies ===<br />
We successfully amplified the following BioBrick standard parts from Spring 2008 DNA Distribution:<br />
{|align="center"<br />
|[[Image:pv_resusp.png|thumb|600px|left|Successful amplifications]]<br />
|}<br />
<br><br />
while we couldn't amplify correctly the following parts:<br />
{|align="center"<br />
|[[Image:pv_notresusp.png|thumb|600px|left|Unsuccessful amplifications]]<br />
|}<br />
<br><br />
To build up our designed devices, we followed and completed this assembly tree schema:<br />
{|align="center"<br />
|[[Image:pv_assemblyschema.png|thumb|600px|left|Assembly tree schema]]<br />
|}<br />
<br />
=== Functional tests ===<br />
We performed these boolean (on/off) fluorescence tests:<br />
<br><br />
<hr><br />
*'''TEST 1'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test1.png|thumb|250px|left|GFP protein generator under Plambda]]<br />
|}<br />
'''Description:''' we assembled an available GFP protein generator (E0240) under Plambda promoter, keeping pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' cI protein was not present, so Plambda should work like a constitutive promoter. A reporter gene downstream of this promoter allows us to validate Plambda costitutive activity. That can be very useful to validate our NOT logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of R0051 and E0240, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test1.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that GFP was expressed in positive transformants, so Plambda activity was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 2'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test2.png|thumb|250px|left|GFP protein generator under Ptet]]<br />
|}<br />
'''Description:''' we assembled E0240 under Ptet promoter, keeping pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' tetR protein was not present, so Ptet should work like a constitutive promoter. A reporter gene downstream of this promoter allows us to validate Ptet costitutive activity. Ptet is useful for specific input building.<br />
<br><br />
'''Methods:''' after ligation reaction of R0040 and E0240, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test2.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that GFP was expressed in positive transformants, so Ptet activity was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 3'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test3.png|thumb|250px|left|RFP protein generator under constitutive promoter]]<br />
|}<br />
'''Description:''' we didn't perform any assembly for this experiment, because the promoter we wanted to test was contained into BBa_J61002 vector, which places a RFP protein generator between SpeI and PstI restriction sites.<br />
<br><br />
'''Motivation:''' J23100, which we call Pcon, should have a strong constitutive activity. A reporter gene downstream of this promoter allows us to validate this activity. Pcon is useful to build our inputs because some sensors like IPTG and Tetracycline sensors need the constitutive production of specific proteins, in this case lacI and tetR respectively.<br />
<br><br />
'''Methods''' we transformed J23100 using Invitrogen TOP10 and plated transformed bacteria. We expected to observe red fluorescence (using transluminator) in all the colonies. We also picked up a colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through TRITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' all the grown colonies glowed under UV rays and red fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test3.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that RFP was expressed in transformed bacteria, so Pcon functionality was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 4'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test4.png|thumb|250px|left|Our GFP protein generator (K081012) under a constitutive promoter]]<br />
|}<br />
'''Description:''' we assembled a constitutive promoter family member (J23100) upstream of K081012, a GFP protein generator we built. We decided to excide J23100 from its plasmid (E-S) and to open K081012 plasmid (pSB1AK3) (E-X).<br />
<br><br />
'''Motivation:''' we already validated qualitatively J23100 constitutive activity, so this promoter can be used to test the GFP protein generator we built. We wanted to check if K081012 actually generates GFP.<br />
<br><br />
'''Methods''' after ligation reaction of J23100 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays (see figure) and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test4bis.jpg|thumb|400px|left|TOP10 colonies under UV]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test4.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that our GFP protein generator actually generates GFP.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 5'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test5.png|thumb|250px|left|Our RFP protein generator (K081014) under a constitutive promoter]]<br />
|}<br />
'''Description:''' we assembled a constitutive promoter family member (J23100) upstream of K081014, a RFP protein generator we built. We decided to excide J23100 from its plasmid (E-S) and to open K081014 plasmid (pSB1AK3) (E-X).<br />
<br><br />
'''Motivation:''' we already validated qualitatively J23100 constitutive activity, so this promoter can be used to test the RFP protein generator we built. We wanted to check if K081014 actually generates RFP.<br />
<br><br />
'''Methods''' after ligation reaction of J23100 and K081014, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through TRITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays (see figure) and red fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test5bis.jpg|thumb|400px|left|TOP10 colonies under UV]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test5.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that our RFP protein generator actually generates RFP.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 6'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test6.png|thumb|350px|left|GFP protein generator under Plux]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081000. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we didn't assemble any input to K081000 device, so we could study only the promoter basic activity. We expected to find a weak basic activity. Plux is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of K081000 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we performed a screening on 5 colonies through colony PCR to search for correctly ligated plasmid. We infected 9 ml of LB + Amp with the chosen colony and incubated the culture at 37°C, 220 rpm for 15 hours. Then we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test6.png|thumb|400px|left|No fluorescent TOP10 cells can be seen at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' the picture above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria. Increasing exposition time, green fluorescence could be observed (picture not reported). So, we can say that GFP was expressed very weakly. This experiment confirmed that Plux promoter has a weak activity without luxI and luxR proteins.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 7'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test7.png|thumb|350px|left|GFP protein generator under Plas]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plas promoter that was contained into K081001. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we didn't assemble any input to K081001 device, so we could study only the promoter basic activity. We expected to find a weak basic activity. Plas is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of K081001 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we performed a screening on 5 colonies through colony PCR to search for correctly ligated plasmid. We infected 9 ml of LB + Amp with the chosen colony and incubated the culture at 37°C, 220 rpm for 15 hours. Then we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test7.png|thumb|400px|left|No fluorescent TOP10 cells can be seen at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' the picture above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria. Increasing exposition time, green fluorescence could be observed (picture not reported). So, we can say that GFP was expressed very weakly. This experiment confirmed that Plas promoter has a weak activity without lasI and lasR proteins.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 8'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test8.png|thumb|600px|left|GFP protein generator under Plux and constitutive expression of luxI and luxR]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081000. We also assembled K081011 upstream of K081000. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' Plux promoter activity was studied in response of a constitutive expression of luxI and luxR. Plambda promoter without cI repressor guaranteed the constitutive expression of these genes. We expected to find a strong activity because Plux is turned on. lux system is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' we ligated K081012 under K081000, transformed ligation, plated transformed bacteria, performed PCR screening on some colonies and extracted correctly ligated plasmids. We repeated these steps to assemble K081011 upstream of K081000-K081012, but we didn't perform PCR screening. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test8.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that Plux promoter can be activated by the contemporary presence of luxI and luxR. This is a crucial result for our AND logic gate.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 9'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test9.png|thumb|500px|left|GFP protein generator under Plux and constitutive expression of luxR]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081022. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' Plux promoter activity was studied in response to a constitutive expression of luxR. Plambda promoter without cI repressor guaranteed the constitutive expression of this gene. We expected to find a weak activity because luxR transcription factor is not active and so Plux cannot be turned on. We also expected to find a strong activity if we induce luxR activation using 3OC6-HSL (this is equivalent to TEST 8 conditions, because 3OC6-HSL is synthesized by luxI). lux system is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' we ligated K081012 downstream of K081022, transformed ligation, plated transformed bacteria and screened three colonies to insulate a colony containing correctly ligated plasmids. We inoculated the positive colony into 9 ml of LB + Amp and incubated the culture at 37°C, 220 rpm for 15 hours. Then we diluted 1:10 the culture in two falcon tubes (5 ml cultures). One of these cultures was induced with 3OC6-HSL 1 uM. We let the two cultures grow for 2 hours and then we watched 50 ul at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture.<br />
{|align="center"<br />
|[[Image:pv_fig_test9.png|thumb|400px|left|Non induced culture: TOP10 cells cannot be seen at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test9bis.png|thumb|400px|left|Induced culture: fluorescent TOP10 cells at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test9superexp.png|thumb|400px|left|Same frames as above, but superexposed: non induced (left) and induced (right) cultures]]<br />
|}<br />
<br><br />
'''Comments:''' the pictures above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria for the non induced culture, while we can see green fluorescent TOP10 in the induced culture. As we wrote for TEST 6 and TEST 7, increasing exposition time green fluorescence could be observed even for the non induced culture (last picture), confirming the weak activity of Plux promoter in response of unactive luxR. This experiment confirmed that Plux promoter has a weak activity without luxI (or 3OC6-HSL) and luxR protein is not sufficient to induce a strong transcription. Adding 3OC6-HSL, luxR becomes active and so Plux is turned on.<br />
<br><br />
NOTE: K081022 has a point mutation in position 349 of C0062 coding sequence. This mutation changes the aminoacid, but luxR seems to work as expected.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 10'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test10.png|thumb|600px|left|RFP protein generator under Plux and constitutive expression of luxR]]<br />
|}<br />
'''Description:''' this test is equivalent to TEST 9: we assembled K081014 (our RFP) under the Plux promoter that was contained into K081004. We kept pSB1AK3 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we wanted to repeat TEST 9 experiment using a longer construct and a different reporter gene.<br />
<br><br />
'''Methods:''' the same as TEST 9, but using K081004 instead of K081022 and using RFP (K081014) instead of GFP (K081012).<br />
<br><br />
'''Results:''' red fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture.<br />
{|align="center"<br />
|[[Image:pv_fig_test10.png|thumb|400px|left|Non induced culture: TOP10 cells cannot be seen at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test10bis.png|thumb|400px|left|Induced culture: fluorescent TOP10 cells at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test10superexp.png|thumb|400px|left|Same frames as above, but superexposed: non induced (left) and induced (right) cultures]]<br />
|}<br />
<br><br />
'''Comments:''' the same as TEST 9.<br />
<br><br />
NOTE: C0062 has the same mutation described in TEST 9. We also performed this test with an old version of K081004 carrying another point mutation (C->T at position 704 of C0062 coding sequence) that changed an aminoacid. Even in this case K081004 seemed to work as expected (results not shown).<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 11'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test11.png|thumb|600px|left|Terminator efficiency test]]<br />
|}<br />
'''Description:''' we assembled K081014 (our RFP) under the artificial 39 bp terminator (B1006) that is at the end of K081022-K081012 (composite part used for TEST 9). We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we wanted to test qualitatively if our terminator actually stops transcription.<br />
<br><br />
'''Methods:''' we assembled K081014 downstream of K081022, transformed ligation, plated transformed bacteria and insulated a colony containing correctly ligated plasmid. We inoculated the colony into 9 ml of LB + Amp and incubated the culture at 37°C, 220 rpm for 15 hours. Then we diluted 1:10 the culture in two falcon tubes (5 ml cultures). One of these cultures was induced with 3OC6-HSL 1 uM. We let the two cultures grow for 2 hours and then we watched 50 ul at microscope through FITC channel (positive control), TRITC channel and DAPI channel (negative control).<br />
<br><br />
'''Results:''' soon<br />
<br><br />
'''Comments:''' soon<br />
<br><br />
<br><br />
<hr><br />
<br><br />
We also performed these quantitative fluorescence tests:<br />
<hr><br />
*'''TEST 1'''<br />
<br><br />
'''Description:''' this test is an extension of TEST 9. We induced six cultures with 3OC6-HSL at different concentrations to study quantitatively HSL-GFP static transfer function.<br />
<br><br />
'''Motivation:''' we want to know cutoff point of this device.<br />
<br><br />
'''Methods:''' the same as TEST 9, but we diluted the overnight 9 ml culture 1:10 in six falcon tubes (5 ml cultures). We induced the six cultures with 3OC6-HSL 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM and 1 uM respectively. We incubated the cultures at 37°C, 220 rpm for 2 hours and then we watched 50 ul at microscope through FITC channel and DAPI channel for negative control. We prepared two 50 ul glasses for each culture. We acquired three frames at 2.5 s (maximum exposition time) and 10 ms exposition time for every sample. We used ImageJ software to count automatically the number of cells for every frame. Then we computed n10/n2.5 (where n is the number of cells) to calculate the percentage of cells that glowed in the 10 ms acquisition, assuming that in the 2.5 s acquisition we can see the total number of cells. For each HSL concentration, we calculated the mean value of the 6 statistics (3 frames for each of the 2 glasses).<br />
<br><br />
'''Results:'''<br />
{|align="center"<br />
|[[Image:pv_fig_test1q.png|thumb|600px|left|GFP (arbitrary units) vs 3OC6-HSL concentration: 1=0nM, 2=0.1nM, 3=1nM, 4=10nM, 5=100nM, 6=1uM]]<br />
|}<br />
<br><br />
'''Comments:''' we computed the statistic n10/n2.5 because we know that when fluorescence is weak, cells cannot be seen at low exposition times. So, we calculate the ratio between the cells we can see at a low exposition time and the total number of cells in the frame. We chose 10 ms because the previous experiments with GFP had been performed using this parameter. We know that this is not an exact statistic, in fact we have to consider the count errors of ImageJ software, especially when cells are superimposed. Further quantitative experiments will have to be performed using standard measurement, in order to characterize parts.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 2'''<br />
<br><br />
'''Description:''' this test is an extension of TEST 10. We induced seven cultures with 3OC6-HSL at different concentrations to study quantitatively HSL-RFP static transfer function.<br />
<br><br />
'''Motivation:''' we want to know cutoff point of this device.<br />
<br><br />
'''Methods:''' the same as TEST 10, but we diluted the overnight 9 ml culture 1:10 in seven falcon tubes (5 ml cultures). We induced the seven cultures with 3OC6-HSL 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 uM and 10 uM respectively. We incubated the cultures at 37°C, 220 rpm for 2 hours and then we watched 50 ul at microscope through TRITC channel and DAPI channel for negative control. We prepared two 50 ul glasses for each culture. We acquired three frames at 2.5 s (maximum exposition time) and 90 ms exposition time for every sample. We used ImageJ software to count automatically the number of cells for every acquisition. Then we computed n90/n2.5 (where n is the number of cells) to calculate the percentage of cells that glowed in the 90 ms acquisition, assuming that in the 2.5 s acquisition we can see the total number of cells. For each HSL concentration, we calculated the mean value of the 6 statistics (3 frames for each of the 2 glasses).<br />
<br><br />
'''Results:'''<br />
{|align="center"<br />
|[[Image:pv_fig_test2q.png|thumb|600px|left|RFP (arbitrary units) vs 3OC6-HSL concentration: 1=0nM, 2=0.1nM, 3=1nM, 4=10nM, 5=100nM, 6=1uM, 7=10uM]]<br />
|}<br />
<br><br />
'''Comments:''' we computed the statistic n90/n2.5 because we know that when fluorescence is weak, cells cannot be seen at low exposition times. So, we calculate the ratio between the cells we can see at a low exposition time and the total number of cells in the frame. We chose 90 ms because the previous experiments with RFP had been performed using this parameter. We know that this is not an exact statistic, in fact we have to consider the count errors of ImageJ software, especially when cells are superimposed. As we wrote for TEST 9, further quantitative experiments will have to be performed using standard measurement, in order to characterize parts.<br />
<br><br />
<hr><br />
<br />
=== Conclusions ===<br />
All the experiments we performed were consistent with our expectations.<br />
<br><br />
Considering the elementary logic gates of our final devices (AND, OR, NOT), we validated some truth table rows:<br />
{|align="center"<br />
|[[Image:pv_summary.png|thumb|600px|left|Biological truth tables]]<br />
|}<br />
<br />
In particular:<br />
*'''TEST 1 validated the first row of NOT logic gate.'''<br />
*'''TEST 6 validated the first row of AND (lux) logic gate.'''<br />
*'''TEST 7 validated the first row of AND (las) logic gate.'''<br />
*'''TEST 8, TEST 9 (with induction) and TEST 10 (with induction) validated the fourth row of AND (lux) logic gate.'''<br />
*'''TEST 9 (without induction) and TEST 10 (without induction) validated the third row of AND (lux) logic gate.'''</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/ProjectTeam:UNIPV-Pavia/Project2008-10-27T14:35:50Z<p>Magni: /* Applications */</p>
<hr />
<div>{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
== '''Overall project''' ==<br />
<br />
We are trying to mimic Multiplexer (Mux) and Demultiplexer (Demux) logic functions in E. coli.<br />
<br><br />
In the following paragraphs project details will be described from both digital electronic and genetic points of view.<br />
<br><br />
<br><br />
<br />
== '''Electronic Implementation''' ==<br />
<br><br />
=== '''What kind of components are Mux and Demux?''' ===<br />
'''Mux''' is a component which conveys one of the two input channels values into a single output channel. The choice of the input channel is made by a selector.<br />
<br><br />
'''Demux''' is a component which conveys the only input channel value into one of the two output channels. The choice of the output channel is made by a selector.<br />
<br><br />
<br><br />
The following pictures show data flow in Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_dataflow0.png|thumb|300px|left|Data flow in Multiplexer - SELECTOR=0]]<br />
|[[Image:pv_mux_dataflow1.png|thumb|300px|left|Data flow in Multiplexer - SELECTOR=1]]<br />
|-<br />
|[[Image:pv_demux_dataflow0.png|thumb|300px|left|Data flow in Demultiplexer - SELECTOR=0]]<br />
|[[Image:pv_demux_dataflow1.png|thumb|300px|left|Data flow in Demultiplexer - SELECTOR=1]]<br />
|}<br />
<br><br />
<br />
=== '''What kind of signals do we process?''' ===<br />
In this project we consider Boolean logic signals, thus every input/output value can assume only the values 0 and 1. A function that processes Boolean values is called logic function.<br />
<br><br />
Mux and Demux can be considered by now as black boxes which implement a logic function that can process input signals to output signals. Here you can see examples of Boolean data flow in Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_bool.png|thumb|300px|left|Example: Mux Boolean data flow]]<br />
|[[Image:pv_demux_bool.png|thumb|300px|left|Example: Demux Boolean data flow]]<br />
|}<br />
In the following documentation we will see what is inside these black boxes.<br />
<br><br />
<br><br />
=== How can we formalize Mux and Demux logic behavior? ===<br />
Logic functions can be formalized writing a truth table; a truth table is a mathematical table in which every row represents a combination of input values and its respective output values. The table has to be filled with every input combination.<br />
<br><br />
<br><br />
Here you can see Mux and Demux truth tables (output columns are gray):<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_truth.png|thumb|300px|left|Mux truth table]]<br />
|[[Image:pv_demux_truth.png|thumb|300px|left|Demux truth table]]<br />
|}<br />
<br><br />
<br />
=== Building a logic circuit from a truth table ===<br />
Our goal in this section is to project two logic gates networks which behave like Mux and Demux truth tables. A very useful tool to transform a truth table into a logic network is Karnaugh map.<br />
<br><br />
It is possible to read about Karnaugh maps at: [http://en.wikipedia.org/wiki/Karnaugh_map]<br />
<br><br />
<br><br />
Following Karnaugh maps method, we can write these two logic networks for Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux.png|thumb|340px|left|Mux - logic circuit]]<br />
|[[Image:pv_mux_example.png|thumb|340px|left|Mux - Example]]<br />
|-<br />
|[[Image:pv_demux.png|thumb|340px|left|Demux - logic circuit]]<br />
|[[Image:pv_demux_example.png|thumb|340px|left|Demux - Example]]<br />
|}<br />
<br />
<br><br />
<br />
== '''Genetic Implementation''' ==<br />
Our goal is to mimic Mux and Demux logic networks in a biological device, such as E. coli. To perform this, we use protein/DNA and protein/protein interactions to build up biological logic gates.<br />
Mux and Demux logic circuits are composed by three fundamental logic gates, AND, OR, NOT: in the next paragraphs genetic implementation of these logic gates will be provided.<br />
<br><br />
<br><br />
=== AND ===<br />
To mimic an AND gate, we need a biological function, such as a promoter activation, which is directly turned on by the interaction between two upstream genes. In our synthetic devices, we use the luxR/luxI system: luxR can activate Plux promoter only upon 3-oxo-hexanoyl-homoserine lactone (HSL) binding; luxI generates HSL; so, only the contemporary expression of LuxR and luxI proteins can activate the downstream Plux-dependent gene expression. Another AND gate we use is the lasR/lasI system, which works in a very similar way but through another chemical intermediate, N-(3-oxododecanoyl) homoserine lactone (PAI-1).<br />
{|<br />
|[[Image:pv_proj_AND.png|thumb|340px|left|Genetic AND: Plux can be turned on only when the two proteins luxI and luxR are present.]]<br />
|}<br />
=== OR ===<br />
To mimic an OR gate in Mux, we need a biological function which can be activated alternatively by two independent upstream signals or by both. Thus, we combine the outputs of the upstream AND gates to assemble directly an OR reporter function, by simply repeating the reporter gene (GFP) under two different promoters (Plux and Plas). It’s sufficient to activate one of the two promoters (or both) to recover the GFP signal from engineered bacteria.<br />
There should not be an over-expression problem for GFP, in fact, in Mux device, only one promoter can be active, either Plux or Plas. Here we considered GFP output, but OR device can be generalized for every output gene.<br />
{|<br />
|[[Image:pv_proj_OR.png|thumb|340px|left|Genetic OR: it is sufficient that one of the two input promoters is active to obtain GFP expression.]]<br />
|}<br />
=== NOT ===<br />
To mimic a NOT gate, we need an efficient and regulated repressor of a specific downstream promoter: in this case, we choose cI repression on Plambda, which should be specific and, upon cI inactivation, quick and efficient.<br />
{|<br />
|[[Image:pv_proj_NOT.png|thumb|340px|left|Genetic NOT: Plambda can be turned on only when cI protein is not present.]]<br />
|}<br />
<br />
According to what above stated, genetic implementation of Mux and Demux can be obtained connecting these basic logic gates and can be summarized in this way:<br />
<br><br />
<br><br />
<br><br />
<br />
=== Genetic Mux ===<br />
Let PA, PB and PS be three generic promoters that can be ACTIVATED respectively by the three exogenous molecules "A", "B" and "S". A genetic Mux with inputs "A" (CH0) and "B" (CH1), selector "S" and the generic protein GOI as output, can be implemented by the following gene network:<br />
{|align="center"<br />
|[[Image:pv_MUX.png|thumb|420px|left|Genetic Mux]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_MUXint.png|thumb|420px|left|Genetic Mux - interactions]]<br />
|}<br />
We want to supply a device that can be generalized to detect every kind of input and to express every kind of genes as output. So, input promoters and output protein generators are not part of the genetic Mux we want to build up. In this way, a hypothetical user can re-use our device assembling the desired input and output elements.<br />
<br><br />
Note that the output genes are two, but, as explained in "OR" section, if the genes are identical, we can say that there is only one output; in fact, the real output is a protein synthesis and so it is not important which of the two identical genes is expressed.<br />
<br><br />
However, it is possible to assemble two different genes downstream of Plux and Plas, for example two reporters. In this way, debugging process becomes easy, because we can discriminate lux system and las system activities.<br />
<br><br />
Here we report the truth table of this network:<br />
{|align="center"<br />
|[[Image:pv_gmux_truth.png|thumb|420px|left|Genetic Mux - truth table]]<br />
|}<br />
<br><br />
Now we report two examples of genetic Mux behavior, in response to generic inputs.<br />
<br><br />
<br><br />
====Genetic Mux behavior - Example 1====<br />
{|<br />
|[[Image:pv_genmux_example1.png|thumb|420px|left|First example of genetic Mux behavior]]<br />
|}<br />
According to Mux truth table, if "A" is not present and "B" and "S" are present, we expect to have GOI synthesis.<br />
<br><br />
"A" molecule is not present, so PA promoter is not active and luxI gene is not expressed.<br />
<br><br />
"B" molecule is present, so PB promoter is active and lasI gene is expressed.<br />
<br><br />
"S" molecule is present, so PS promoter is active and lasR and cI genes are expressed.<br />
<br><br />
cI protein represses transcription of the gene downstream of Plambda promoter, which is luxR.<br />
<br><br />
lasI protein can synthesize 3-OC12-HSL. This molecule activates transcription factor lasR, which can activate Plas promoter. So, the copy of GOI gene under Plas regulation can be expressed.<br />
<br><br />
The other copy of GOI gene, which is under Plux promoter regulation, is not expressed. In fact Plux is not active, because both luxI and luxR proteins are not present.<br />
<br><br />
Only one of the two GOI genes is expressed, but this is sufficient to synthesize the output protein GOI.<br />
<br><br />
<br><br />
====Genetic Mux behavior - Example 2====<br />
{|<br />
|[[Image:pv_genmux_example2.png|thumb|420px|left|Second example of genetic Mux behavior]]<br />
|}<br />
We expect to have no GOI synthesis in response to this input combination.<br />
<br><br />
"A" molecule is not present, so PA promoter is not active and luxI gene is not expressed.<br />
<br><br />
"B" molecule is present, so PB promoter is active and lasI gene is expressed.<br />
<br><br />
"S" molecule is not present, so PS promoter can't transcribe cI and lasR genes.<br />
<br><br />
cI protein is not present, so Plambda promoter can transcribe luxR gene.<br />
<br><br />
None of the two logic AND systems is active, because lux system lacks of luxI and las system lacks of lasR. So, Plux and Plas promoters are unactive and can't express GOI genes. For this reason, GOI protein is not synthesized.<br />
<br><br />
<br><br />
These two examples show how "S" molecule can select the input to be conveyed into the single output channel: in fact its presence allows lasR expression and represses luxR expression, while "S" absence represses lasR expression and allows luxR expression.<br />
<br><br />
<br><br />
<br />
=== Genetic Demux ===<br />
Let PI and PS be two generic promoters that can be ACTIVATED respectively by the two exogenous molecules "I" and "S". A genetic Demux with input "I", selector "S" and the generic proteins GOI0 and GOI1 as outputs (OUT0 and OUT1 respectively), can be implemented by the following gene network:<br />
{|align="center"<br />
|[[Image:pv_DEMUX.png|thumb|420px|left|Genetic Demux]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_DEMUXint.png|thumb|420px|left|Genetic Demux - interactions]]<br />
|}<br />
As described for genetic Mux, we want to supply a device that can be generalized to detect every kind of inputs and to express every kind of genes as output. So, input promoters and output protein generators are not part of the genetic Demux. In this way, a hypothetical user can re-use our device assembling the desired input and output elements.<br />
<br><br />
Here we report the truth table of this network:<br />
{|align="center"<br />
|[[Image:pv_gdemux_truth.png|thumb|420px|left|Genetic Demux - truth table]]<br />
|}<br />
<br><br />
Now we report two examples of genetic Demux behavior, in response to generic inputs.<br />
<br><br />
<br><br />
====Genetic Demux behavior - Example 1====<br />
{|<br />
|[[Image:pv_gendemux_example1.png|thumb|420px|left|First example of genetic Demux behavior]]<br />
|}<br />
According to Demux truth table, if "I" molecule is present and "S" molecule is absent, we expect to have GOI0 protein synthesis and no GOI1 protein synthesis.<br />
<br><br />
"I" is present, so PI promoter is active and luxI and lasI genes are expressed.<br />
<br><br />
"S" is not present, so PS promoter can't transcribe lasR and cI genes.<br />
<br><br />
cI protein is not present, so Plambda promoter is not inhibited and luxR gene is expressed.<br />
<br><br />
luxI protein can synthesize 3-OC6-HSL lactone. This molecule activates luxR transcription factor which can activate Plux promoter. In this way, GOI0 gene, which is under Plux regulation, is expressed.<br />
<br><br />
On the other hand, lasI protein can synthesize 3-OC12-HSL, but lasR transcription factor is not present and so Plas promoter cannot be activated. In this way, GOI1 gene, which is under Plas regulation, is not expressed.<br />
<br><br />
So, we have GOI0 protein synthesis and no GOI1 protein synthesis.<br />
<br><br />
<br><br />
====Genetic Demux behavior - Example 2====<br />
{|<br />
|[[Image:pv_gendemux_example2.png|thumb|420px|left|Second example of genetic Demux behavior]]<br />
|}<br />
We expect to have GOI1 synthesis and no GOI0 synthesis in response to this input combination.<br />
<br><br />
"I" is present, so PI promoter is active and luxI and lasI genes are expressed.<br />
<br><br />
"S" is present, so PS promoter lasR and cI genes are expressed.<br />
<br><br />
cI protein is present, so Plambda promoter is inhibited and luxR gene is not expressed.<br />
<br><br />
lasI protein can synthesize 3-OC12-HSL lactone. This molecule activates lasR transcription factor which can activate Plas promoter. In this way, GOI1 gene, which is under Plas regulation, is expressed.<br />
<br><br />
On the other hand, luxI protein can synthesize 3-OC6-HSL, but luxR transcription factor is not present and so Plux promoter cannot be activated. In this way, GOI0 gene, which is under Plux regulation, is not expressed.<br />
<br><br />
So, we have GOI1 protein synthesis and no GOI0 protein synthesis.<br />
<br />
<br />
=== A complete genetic Mux===<br />
In this section a complete Mux gene network is described.<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_implementation.png|thumb|420px|left|Example of a complete genetic Mux]]<br />
|[[Image:pv_mux_gn_implementation.png|thumb|420px|left|Example of a complete genetic Mux - Gene network]]<br />
|}<br />
We want to build up a device in which Channel 0, Channel 1 and Selector are respectively sensitive to Tetracycline, IPTG and red light. The presence of each input corresponds to logic 1.<br />
We chose green fluorescence as Mux output: expression of GFP corresponds to logic 1, while absence of fluorescence corresponds to logic 0.<br />
<br><br />
Tetracycline and IPTG can be considered as "A" and "B" molecules, introduced in "Genetic Mux" section, because both Tetracycline and IPTG are indirect ACTIVATORS of Ptet and Plac promoters respectively.<br />
On the other hand, red light is quite different from "S" molecule, because red light is an indirect REPRESSOR of Pomp promoter and not an activator as required by the original schema. For this reason, if we want a device in which the presence of Tetracycline, IPTG and red light correspond to logic 1, red light input should be ''inverted''. The simplest way to do this, is to cross-exchange the two input channels. So, Tetracycline sensor (CH0) has to be assembled to las system and IPTG sensor (CH1) has to be assembled to lux system.<br />
<br />
<br />
====Complete genetic Mux - Example====<br />
{|<br />
|[[Image:pv_mux_example1.png|thumb|420px|left|Example of a complete genetic Mux behavior]]<br />
|}<br />
According to Mux truth table, if SEL=0, CH0=1, CH1=0, output channel must be logic 1.<br />
Red light is not present, so it can't dephosphorylate cph8-ho1-pcyA complex, which is constitutively expressed. cph8-ho1-pcyA complex activates endogenous ompR, which can activate Pomp promoter, so cI and lasR genes are transcribed.<br />
cI protein binds Plambda promoter and so luxR expression is inhibited.<br />
TetR is a repressor for Ptet promoter, but Tetracycline is present, so it can bind tetR protein, which is constitutively expressed, and can activate transcription of downstream gene: lasI.<br />
Simultaneous expression of lasI and lasR can activate GFP, which is under Plas promoter.<br />
IPTG is not present, so lacI protein binds Plac promoter and represses luxI transcription.<br />
<br />
<br />
=== A complete genetic Demux===<br />
In this section a complete Demux gene network is described.<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_demux_implementation.png|thumb|420px|left|Example of a complete genetic Demux]]<br />
|[[Image:pv_demux_gn_implementation.png|thumb|420px|left|Example of a complete genetic Demux - Gene network]]<br />
|}<br />
<br />
We want to build up a device in which Input and Selector are respectively IPTG and Tetracycline.<br><br />
In Demux we have two output channels: red fluorescence corresponds to logic 1 at Channel 0, while green fluorescence corresponds to logic 1 at Channel 1.<br><br />
Absence of reporters expression corresponds to logic 0 at Channel 0 and Channel 1.<br><br />
There isn't any input combination that corresponds to logic 1 at Channel 0 and Channel 1 together.<br />
<br />
====Complete genetic Demux - Example====<br />
{|<br />
|[[Image:pv_demux_example1.png|thumb|420px|left|Example of a complete genetic Demux behavior]]<br />
|}<br />
According to Demux truth table, if IN=1 and SEL=1, output channel 0 is logic 0 and output channel 1 is logic 1.<br><br />
IPTG is present, so Plac promoter is active because IPTG binds lacI protein. This allows lasI and luxI transcription.<br><br />
Tetracycline is present, so Ptet promoter is active because Tetracycline binds tetR protein. This allows cI and lasR transcription.<br><br />
cI protein binds Plambda promoter and so luxR expression is inhibited.<br><br />
Simultaneous expression of lasI and lasR can activate GFP, which is under Plas promoter.<br><br />
There is no simultaneous expression of luxI and luxR, so RFP (which is under Plux regulation) cannot be expressed.<br />
<br />
== Final devices==<br />
BioBrick standard parts for genetic Mux and Demux are summarized in the following schemas:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_final.png|thumb|420px|left|Three standard parts for mux]]<br />
|[[Image:pv_demux_final.png|thumb|450px|left|Two standard parts for demux]]<br />
|}<br />
<br />
A hypothetical user of Mux or Demux has to ligate our standard parts with desired inputs and outputs, as shown in the pictures above.<br />
<br><br />
The structure of our devices show that Mux and Demux systems both conform to the PoPS device boundary standard.<br />
<br><br />
<br />
==Applications==<br />
Mux and Demux are two fundamental devices in electronics. They are used in several applications, for example in communication devices, in Arithmetic Logic Units (ALUs), or in general in applications that involve channel sharing.<br />
<br><br />
Analogously, they could play a crucial role in the building of complex genetic circuits. In fact, both of them can be used as controlled genetic switches.<br />
<br><br />
Because our devices had been designed to be general, their application field is very wide. For example, genetic Mux can be used to integrate signals from the environment in a two-inputs and one-output biosensor; once detected, the selector controls which of the two inputs must be transferred in output. In this way, two sensing devices can be integrated in only one circuit that can compute multiplexing logic function.<br />
On the other hand, genetic Demux can be used for controlled protein productions, where the choice of the protein to produce is made by the selector. In this case, we can imagine an industrial process where two consequent enzyme reactions are necessary to build a final product. Using a Demux, we can consider the two enzymes as system outputs and then we can easily switch on and off their production modulating selection signal.<br />
<br><br />
Selector in Mux and Demux can be controlled manually, but also automatically. In fact, it is possible to use feedback control to pilot the selector in order, according with Demux example, to switch enzyme production when a specific condition (for example a pH threshold) is reached.<br />
<br><br />
Switching activity in these two devices is a very powerful tool to manage multi-input and multi-output systems.<br />
<br />
== Experiments and results ==<br />
=== Assemblies ===<br />
We successfully amplified the following BioBrick standard parts from Spring 2008 DNA Distribution:<br />
{|align="center"<br />
|[[Image:pv_resusp.png|thumb|600px|left|Successful amplifications]]<br />
|}<br />
<br><br />
while we couldn't amplify correctly the following parts:<br />
{|align="center"<br />
|[[Image:pv_notresusp.png|thumb|600px|left|Unsuccessful amplifications]]<br />
|}<br />
<br><br />
To build up our designed devices, we followed and completed this assembly tree schema:<br />
{|align="center"<br />
|[[Image:pv_assemblyschema.png|thumb|600px|left|Assembly tree schema]]<br />
|}<br />
<br />
=== Functional tests ===<br />
We performed these boolean (on/off) fluorescence tests:<br />
<br><br />
<hr><br />
*'''TEST 1'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test1.png|thumb|250px|left|GFP protein generator under Plambda]]<br />
|}<br />
'''Description:''' we assembled an available GFP protein generator (E0240) under Plambda promoter, keeping pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' cI protein was not present, so Plambda should work like a constitutive promoter. A reporter gene downstream of this promoter allows us to validate Plambda costitutive activity. That can be very useful to validate our NOT logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of R0051 and E0240, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test1.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that GFP was expressed in positive transformants, so Plambda activity was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 2'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test2.png|thumb|250px|left|GFP protein generator under Ptet]]<br />
|}<br />
'''Description:''' we assembled E0240 under Ptet promoter, keeping pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' tetR protein was not present, so Ptet should work like a constitutive promoter. A reporter gene downstream of this promoter allows us to validate Ptet costitutive activity. Ptet is useful for specific input building.<br />
<br><br />
'''Methods:''' after ligation reaction of R0040 and E0240, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test2.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that GFP was expressed in positive transformants, so Ptet activity was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 3'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test3.png|thumb|250px|left|RFP protein generator under constitutive promoter]]<br />
|}<br />
'''Description:''' we didn't perform any assembly for this experiment, because the promoter we wanted to test was contained into BBa_J61002 vector, which places a RFP protein generator between SpeI and PstI restriction sites.<br />
<br><br />
'''Motivation:''' J23100, which we call Pcon, should have a strong constitutive activity. A reporter gene downstream of this promoter allows us to validate this activity. Pcon is useful to build our inputs because some sensors like IPTG and Tetracycline sensors need the constitutive production of specific proteins, in this case lacI and tetR respectively.<br />
<br><br />
'''Methods''' we transformed J23100 using Invitrogen TOP10 and plated transformed bacteria. We expected to observe red fluorescence (using transluminator) in all the colonies. We also picked up a colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through TRITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' all the grown colonies glowed under UV rays and red fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test3.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that RFP was expressed in transformed bacteria, so Pcon functionality was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 4'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test4.png|thumb|250px|left|Our GFP protein generator (K081012) under a constitutive promoter]]<br />
|}<br />
'''Description:''' we assembled a constitutive promoter family member (J23100) upstream of K081012, a GFP protein generator we built. We decided to excide J23100 from its plasmid (E-S) and to open K081012 plasmid (pSB1AK3) (E-X).<br />
<br><br />
'''Motivation:''' we already validated qualitatively J23100 constitutive activity, so this promoter can be used to test the GFP protein generator we built. We wanted to check if K081012 actually generates GFP.<br />
<br><br />
'''Methods''' after ligation reaction of J23100 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays (see figure) and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test4bis.jpg|thumb|400px|left|TOP10 colonies under UV]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test4.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that our GFP protein generator actually generates GFP.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 5'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test5.png|thumb|250px|left|Our RFP protein generator (K081014) under a constitutive promoter]]<br />
|}<br />
'''Description:''' we assembled a constitutive promoter family member (J23100) upstream of K081014, a RFP protein generator we built. We decided to excide J23100 from its plasmid (E-S) and to open K081014 plasmid (pSB1AK3) (E-X).<br />
<br><br />
'''Motivation:''' we already validated qualitatively J23100 constitutive activity, so this promoter can be used to test the RFP protein generator we built. We wanted to check if K081014 actually generates RFP.<br />
<br><br />
'''Methods''' after ligation reaction of J23100 and K081014, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through TRITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays (see figure) and red fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test5bis.jpg|thumb|400px|left|TOP10 colonies under UV]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test5.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that our RFP protein generator actually generates RFP.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 6'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test6.png|thumb|350px|left|GFP protein generator under Plux]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081000. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we didn't assemble any input to K081000 device, so we could study only the promoter basic activity. We expected to find a weak basic activity. Plux is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of K081000 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we performed a screening on 5 colonies through colony PCR to search for correctly ligated plasmid. We infected 9 ml of LB + Amp with the chosen colony and incubated the culture at 37°C, 220 rpm for 15 hours. Then we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test6.png|thumb|400px|left|No fluorescent TOP10 cells can be seen at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' the picture above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria. Increasing exposition time, green fluorescence could be observed (picture not reported). So, we can say that GFP was expressed very weakly. This experiment confirmed that Plux promoter has a weak activity without luxI and luxR proteins.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 7'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test7.png|thumb|350px|left|GFP protein generator under Plas]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plas promoter that was contained into K081001. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we didn't assemble any input to K081001 device, so we could study only the promoter basic activity. We expected to find a weak basic activity. Plas is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of K081001 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we performed a screening on 5 colonies through colony PCR to search for correctly ligated plasmid. We infected 9 ml of LB + Amp with the chosen colony and incubated the culture at 37°C, 220 rpm for 15 hours. Then we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test7.png|thumb|400px|left|No fluorescent TOP10 cells can be seen at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' the picture above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria. Increasing exposition time, green fluorescence could be observed (picture not reported). So, we can say that GFP was expressed very weakly. This experiment confirmed that Plas promoter has a weak activity without lasI and lasR proteins.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 8'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test8.png|thumb|600px|left|GFP protein generator under Plux and constitutive expression of luxI and luxR]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081000. We also assembled K081011 upstream of K081000. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' Plux promoter activity was studied in response of a constitutive expression of luxI and luxR. Plambda promoter without cI repressor guaranteed the constitutive expression of these genes. We expected to find a strong activity because Plux is turned on. lux system is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' we ligated K081012 under K081000, transformed ligation, plated transformed bacteria, performed PCR screening on some colonies and extracted correctly ligated plasmids. We repeated these steps to assemble K081011 upstream of K081000-K081012, but we didn't perform PCR screening. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test8.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that Plux promoter can be activated by the contemporary presence of luxI and luxR. This is a crucial result for our AND logic gate.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 9'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test9.png|thumb|500px|left|GFP protein generator under Plux and constitutive expression of luxR]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081022. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' Plux promoter activity was studied in response to a constitutive expression of luxR. Plambda promoter without cI repressor guaranteed the constitutive expression of this gene. We expected to find a weak activity because luxR transcription factor is not active and so Plux cannot be turned on. We also expected to find a strong activity if we induce luxR activation using 3OC6-HSL (this is equivalent to TEST 8 conditions, because 3OC6-HSL is synthesized by luxI). lux system is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' we ligated K081012 downstream of K081022, transformed ligation, plated transformed bacteria and screened three colonies to insulate a colony containing correctly ligated plasmids. We inoculated the positive colony into 9 ml of LB + Amp and incubated the culture at 37°C, 220 rpm for 15 hours. Then we diluted 1:10 the culture in two falcon tubes (5 ml cultures). One of these cultures was induced with 3OC6-HSL 1 uM. We let the two cultures grow for 2 hours and then we watched 50 ul at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture.<br />
{|align="center"<br />
|[[Image:pv_fig_test9.png|thumb|400px|left|Non induced culture: TOP10 cells cannot be seen at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test9bis.png|thumb|400px|left|Induced culture: fluorescent TOP10 cells at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test9superexp.png|thumb|400px|left|Same frames as above, but superexposed: non induced (left) and induced (right) cultures]]<br />
|}<br />
<br><br />
'''Comments:''' the pictures above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria for the non induced culture, while we can see green fluorescent TOP10 in the induced culture. As we wrote for TEST 6 and TEST 7, increasing exposition time green fluorescence could be observed even for the non induced culture (last picture), confirming the weak activity of Plux promoter in response of unactive luxR. This experiment confirmed that Plux promoter has a weak activity without luxI (or 3OC6-HSL) and luxR protein is not sufficient to induce a strong transcription. Adding 3OC6-HSL, luxR becomes active and so Plux is turned on.<br />
<br><br />
NOTE: K081022 has a point mutation in position 349 of C0062 coding sequence. This mutation changes the aminoacid, but luxR seems to work as expected.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 10'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test10.png|thumb|600px|left|RFP protein generator under Plux and constitutive expression of luxR]]<br />
|}<br />
'''Description:''' this test is equivalent to TEST 9: we assembled K081014 (our RFP) under the Plux promoter that was contained into K081004. We kept pSB1AK3 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we wanted to repeat TEST 9 experiment using a longer construct and a different reporter gene.<br />
<br><br />
'''Methods:''' the same as TEST 9, but using K081004 instead of K081022 and using RFP (K081014) instead of GFP (K081012).<br />
<br><br />
'''Results:''' red fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture.<br />
{|align="center"<br />
|[[Image:pv_fig_test10.png|thumb|400px|left|Non induced culture: TOP10 cells cannot be seen at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test10bis.png|thumb|400px|left|Induced culture: fluorescent TOP10 cells at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test10superexp.png|thumb|400px|left|Same frames as above, but superexposed: non induced (left) and induced (right) cultures]]<br />
|}<br />
<br><br />
'''Comments:''' the same as TEST 9.<br />
<br><br />
NOTE: C0062 has the same mutation described in TEST 9. We also performed this test with an old version of K081004 carrying another point mutation (C->T at position 704 of C0062 coding sequence) that changed an aminoacid. Even in this case K081004 seemed to work as expected (results not shown).<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 11'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test11.png|thumb|600px|left|Terminator efficiency test]]<br />
|}<br />
'''Description:''' we assembled K081014 (our RFP) under the artificial 39 bp terminator (B1006) that is at the end of K081022-K081012 (composite part used for TEST 9). We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we wanted to test qualitatively if our terminator actually stops transcription.<br />
<br><br />
'''Methods:''' we assembled K081014 downstream of K081022, transformed ligation, plated transformed bacteria and insulated a colony containing correctly ligated plasmid. We inoculated the colony into 9 ml of LB + Amp and incubated the culture at 37°C, 220 rpm for 15 hours. Then we diluted 1:10 the culture in two falcon tubes (5 ml cultures). One of these cultures was induced with 3OC6-HSL 1 uM. We let the two cultures grow for 2 hours and then we watched 50 ul at microscope through FITC channel (positive control), TRITC channel and DAPI channel (negative control).<br />
<br><br />
'''Results:''' soon<br />
<br><br />
'''Comments:''' soon<br />
<br><br />
<br><br />
<hr><br />
<br><br />
We also performed these quantitative fluorescence tests:<br />
<hr><br />
*'''TEST 1'''<br />
<br><br />
'''Description:''' this test is an extension of TEST 9. We induced six cultures with 3OC6-HSL at different concentrations to study quantitatively HSL-GFP static transfer function.<br />
<br><br />
'''Motivation:''' we want to know cutoff point of this device.<br />
<br><br />
'''Methods:''' the same as TEST 9, but we diluted the overnight 9 ml culture 1:10 in six falcon tubes (5 ml cultures). We induced the six cultures with 3OC6-HSL 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM and 1 uM respectively. We incubated the cultures at 37°C, 220 rpm for 2 hours and then we watched 50 ul at microscope through FITC channel and DAPI channel for negative control. We prepared two 50 ul glasses for each culture. We acquired three frames at 2.5 s (maximum exposition time) and 10 ms exposition time for every sample. We used ImageJ software to count automatically the number of cells for every frame. Then we computed n10/n2.5 (where n is the number of cells) to calculate the percentage of cells that glowed in the 10 ms acquisition, assuming that in the 2.5 s acquisition we can see the total number of cells. For each HSL concentration, we calculated the mean value of the 6 statistics (3 frames for each of the 2 glasses).<br />
<br><br />
'''Results:'''<br />
{|align="center"<br />
|[[Image:pv_fig_test1q.png|thumb|600px|left|GFP (arbitrary units) vs 3OC6-HSL concentration: 1=0nM, 2=0.1nM, 3=1nM, 4=10nM, 5=100nM, 6=1uM]]<br />
|}<br />
<br><br />
'''Comments:''' we computed the statistic n10/n2.5 because we know that when fluorescence is weak, cells cannot be seen at low exposition times. So, we calculate the ratio between the cells we can see at a low exposition time and the total number of cells in the frame. We chose 10 ms because the previous experiments with GFP had been performed using this parameter. We know that this is not an exact statistic, in fact we have to consider the count errors of ImageJ software, especially when cells are superimposed. Further quantitative experiments will have to be performed using standard measurement, in order to characterize parts.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 2'''<br />
<br><br />
'''Description:''' this test is an extension of TEST 10. We induced seven cultures with 3OC6-HSL at different concentrations to study quantitatively HSL-RFP static transfer function.<br />
<br><br />
'''Motivation:''' we want to know cutoff point of this device.<br />
<br><br />
'''Methods:''' the same as TEST 10, but we diluted the overnight 9 ml culture 1:10 in seven falcon tubes (5 ml cultures). We induced the seven cultures with 3OC6-HSL 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 uM and 10 uM respectively. We incubated the cultures at 37°C, 220 rpm for 2 hours and then we watched 50 ul at microscope through TRITC channel and DAPI channel for negative control. We prepared two 50 ul glasses for each culture. We acquired three frames at 2.5 s (maximum exposition time) and 90 ms exposition time for every sample. We used ImageJ software to count automatically the number of cells for every acquisition. Then we computed n90/n2.5 (where n is the number of cells) to calculate the percentage of cells that glowed in the 90 ms acquisition, assuming that in the 2.5 s acquisition we can see the total number of cells. For each HSL concentration, we calculated the mean value of the 6 statistics (3 frames for each of the 2 glasses).<br />
<br><br />
'''Results:'''<br />
{|align="center"<br />
|[[Image:pv_fig_test2q.png|thumb|600px|left|RFP (arbitrary units) vs 3OC6-HSL concentration: 1=0nM, 2=0.1nM, 3=1nM, 4=10nM, 5=100nM, 6=1uM, 7=10uM]]<br />
|}<br />
<br><br />
'''Comments:''' we computed the statistic n90/n2.5 because we know that when fluorescence is weak, cells cannot be seen at low exposition times. So, we calculate the ratio between the cells we can see at a low exposition time and the total number of cells in the frame. We chose 90 ms because the previous experiments with RFP had been performed using this parameter. We know that this is not an exact statistic, in fact we have to consider the count errors of ImageJ software, especially when cells are superimposed. As we wrote for TEST 9, further quantitative experiments will have to be performed using standard measurement, in order to characterize parts.<br />
<br><br />
<hr><br />
<br />
=== Conclusions ===<br />
All the experiments we performed were consistent with our expectations.<br />
<br><br />
Considering the elementary logic gates of our final devices (AND, OR, NOT), we validated some truth table rows:<br />
{|align="center"<br />
|[[Image:pv_summary.png|thumb|600px|left|Biological truth tables]]<br />
|}<br />
<br />
In particular:<br />
*'''TEST 1 validated the first row of NOT logic gate.'''<br />
*'''TEST 6 validated the first row of AND (lux) logic gate.'''<br />
*'''TEST 7 validated the first row of AND (las) logic gate.'''<br />
*'''TEST 8, TEST 9 (with induction) and TEST 10 (with induction) validated the fourth row of AND (lux) logic gate.'''<br />
*'''TEST 9 (without induction) and TEST 10 (without induction) validated the third row of AND (lux) logic gate.'''</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/TeamTeam:UNIPV-Pavia/Team2008-10-27T14:05:34Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
== '''Who we are''' ==<br />
{|border = "0"<br />
|-<br />
|rowspan="3"|<br />
<br />
<br />
<br />
'''Instructors:'''<br />
<br />
*''' Maria Gabriella Cusella''': professor of Anatomy<br />
*''' Paolo Magni''': professor of Bioinformatics<br />
<br />
<br />
'''Advisor:'''<br />
<br />
*''' Daniela Galli''': post-doc researcher working on Tissue Engineering<br />
<br />
<br />
'''Undergrads:'''<br />
<br />
*'''Lorenzo Pasotti''': Master student in Biomedical Engineering.<br />
*'''Mattia Quattrocelli''': Master student in Molecular Biology<br />
<br />
<!--project design, wet lab activities, wiki updating, modeling, trained Mattia about logic gates and networks--><br />
<!--project design, BioBrick parts choice, wet lab activities coordinator and operator, training Lorenzo about vectors and Molecular Biology principles--><br />
<br />
<br />
|<br />
<table><br />
<tr><br />
<td><br />
[[Image:cusella.jpg|thumb|75px|center|M.G.Cusella]]<br />
</td><br />
<td><br />
[[Image:magni.jpg|thumb|120px|center|P.Magni]]<br />
</td><br />
<td><br />
[[Image:galli.jpg|thumb|140px|center|D.Galli]]<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
[[Image:pasotti.jpg|thumb|100px|center|Paso]]<br />
</td><br />
<td><br />
[[Image:quattrocelli.jpg|thumb|100px|center|Mattia]]<br />
</td><br />
</tr><br />
</table><br />
<br />
<!--<br />
<gallery><br />
Image:cusella.png|Maria Gabriella Cusella<br />
Image:magni.jpg|Paolo Magni<br />
Image:galli.jpg|Daniela Galli<br />
Image:pasotti.jpg|Lorenzo Pasotti<br />
<br />
Image:quattrocelli.jpg|Mattia Quattrocelli<br />
</gallery><br />
--><br />
|}<br />
<br />
<br><br />
<br />
== '''What we did & collaboration with other iGEM 2008 Teams''' ==<br />
<br />
Paolo Magni and colleagues wanted to introduce Biomedical Engineering students into Synthetic Biology. So, he decided to get some information from Silvio Cavalcanti group (Bologna University), who already worked in this field.<br />
At the beginning of 2008 Paolo Magni visited Bologna University labs, located in Cesena, to understand what kind of wet lab resources were necessary to develop a Synthetic Biology project and, discussing with Silvio Cavalcanti and Francesca Ceroni from Bologna group, the iGEM competition seemed to be the right opportunity to start a project.<br />
Because Engineering faculty in Pavia had not the required facilities, it was necessary to involve Maria Gabriella Cusella, who leads a Molecular Biology lab in a structure that already worked with Engineering faculty (Center for Tissue Engineering).<br />
Then they started selecting students to build up the iGEM team. They chose one Biomedical Engineering master student, Lorenzo Pasotti, one Molecular Biology master student, Mattia Quattrocelli, and a lab advisor, Daniela Galli.<br />
In the first weeks of April, Lorenzo trained Mattia about Digital Electronics principles and mathematical modeling.<br />
In the meanwhile, Mattia trained Lorenzo about wet lab work. The training included a presentation of all the lab instrumentation, basic Microbiology and Molecular Biology protocols and safety issues.<br />
These activities allowed the two students to learn the preliminary skills to start a multidisciplinary collaboration and then they started to get information from literature and from the Registry, to understand what kind of original device they could design.<br />
After some weeks they two chose a project and, after some meetings with the other team members, they begun project implementation when Spring 2008 DNA Distribution arrived.<br />
The two students worked together in lab to build up and test the designed devices. They also organized periodic meetings with the team instructors to discuss about project progress. Daniela Galli supervised students' wet lab activity and was always ready to help to solve specific biological problems.<br />
During our activities, we found very useful to keep in touch with Bologna iGEM Team to share doubts and issues about the work. In particular, several conference calls were organized and two meetings were scheduled in Pisa and Bressanone (Italy).<br />
It was fundamental to compare lab protocols and techniques to help each other avoiding mistakes and speeding up project progress. The main topics of our discussion were the optimization of plasmid resuspension and ligation reaction steps as well as how to measure fluorescence. Finally, before DNA Repository quality control publication on the Registry web site, we cross-checked some parts that showed problems after DNA transformation. Problems had been confirmed by quality control results (parts' sequences classified as "inconsistent"). In particular, we were interested in red light sensor parts (see "notebook" section for more information).<br />
<br />
== '''Where we're from''' ==<br />
<br />
Pavia is a town of south-western Lombardy, northern Italy, 35 km south of Milan on the lower Ticino river near its confluence with the Po. It has a population of about 71,000.<br />
<br />
Pavia is the capital of a fertile province known for agricultural products including wine, rice, cereals, and dairy products. Some industries located in the suburbs do not disturb the peaceful atmosphere which comes from the preservation of the city's past and the climate of study and meditation associated with its ancient University. The University of Pavia, together with the IUSS (Institute for Advanced Studies of Pavia), the Ghislieri College, the Borromeo College, the Nuovo College, the Santa Caterina College and the EDiSU, belongs to the Pavia Study System.<br />
<br />
Pavia University is one of the oldest universities in Europe and the oldest one in Lombardy. Since its foundation in 1361 it has been a good place to study for both Italian and foreign students.<br />
<br />
Each year, thousands of students can appreciate the multidisciplinary vocation of our University and the hospitality of its campus, really unique in Italy for the possibility it offers of living and studying in a lively, intellectually challenging environment.<br />
<br />
{|align="center"<br />
|[[Image:pavialocation.jpg|300px]]<br />
|}<br />
<br />
The University of Pavia today:<br />
<br><br />
3 hospitals<br />
<br><br />
9 faculties<br />
<br><br />
15 colleges<br />
<br><br />
20 master degrees<br />
<br><br />
35 libraries<br />
<br><br />
49 departements<br />
<br><br />
54 specialization schools<br />
<br><br />
104 degree couses<br />
<br><br />
310 exchange relationships over the world<br />
<br><br />
1.120 professors<br />
<br><br />
1.600 study grants<br />
<br><br />
4.000 graduated each year<br />
<br><br />
23.000 students</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/PartsTeam:UNIPV-Pavia/Parts2008-10-27T14:03:50Z<p>Magni: /* Assembled Parts Notation */</p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
===Assembled Parts Notation===<br />
In notebook section we sometimes use abbreviations to name assembled parts. New parts' official name, notebook abbreviations and new parts description are reported in the following table:<br />
<br />
<br />
{|border="1" width="60%"<br />
!New Part<br />
!Notebook Name<br />
!Assembled Parts(code)<br />
!Assembled Parts(name)<br />
!Vector<br />
!Description<br />
|-<br />
|''-''<br />
|01<br />
|J23100-E0240<br />
|Pcon-GFP_protein_generator<br />
|J61002<br />
|Test<br />
|-<br />
|<font color="green">'''K081005'''</font><br />
|<font color="green">'''02'''</font><br />
|<font color="green">'''J23100-B0030'''</font><br />
|<font color="green">'''Pcon-RBS'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081006'''</font><br />
|<font color="green">'''03'''</font><br />
|<font color="green">'''R0051-B0030'''</font><br />
|<font color="green">'''Plambda-RBS'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''S04119'''</font><br />
|<font color="green">'''04'''</font><br />
|<font color="green">'''B0030-E0040'''</font><br />
|<font color="green">'''RBS-GFP'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081007'''</font><br />
|<font color="green">'''05'''</font><br />
|<font color="green">'''B0030-C0051'''</font><br />
|<font color="green">'''RBS-cI'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''S04120'''</font><br />
|<font color="green">'''06'''</font><br />
|<font color="green">'''23100-E1010'''</font><br />
|<font color="green">'''RBS-RFP'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081008'''</font><br />
|<font color="green">'''07'''</font><br />
|<font color="green">'''B0030-C0061'''</font><br />
|<font color="green">'''RBS-luxI'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081009'''</font><br />
|<font color="green">'''08'''</font><br />
|<font color="green">'''B0030-C0078'''</font><br />
|<font color="green">'''RBS-lasI'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|style="background:gray"|''-''<br />
|style="background:gray"|09<br />
|style="background:gray"|J23100-B0030-C0012<br />
|style="background:gray"|Pcon-RBS-lacI<br />
|style="background:gray"|pSB1A2<br />
|style="background:gray"|Intermediate<br />
|-<br />
|''K081010''<br />
|10<br />
|J23100-B0030-C0040<br />
|Pcon-RBS-tetR<br />
|pSB1A2<br />
|Intermediate<br />
|-<br />
|style="background:red; color:white"|''-''<br />
|style="background:red; color:white"|11<br />
|style="background:red; color:white"|J23100-B0030-I15010<br />
|style="background:red; color:white"|Pcon-RBS-cph8<br />
|style="background:red; color:white"|pSB1A2<br />
|style="background:red; color:white"|Intermediate<br />
|-<br />
|<font color="green">'''K081011'''</font><br />
|<font color="green">'''12'''</font><br />
|<font color="green">'''R0051-B0030-C0062'''</font><br />
|<font color="green">'''Plambda-RBS-luxR'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081012'''</font><br />
|<font color="green">'''13'''</font><br />
|<font color="green">'''B0030-E0040-B1006'''</font><br />
|<font color="green">'''RBS-GFP-T'''</font><br />
|<font color="green">'''pSB1AK3'''</font><br />
|<font color="green">'''GFP protein generator with 39bp artificial terminator'''</font><br />
|-<br />
|<font color="green">'''K081013'''</font><br />
|<font color="green">'''14'''</font><br />
|<font color="green">'''B0030-C0051-B0030'''</font><br />
|<font color="green">'''RBS-cI-RBS'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081014'''</font><br />
|<font color="green">'''15'''</font><br />
|<font color="green">'''B0030-E1010-B1006'''</font><br />
|<font color="green">'''RBS-RFP-T'''</font><br />
|<font color="green">'''pSB1AK3'''</font><br />
|<font color="green">'''GFP protein generator with 39bp artificial terminator'''</font><br />
|-<br />
|''K081015''<br />
|16<br />
|B0030-C0061-B1006<br />
|RBS-luxI-T<br />
|pSB1AK3<br />
|luxI protein generator RCT<br />
|-<br />
|''K081016''<br />
|17<br />
|B0030-C0078-B1006<br />
|RBS-lasI-T<br />
|pSB1AK3<br />
|lasI protein generator RCT<br />
|-<br />
|''K081017''<br />
|18<br />
|B0030-I15009<br />
|RBS-pcyA<br />
|pSB1A2<br />
|Intermediate<br />
|-<br />
|style="background:gray"|''-''<br />
|style="background:gray"|19<br />
|style="background:gray"|J23100-B0030-C0012-B1006<br />
|style="background:gray"|Pcon-RBS-lacI-T<br />
|style="background:gray"|pSB1AK3<br />
|style="background:gray"|lacI protein generator PRCT<br />
|-<br />
|<font color="green">'''K081018'''</font><br />
|<font color="green">'''20'''</font><br />
|<font color="green">'''J23100-B0030-C0040-B1006'''</font><br />
|<font color="green">'''Pcon-RBS-tetR-T'''</font><br />
|<font color="green">'''pSB1AK3'''</font><br />
|<font color="green">'''tetR protein generator PCRT'''</font><br />
|-<br />
|style="background:red; color:white"|''-''<br />
|style="background:red; color:white"|21<br />
|style="background:red; color:white"|J23100-B0030-I15010-B0030<br />
|style="background:red; color:white"|Pcon-RBS-cph8-RBS<br />
|style="background:red; color:white"|?<br />
|style="background:red; color:white"|Intermediate<br />
|-<br />
|<font color="green">'''K081019'''</font><br />
|<font color="green">'''22'''</font><br />
|<font color="green">'''R0051-B0030-C0062-B1006'''</font><br />
|<font color="green">'''Plambda-RBS-luxR-T'''</font><br />
|<font color="green">'''pSB1AK3'''</font><br />
|<font color="green">'''Plambda regulated luxR protein generator PRCT'''</font><br />
|-<br />
|''K081020''<br />
|23<br />
|B0030-C0051-B0030-C0079<br />
|RBS-cI-RBS-lasR<br />
|pSB1A2<br />
|Intermediate<br />
|-<br />
|<font color="green">'''K081000'''</font><br />
|<font color="green">'''24'''</font><br />
|<font color="green">'''B0030-C0061-B1006-R0062'''</font><br />
|<font color="green">'''RBS-luxI-T-Plux'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Mux - Channel 0'''</font><br />
|-<br />
|<font color="green">'''K081001'''</font><br />
|<font color="green">'''25'''</font><br />
|<font color="green">'''B0030-C0078-B1006-R0079'''</font><br />
|<font color="green">'''RBS-lasI-T-Plas'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Mux - Channel 1'''</font><br />
|-<br />
|''K081021''<br />
|26<br />
|B0030-I15009-B1006<br />
|RBS-pcyA-T<br />
|pSB1AK3<br />
|pcyA protein generator RCT<br />
|-<br />
|style="background:gray"|''-''<br />
|style="background:gray"|27<br />
|style="background:gray"|J23100-B0030-C0012-B1006-R0010<br />
|style="background:gray"|Pcon-RBS-lacI-T-Plac<br />
|style="background:gray"|pSB1AK3<br />
|style="background:gray"|lacI composite part: PRCT-Plac<br />
|-<br />
|style="background:gray"|''-''<br />
|style="background:gray"|28<br />
|style="background:gray"|J23100-B0030-C0040-B1006-R0040<br />
|style="background:gray"|Pcon-RBS-tetR-T-Ptet<br />
|style="background:gray"|pSB1A2<br />
|style="background:gray"|tetR composite part: PRCT-Ptet<br />
|-<br />
|style="background:red; color:white"|''-''<br />
|style="background:red; color:white"|29<br />
|style="background:red; color:white"|J23100-B0030-I15010-B0030-I15008<br />
|style="background:red; color:white"|Pcon-RBS-cph8-RBS-ho1<br />
|style="background:red; color:white"|?<br />
|style="background:red; color:white"|Intermediate<br />
|-<br />
|<font color="red">'''K081022'''</font><br />
|<font color="red">'''30'''</font><br />
|<font color="red">'''R0051-B0030-C0062-B1006-R0062'''</font><br />
|<font color="red">'''Plambda-RBS-luxR-T-Plux'''</font><br />
|<font color="red">'''pSB1A2'''</font><br />
|<font color="red">'''luxR composite part: Plambda regulated PRCT-Plux'''</font><br />
|-<br />
|<font color="blue">'''K081023'''</font><br />
|<font color="blue">'''31'''</font><br />
|<font color="blue">'''B0030-C0051-B0030-C0079-B1006'''</font><br />
|<font color="blue">'''RBS-cI-RBS-lasR-T'''</font><br />
|<font color="blue">'''pSB1AK3'''</font><br />
|<font color="blue">'''cI-lasR protein generator RCRCT'''</font><br />
|-<br />
|''K081024''<br />
|32<br />
|B0030-I15009-B1006-R0082<br />
|RBS-pcyA-T-Pomp<br />
|pSB1A2<br />
|pcyA composite part: RCT-Pomp<br />
|-<br />
|style="background:red; color:white"|''-''<br />
|style="background:red; color:white"|33<br />
|style="background:red; color:white"|J23100-B0030-I15010-B0030-I15008-B0030-I15009-B1006-R0082<br />
|style="background:red; color:white"|Pcon-RBS-cph8-RBS-ho1-RBS-pcyA-T-Pomp<br />
|style="background:red; color:white"|?<br />
|style="background:red; color:white"|Light responsive system under constitutive promoter: PRCRCRCT-Pomp<br />
|-<br />
|<font color="red">'''K081004'''</font><br />
|<font color="red">'''34'''</font><br />
|<font color="red">'''B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062'''</font><br />
|<font color="red">'''RBS-cI-RBS-lasR-T-Plambda-RBS-luxR-T-Plux'''</font><br />
|<font color="red">'''pSB1AK3'''</font><br />
|<font color="red">'''Demux - Selector'''</font><br />
|-<br />
|<font color="blue">'''K081002'''</font><br />
|<font color="blue">'''35'''</font><br />
|<font color="blue">'''B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006'''</font><br />
|<font color="blue">'''RBS-cI-RBS-lasR-T-Plambda-RBS-luxR-T'''</font><br />
|<font color="blue">'''pSB1AK3'''</font><br />
|<font color="blue">'''Mux - Selector'''</font><br />
|-<br />
|<font color="blue">'''K081003'''</font><br />
|<font color="blue">'''36'''</font><br />
|<font color="blue">'''B0030-C0061-B0030-C0078-B1006-R0079'''</font><br />
|<font color="blue">'''RBS-luxI-RBS-lasI-T-Plas'''</font><br />
|<font color="blue">'''pSB1A2'''</font><br />
|<font color="blue">'''Demux - Input'''</font><br />
|}<br />
<br />
<br><br />
*01 part has not been submitted to the registry because it was a test assembly.<br />
*<font color="red">'''Red'''</font>-highlighted parts (11, 21, 29, 33) contain I15010 (cph8): according to IGEM DNA Repositories Quality Control (and according to our results) the sequence is inconsistent and so these parts cannot be assembled.<br />
*<font color="green">'''Green'''</font>-coloured parts have been succesfully sequenced.<br />
*<font color="blue">'''Blue'''</font>-coloured parts are long parts and have not been totally sequenced. The sequenced portions were correct.<br />
*<font color="red">'''Red'''</font>-coloured parts have been sequenced and a point mutation in C0062 coding sequence (position 349, G->C) was noticed, but these parts seemed to work and so we decided to submit them.<br />
*24, 25, 34, 35, 36 are parts of our final Mux or Demux systems.<br />
*Sequencing results of all the sequenced parts containing C0061 and C0078 showed that these two coding sequences have an undocumented barcode downstream. This should be documented on C0061 and C0078 Registry pages!<br />
*Parts in <font color="gray">'''gray'''</font> were programmed, but they have not been assembled and so they haven't been submitted.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/PartsTeam:UNIPV-Pavia/Parts2008-10-27T13:59:47Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
===Assembled Parts Notation===<br />
In notebook section we sometimes use abbreviations to name assembled parts. New parts' official name, notebook abbreviations and new parts description are reported in the following table:<br />
<br />
<br />
{|border="1" width="60%"<br />
!New Part<br />
!Notebook Name<br />
!Assembled Parts(code)<br />
!Assembled Parts(name)<br />
!Vector<br />
!Description<br />
|-<br />
|''-''<br />
|01<br />
|J23100-E0240<br />
|Pcon-GFP_protein_generator<br />
|J61002<br />
|Test<br />
|-<br />
|<font color="green">'''K081005'''</font><br />
|<font color="green">'''02'''</font><br />
|<font color="green">'''J23100-B0030'''</font><br />
|<font color="green">'''Pcon-RBS'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081006'''</font><br />
|<font color="green">'''03'''</font><br />
|<font color="green">'''R0051-B0030'''</font><br />
|<font color="green">'''Plambda-RBS'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''S04119'''</font><br />
|<font color="green">'''04'''</font><br />
|<font color="green">'''B0030-E0040'''</font><br />
|<font color="green">'''RBS-GFP'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081007'''</font><br />
|<font color="green">'''05'''</font><br />
|<font color="green">'''B0030-C0051'''</font><br />
|<font color="green">'''RBS-cI'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''S04120'''</font><br />
|<font color="green">'''06'''</font><br />
|<font color="green">'''23100-E1010'''</font><br />
|<font color="green">'''RBS-RFP'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081008'''</font><br />
|<font color="green">'''07'''</font><br />
|<font color="green">'''B0030-C0061'''</font><br />
|<font color="green">'''RBS-luxI'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081009'''</font><br />
|<font color="green">'''08'''</font><br />
|<font color="green">'''B0030-C0078'''</font><br />
|<font color="green">'''RBS-lasI'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|style="background:gray"|''-''<br />
|style="background:gray"|09<br />
|style="background:gray"|J23100-B0030-C0012<br />
|style="background:gray"|Pcon-RBS-lacI<br />
|style="background:gray"|pSB1A2<br />
|style="background:gray"|Intermediate<br />
|-<br />
|''K081010''<br />
|10<br />
|J23100-B0030-C0040<br />
|Pcon-RBS-tetR<br />
|pSB1A2<br />
|Intermediate<br />
|-<br />
|style="background:red; color:white"|''-''<br />
|style="background:red; color:white"|11<br />
|style="background:red; color:white"|J23100-B0030-I15010<br />
|style="background:red; color:white"|Pcon-RBS-cph8<br />
|style="background:red; color:white"|pSB1A2<br />
|style="background:red; color:white"|Intermediate<br />
|-<br />
|<font color="green">'''K081011'''</font><br />
|<font color="green">'''12'''</font><br />
|<font color="green">'''R0051-B0030-C0062'''</font><br />
|<font color="green">'''Plambda-RBS-luxR'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081012'''</font><br />
|<font color="green">'''13'''</font><br />
|<font color="green">'''B0030-E0040-B1006'''</font><br />
|<font color="green">'''RBS-GFP-T'''</font><br />
|<font color="green">'''pSB1AK3'''</font><br />
|<font color="green">'''GFP protein generator with 39bp artificial terminator'''</font><br />
|-<br />
|<font color="green">'''K081013'''</font><br />
|<font color="green">'''14'''</font><br />
|<font color="green">'''B0030-C0051-B0030'''</font><br />
|<font color="green">'''RBS-cI-RBS'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081014'''</font><br />
|<font color="green">'''15'''</font><br />
|<font color="green">'''B0030-E1010-B1006'''</font><br />
|<font color="green">'''RBS-RFP-T'''</font><br />
|<font color="green">'''pSB1AK3'''</font><br />
|<font color="green">'''GFP protein generator with 39bp artificial terminator'''</font><br />
|-<br />
|''K081015''<br />
|16<br />
|B0030-C0061-B1006<br />
|RBS-luxI-T<br />
|pSB1AK3<br />
|luxI protein generator RCT<br />
|-<br />
|''K081016''<br />
|17<br />
|B0030-C0078-B1006<br />
|RBS-lasI-T<br />
|pSB1AK3<br />
|lasI protein generator RCT<br />
|-<br />
|''K081017''<br />
|18<br />
|B0030-I15009<br />
|RBS-pcyA<br />
|pSB1A2<br />
|Intermediate<br />
|-<br />
|style="background:gray"|''-''<br />
|style="background:gray"|19<br />
|style="background:gray"|J23100-B0030-C0012-B1006<br />
|style="background:gray"|Pcon-RBS-lacI-T<br />
|style="background:gray"|pSB1AK3<br />
|style="background:gray"|lacI protein generator PRCT<br />
|-<br />
|<font color="green">'''K081018'''</font><br />
|<font color="green">'''20'''</font><br />
|<font color="green">'''J23100-B0030-C0040-B1006'''</font><br />
|<font color="green">'''Pcon-RBS-tetR-T'''</font><br />
|<font color="green">'''pSB1AK3'''</font><br />
|<font color="green">'''tetR protein generator PCRT'''</font><br />
|-<br />
|style="background:red; color:white"|''-''<br />
|style="background:red; color:white"|21<br />
|style="background:red; color:white"|J23100-B0030-I15010-B0030<br />
|style="background:red; color:white"|Pcon-RBS-cph8-RBS<br />
|style="background:red; color:white"|?<br />
|style="background:red; color:white"|Intermediate<br />
|-<br />
|<font color="green">'''K081019'''</font><br />
|<font color="green">'''22'''</font><br />
|<font color="green">'''R0051-B0030-C0062-B1006'''</font><br />
|<font color="green">'''Plambda-RBS-luxR-T'''</font><br />
|<font color="green">'''pSB1AK3'''</font><br />
|<font color="green">'''Plambda regulated luxR protein generator PRCT'''</font><br />
|-<br />
|''K081020''<br />
|23<br />
|B0030-C0051-B0030-C0079<br />
|RBS-cI-RBS-lasR<br />
|pSB1A2<br />
|Intermediate<br />
|-<br />
|<font color="green">'''K081000'''</font><br />
|<font color="green">'''24'''</font><br />
|<font color="green">'''B0030-C0061-B1006-R0062'''</font><br />
|<font color="green">'''RBS-luxI-T-Plux'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Mux - Channel 0'''</font><br />
|-<br />
|<font color="green">'''K081001'''</font><br />
|<font color="green">'''25'''</font><br />
|<font color="green">'''B0030-C0078-B1006-R0079'''</font><br />
|<font color="green">'''RBS-lasI-T-Plas'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Mux - Channel 1'''</font><br />
|-<br />
|''K081021''<br />
|26<br />
|B0030-I15009-B1006<br />
|RBS-pcyA-T<br />
|pSB1AK3<br />
|pcyA protein generator RCT<br />
|-<br />
|style="background:gray"|''-''<br />
|style="background:gray"|27<br />
|style="background:gray"|J23100-B0030-C0012-B1006-R0010<br />
|style="background:gray"|Pcon-RBS-lacI-T-Plac<br />
|style="background:gray"|pSB1AK3<br />
|style="background:gray"|lacI composite part: PRCT-Plac<br />
|-<br />
|style="background:gray"|''-''<br />
|style="background:gray"|28<br />
|style="background:gray"|J23100-B0030-C0040-B1006-R0040<br />
|style="background:gray"|Pcon-RBS-tetR-T-Ptet<br />
|style="background:gray"|pSB1A2<br />
|style="background:gray"|tetR composite part: PRCT-Ptet<br />
|-<br />
|style="background:red; color:white"|''-''<br />
|style="background:red; color:white"|29<br />
|style="background:red; color:white"|J23100-B0030-I15010-B0030-I15008<br />
|style="background:red; color:white"|Pcon-RBS-cph8-RBS-ho1<br />
|style="background:red; color:white"|?<br />
|style="background:red; color:white"|Intermediate<br />
|-<br />
|<font color="red">'''K081022'''</font><br />
|<font color="red">'''30'''</font><br />
|<font color="red">'''R0051-B0030-C0062-B1006-R0062'''</font><br />
|<font color="red">'''Plambda-RBS-luxR-T-Plux'''</font><br />
|<font color="red">'''pSB1A2'''</font><br />
|<font color="red">'''luxR composite part: Plambda regulated PRCT-Plux'''</font><br />
|-<br />
|<font color="blue">'''K081023'''</font><br />
|<font color="blue">'''31'''</font><br />
|<font color="blue">'''B0030-C0051-B0030-C0079-B1006'''</font><br />
|<font color="blue">'''RBS-cI-RBS-lasR-T'''</font><br />
|<font color="blue">'''pSB1AK3'''</font><br />
|<font color="blue">'''cI-lasR protein generator RCRCT'''</font><br />
|-<br />
|''K081024''<br />
|32<br />
|B0030-I15009-B1006-R0082<br />
|RBS-pcyA-T-Pomp<br />
|pSB1A2<br />
|pcyA composite part: RCT-Pomp<br />
|-<br />
|style="background:red; color:white"|''-''<br />
|style="background:red; color:white"|33<br />
|style="background:red; color:white"|J23100-B0030-I15010-B0030-I15008-B0030-I15009-B1006-R0082<br />
|style="background:red; color:white"|Pcon-RBS-cph8-RBS-ho1-RBS-pcyA-T-Pomp<br />
|style="background:red; color:white"|?<br />
|style="background:red; color:white"|Light responsive system under constitutive promoter: PRCRCRCT-Pomp<br />
|-<br />
|<font color="red">'''K081004'''</font><br />
|<font color="red">'''34'''</font><br />
|<font color="red">'''B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062'''</font><br />
|<font color="red">'''RBS-cI-RBS-lasR-T-Plambda-RBS-luxR-T-Plux'''</font><br />
|<font color="red">'''pSB1AK3'''</font><br />
|<font color="red">'''Demux - Selector'''</font><br />
|-<br />
|<font color="blue">'''K081002'''</font><br />
|<font color="blue">'''35'''</font><br />
|<font color="blue">'''B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006'''</font><br />
|<font color="blue">'''RBS-cI-RBS-lasR-T-Plambda-RBS-luxR-T'''</font><br />
|<font color="blue">'''pSB1AK3'''</font><br />
|<font color="blue">'''Mux - Selector'''</font><br />
|-<br />
|<font color="blue">'''K081003'''</font><br />
|<font color="blue">'''36'''</font><br />
|<font color="blue">'''B0030-C0061-B0030-C0078-B1006-R0079'''</font><br />
|<font color="blue">'''RBS-luxI-RBS-lasI-T-Plas'''</font><br />
|<font color="blue">'''pSB1A2'''</font><br />
|<font color="blue">'''Demux - Input'''</font><br />
|}<br />
<br />
<br><br />
*01 part has not been submitted to the registry because it was a test assembly.<br />
*<font color="red">'''Red'''</font>-highlighted parts (11, 21, 29, 33) contain I15010 (cph8): according to IGEM DNA Repositories Quality Control (and according to our results) the sequence is inconsistent and so these parts cannot be assembled.<br />
*<font color="green">'''Green'''</font>-coloured parts have been succesfully sequenced.<br />
*<font color="blue">'''Blue'''</font>-coloured parts are long parts and have not been totally sequenced. The sequenced portions were correct.<br />
*<font color="red">'''Red'''</font>-coloured parts have been sequenced and a point mutation in C0062 coding sequence (position 349, G->C) was noticed, but these parts seemed to work and so we decided to submit them.<br />
*24, 25, 34, 35, 36 are part of our final Mux or Demux systems.<br />
*Sequencing results of all the sequenced parts containing C0061 and C0078 showed that these two coding sequences have an undocumented barcode downstream. This should be documented on C0061 and C0078 Registry pages!<br />
*Parts in <font color="gray">'''gray'''</font> were programmed, but they have not been assembled and so they haven't been submitted.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/PartsTeam:UNIPV-Pavia/Parts2008-10-27T13:59:27Z<p>Magni: /* Assembled Parts Notation */</p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
===Assembled Parts Notation===<br />
In notebook section we sometimes use abbreviations to name assembled parts. New parts' official name, notebook abbreviations and new parts description are reported in the following table:<br />
<br />
<br />
{|border="1" width="80%"<br />
!New Part<br />
!Notebook Name<br />
!Assembled Parts(code)<br />
!Assembled Parts(name)<br />
!Vector<br />
!Description<br />
|-<br />
|''-''<br />
|01<br />
|J23100-E0240<br />
|Pcon-GFP_protein_generator<br />
|J61002<br />
|Test<br />
|-<br />
|<font color="green">'''K081005'''</font><br />
|<font color="green">'''02'''</font><br />
|<font color="green">'''J23100-B0030'''</font><br />
|<font color="green">'''Pcon-RBS'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081006'''</font><br />
|<font color="green">'''03'''</font><br />
|<font color="green">'''R0051-B0030'''</font><br />
|<font color="green">'''Plambda-RBS'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''S04119'''</font><br />
|<font color="green">'''04'''</font><br />
|<font color="green">'''B0030-E0040'''</font><br />
|<font color="green">'''RBS-GFP'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081007'''</font><br />
|<font color="green">'''05'''</font><br />
|<font color="green">'''B0030-C0051'''</font><br />
|<font color="green">'''RBS-cI'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''S04120'''</font><br />
|<font color="green">'''06'''</font><br />
|<font color="green">'''23100-E1010'''</font><br />
|<font color="green">'''RBS-RFP'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081008'''</font><br />
|<font color="green">'''07'''</font><br />
|<font color="green">'''B0030-C0061'''</font><br />
|<font color="green">'''RBS-luxI'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081009'''</font><br />
|<font color="green">'''08'''</font><br />
|<font color="green">'''B0030-C0078'''</font><br />
|<font color="green">'''RBS-lasI'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|style="background:gray"|''-''<br />
|style="background:gray"|09<br />
|style="background:gray"|J23100-B0030-C0012<br />
|style="background:gray"|Pcon-RBS-lacI<br />
|style="background:gray"|pSB1A2<br />
|style="background:gray"|Intermediate<br />
|-<br />
|''K081010''<br />
|10<br />
|J23100-B0030-C0040<br />
|Pcon-RBS-tetR<br />
|pSB1A2<br />
|Intermediate<br />
|-<br />
|style="background:red; color:white"|''-''<br />
|style="background:red; color:white"|11<br />
|style="background:red; color:white"|J23100-B0030-I15010<br />
|style="background:red; color:white"|Pcon-RBS-cph8<br />
|style="background:red; color:white"|pSB1A2<br />
|style="background:red; color:white"|Intermediate<br />
|-<br />
|<font color="green">'''K081011'''</font><br />
|<font color="green">'''12'''</font><br />
|<font color="green">'''R0051-B0030-C0062'''</font><br />
|<font color="green">'''Plambda-RBS-luxR'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081012'''</font><br />
|<font color="green">'''13'''</font><br />
|<font color="green">'''B0030-E0040-B1006'''</font><br />
|<font color="green">'''RBS-GFP-T'''</font><br />
|<font color="green">'''pSB1AK3'''</font><br />
|<font color="green">'''GFP protein generator with 39bp artificial terminator'''</font><br />
|-<br />
|<font color="green">'''K081013'''</font><br />
|<font color="green">'''14'''</font><br />
|<font color="green">'''B0030-C0051-B0030'''</font><br />
|<font color="green">'''RBS-cI-RBS'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Intermediate'''</font><br />
|-<br />
|<font color="green">'''K081014'''</font><br />
|<font color="green">'''15'''</font><br />
|<font color="green">'''B0030-E1010-B1006'''</font><br />
|<font color="green">'''RBS-RFP-T'''</font><br />
|<font color="green">'''pSB1AK3'''</font><br />
|<font color="green">'''GFP protein generator with 39bp artificial terminator'''</font><br />
|-<br />
|''K081015''<br />
|16<br />
|B0030-C0061-B1006<br />
|RBS-luxI-T<br />
|pSB1AK3<br />
|luxI protein generator RCT<br />
|-<br />
|''K081016''<br />
|17<br />
|B0030-C0078-B1006<br />
|RBS-lasI-T<br />
|pSB1AK3<br />
|lasI protein generator RCT<br />
|-<br />
|''K081017''<br />
|18<br />
|B0030-I15009<br />
|RBS-pcyA<br />
|pSB1A2<br />
|Intermediate<br />
|-<br />
|style="background:gray"|''-''<br />
|style="background:gray"|19<br />
|style="background:gray"|J23100-B0030-C0012-B1006<br />
|style="background:gray"|Pcon-RBS-lacI-T<br />
|style="background:gray"|pSB1AK3<br />
|style="background:gray"|lacI protein generator PRCT<br />
|-<br />
|<font color="green">'''K081018'''</font><br />
|<font color="green">'''20'''</font><br />
|<font color="green">'''J23100-B0030-C0040-B1006'''</font><br />
|<font color="green">'''Pcon-RBS-tetR-T'''</font><br />
|<font color="green">'''pSB1AK3'''</font><br />
|<font color="green">'''tetR protein generator PCRT'''</font><br />
|-<br />
|style="background:red; color:white"|''-''<br />
|style="background:red; color:white"|21<br />
|style="background:red; color:white"|J23100-B0030-I15010-B0030<br />
|style="background:red; color:white"|Pcon-RBS-cph8-RBS<br />
|style="background:red; color:white"|?<br />
|style="background:red; color:white"|Intermediate<br />
|-<br />
|<font color="green">'''K081019'''</font><br />
|<font color="green">'''22'''</font><br />
|<font color="green">'''R0051-B0030-C0062-B1006'''</font><br />
|<font color="green">'''Plambda-RBS-luxR-T'''</font><br />
|<font color="green">'''pSB1AK3'''</font><br />
|<font color="green">'''Plambda regulated luxR protein generator PRCT'''</font><br />
|-<br />
|''K081020''<br />
|23<br />
|B0030-C0051-B0030-C0079<br />
|RBS-cI-RBS-lasR<br />
|pSB1A2<br />
|Intermediate<br />
|-<br />
|<font color="green">'''K081000'''</font><br />
|<font color="green">'''24'''</font><br />
|<font color="green">'''B0030-C0061-B1006-R0062'''</font><br />
|<font color="green">'''RBS-luxI-T-Plux'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Mux - Channel 0'''</font><br />
|-<br />
|<font color="green">'''K081001'''</font><br />
|<font color="green">'''25'''</font><br />
|<font color="green">'''B0030-C0078-B1006-R0079'''</font><br />
|<font color="green">'''RBS-lasI-T-Plas'''</font><br />
|<font color="green">'''pSB1A2'''</font><br />
|<font color="green">'''Mux - Channel 1'''</font><br />
|-<br />
|''K081021''<br />
|26<br />
|B0030-I15009-B1006<br />
|RBS-pcyA-T<br />
|pSB1AK3<br />
|pcyA protein generator RCT<br />
|-<br />
|style="background:gray"|''-''<br />
|style="background:gray"|27<br />
|style="background:gray"|J23100-B0030-C0012-B1006-R0010<br />
|style="background:gray"|Pcon-RBS-lacI-T-Plac<br />
|style="background:gray"|pSB1AK3<br />
|style="background:gray"|lacI composite part: PRCT-Plac<br />
|-<br />
|style="background:gray"|''-''<br />
|style="background:gray"|28<br />
|style="background:gray"|J23100-B0030-C0040-B1006-R0040<br />
|style="background:gray"|Pcon-RBS-tetR-T-Ptet<br />
|style="background:gray"|pSB1A2<br />
|style="background:gray"|tetR composite part: PRCT-Ptet<br />
|-<br />
|style="background:red; color:white"|''-''<br />
|style="background:red; color:white"|29<br />
|style="background:red; color:white"|J23100-B0030-I15010-B0030-I15008<br />
|style="background:red; color:white"|Pcon-RBS-cph8-RBS-ho1<br />
|style="background:red; color:white"|?<br />
|style="background:red; color:white"|Intermediate<br />
|-<br />
|<font color="red">'''K081022'''</font><br />
|<font color="red">'''30'''</font><br />
|<font color="red">'''R0051-B0030-C0062-B1006-R0062'''</font><br />
|<font color="red">'''Plambda-RBS-luxR-T-Plux'''</font><br />
|<font color="red">'''pSB1A2'''</font><br />
|<font color="red">'''luxR composite part: Plambda regulated PRCT-Plux'''</font><br />
|-<br />
|<font color="blue">'''K081023'''</font><br />
|<font color="blue">'''31'''</font><br />
|<font color="blue">'''B0030-C0051-B0030-C0079-B1006'''</font><br />
|<font color="blue">'''RBS-cI-RBS-lasR-T'''</font><br />
|<font color="blue">'''pSB1AK3'''</font><br />
|<font color="blue">'''cI-lasR protein generator RCRCT'''</font><br />
|-<br />
|''K081024''<br />
|32<br />
|B0030-I15009-B1006-R0082<br />
|RBS-pcyA-T-Pomp<br />
|pSB1A2<br />
|pcyA composite part: RCT-Pomp<br />
|-<br />
|style="background:red; color:white"|''-''<br />
|style="background:red; color:white"|33<br />
|style="background:red; color:white"|J23100-B0030-I15010-B0030-I15008-B0030-I15009-B1006-R0082<br />
|style="background:red; color:white"|Pcon-RBS-cph8-RBS-ho1-RBS-pcyA-T-Pomp<br />
|style="background:red; color:white"|?<br />
|style="background:red; color:white"|Light responsive system under constitutive promoter: PRCRCRCT-Pomp<br />
|-<br />
|<font color="red">'''K081004'''</font><br />
|<font color="red">'''34'''</font><br />
|<font color="red">'''B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062'''</font><br />
|<font color="red">'''RBS-cI-RBS-lasR-T-Plambda-RBS-luxR-T-Plux'''</font><br />
|<font color="red">'''pSB1AK3'''</font><br />
|<font color="red">'''Demux - Selector'''</font><br />
|-<br />
|<font color="blue">'''K081002'''</font><br />
|<font color="blue">'''35'''</font><br />
|<font color="blue">'''B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006'''</font><br />
|<font color="blue">'''RBS-cI-RBS-lasR-T-Plambda-RBS-luxR-T'''</font><br />
|<font color="blue">'''pSB1AK3'''</font><br />
|<font color="blue">'''Mux - Selector'''</font><br />
|-<br />
|<font color="blue">'''K081003'''</font><br />
|<font color="blue">'''36'''</font><br />
|<font color="blue">'''B0030-C0061-B0030-C0078-B1006-R0079'''</font><br />
|<font color="blue">'''RBS-luxI-RBS-lasI-T-Plas'''</font><br />
|<font color="blue">'''pSB1A2'''</font><br />
|<font color="blue">'''Demux - Input'''</font><br />
|}<br />
<br />
<br><br />
*01 part has not been submitted to the registry because it was a test assembly.<br />
*<font color="red">'''Red'''</font>-highlighted parts (11, 21, 29, 33) contain I15010 (cph8): according to IGEM DNA Repositories Quality Control (and according to our results) the sequence is inconsistent and so these parts cannot be assembled.<br />
*<font color="green">'''Green'''</font>-coloured parts have been succesfully sequenced.<br />
*<font color="blue">'''Blue'''</font>-coloured parts are long parts and have not been totally sequenced. The sequenced portions were correct.<br />
*<font color="red">'''Red'''</font>-coloured parts have been sequenced and a point mutation in C0062 coding sequence (position 349, G->C) was noticed, but these parts seemed to work and so we decided to submit them.<br />
*24, 25, 34, 35, 36 are part of our final Mux or Demux systems.<br />
*Sequencing results of all the sequenced parts containing C0061 and C0078 showed that these two coding sequences have an undocumented barcode downstream. This should be documented on C0061 and C0078 Registry pages!<br />
*Parts in <font color="gray">'''gray'''</font> were programmed, but they have not been assembled and so they haven't been submitted.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Dry_LabTeam:UNIPV-Pavia/Dry Lab2008-10-27T13:55:52Z<p>Magni: /* Biomedical Informatics Lab - DIS */</p>
<hr />
<div>{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
===Biomedical Informatics Lab - DIS===<br />
<br />
Our lab is distributed over 7 rooms, in which 6 professors and 30 researchers and students work. The main research activities are mathemathical modeling of biomedical systems, bioinformatics, medical informatics, telemedicine, clinical data mining and knowledge management.<br />
<br />
We have about 30 PCs, two Unix workstations and a Linux cluster of 15 nodes. On these machines, some software tools are running, such as MathWorks MatLab and its toolboxes for which we have a complete Campus License.<br />
We also have free access to the most important journals of IEEE, Elsevier, Blackwell, Kluwer, Nature, Springer and Wiley.<br />
<br />
Click [http://www.labmedinfo.org HERE] for more details about our dry lab.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/ProjectTeam:UNIPV-Pavia/Project2008-10-27T00:56:08Z<p>Magni: </p>
<hr />
<div>{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
== '''Overall project''' ==<br />
<br />
We are trying to mimic Multiplexer (Mux) and Demultiplexer (Demux) logic functions in E. coli.<br />
<br><br />
In the following paragraphs project details will be described from both digital electronic and genetic points of view.<br />
<br><br />
<br><br />
<br />
== '''Electronic Implementation''' ==<br />
<br><br />
=== '''What kind of components are Mux and Demux?''' ===<br />
'''Mux''' is a component which conveys one of the two input channels values into a single output channel. The choice of the input channel is made by a selector.<br />
<br><br />
'''Demux''' is a component which conveys the only input channel value into one of the two output channels. The choice of the output channel is made by a selector.<br />
<br><br />
<br><br />
The following pictures show data flow in Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_dataflow0.png|thumb|300px|left|Data flow in Multiplexer - SELECTOR=0]]<br />
|[[Image:pv_mux_dataflow1.png|thumb|300px|left|Data flow in Multiplexer - SELECTOR=1]]<br />
|-<br />
|[[Image:pv_demux_dataflow0.png|thumb|300px|left|Data flow in Demultiplexer - SELECTOR=0]]<br />
|[[Image:pv_demux_dataflow1.png|thumb|300px|left|Data flow in Demultiplexer - SELECTOR=1]]<br />
|}<br />
<br><br />
<br />
=== '''What kind of signals do we process?''' ===<br />
In this project we consider Boolean logic signals, thus every input/output value can assume only the values 0 and 1. A function that processes Boolean values is called logic function.<br />
<br><br />
Mux and Demux can be considered by now as black boxes which implement a logic function that can process input signals to output signals. Here you can see examples of Boolean data flow in Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_bool.png|thumb|300px|left|Example: Mux Boolean data flow]]<br />
|[[Image:pv_demux_bool.png|thumb|300px|left|Example: Demux Boolean data flow]]<br />
|}<br />
In the following documentation we will see what is inside these black boxes.<br />
<br><br />
<br><br />
=== How can we formalize Mux and Demux logic behavior? ===<br />
Logic functions can be formalized writing a truth table; a truth table is a mathematical table in which every row represents a combination of input values and its respective output values. The table has to be filled with every input combination.<br />
<br><br />
<br><br />
Here you can see Mux and Demux truth tables (output columns are gray):<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_truth.png|thumb|300px|left|Mux truth table]]<br />
|[[Image:pv_demux_truth.png|thumb|300px|left|Demux truth table]]<br />
|}<br />
<br><br />
<br />
=== Building a logic circuit from a truth table ===<br />
Our goal in this section is to project two logic gates networks which behave like Mux and Demux truth tables. A very useful tool to transform a truth table into a logic network is Karnaugh map.<br />
<br><br />
It is possible to read about Karnaugh maps at: [http://en.wikipedia.org/wiki/Karnaugh_map]<br />
<br><br />
<br><br />
Following Karnaugh maps method, we can write these two logic networks for Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux.png|thumb|340px|left|Mux - logic circuit]]<br />
|[[Image:pv_mux_example.png|thumb|340px|left|Mux - Example]]<br />
|-<br />
|[[Image:pv_demux.png|thumb|340px|left|Demux - logic circuit]]<br />
|[[Image:pv_demux_example.png|thumb|340px|left|Demux - Example]]<br />
|}<br />
<br />
<br><br />
<br />
== '''Genetic Implementation''' ==<br />
Our goal is to mimic Mux and Demux logic networks in a biological device, such as E. coli. To perform this, we use protein/DNA and protein/protein interactions to build up biological logic gates.<br />
Mux and Demux logic circuits are composed by three fundamental logic gates, AND, OR, NOT: in the next paragraphs genetic implementation of these logic gates will be provided.<br />
<br><br />
<br><br />
=== AND ===<br />
To mimic an AND gate, we need a biological function, such as a promoter activation, which is directly turned on by the interaction between two upstream genes. In our synthetic devices, we use the luxR/luxI system: luxR can activate Plux promoter only upon 3-oxo-hexanoyl-homoserine lactone (HSL) binding; luxI generates HSL; so, only the contemporary expression of LuxR and luxI proteins can activate the downstream Plux-dependent gene expression. Another AND gate we use is the lasR/lasI system, which works in a very similar way but through another chemical intermediate, N-(3-oxododecanoyl) homoserine lactone (PAI-1).<br />
{|<br />
|[[Image:pv_proj_AND.png|thumb|340px|left|Genetic AND: Plux can be turned on only when the two proteins luxI and luxR are present.]]<br />
|}<br />
=== OR ===<br />
To mimic an OR gate in Mux, we need a biological function which can be activated alternatively by two independent upstream signals or by both. Thus, we combine the outputs of the upstream AND gates to assemble directly an OR reporter function, by simply repeating the reporter gene (GFP) under two different promoters (Plux and Plas). It’s sufficient to activate one of the two promoters (or both) to recover the GFP signal from engineered bacteria.<br />
There should not be an over-expression problem for GFP, in fact, in Mux device, only one promoter can be active, either Plux or Plas. Here we considered GFP output, but OR device can be generalized for every output gene.<br />
{|<br />
|[[Image:pv_proj_OR.png|thumb|340px|left|Genetic OR: it is sufficient that one of the two input promoters is active to obtain GFP expression.]]<br />
|}<br />
=== NOT ===<br />
To mimic a NOT gate, we need an efficient and regulated repressor of a specific downstream promoter: in this case, we choose cI repression on Plambda, which should be specific and, upon cI inactivation, quick and efficient.<br />
{|<br />
|[[Image:pv_proj_NOT.png|thumb|340px|left|Genetic NOT: Plambda can be turned on only when cI protein is not present.]]<br />
|}<br />
<br />
According to what above stated, genetic implementation of Mux and Demux can be obtained connecting these basic logic gates and can be summarized in this way:<br />
<br><br />
<br><br />
<br><br />
<br />
=== Genetic Mux ===<br />
Let PA, PB and PS be three generic promoters that can be ACTIVATED respectively by the three exogenous molecules "A", "B" and "S". A genetic Mux with inputs "A" (CH0) and "B" (CH1), selector "S" and the generic protein GOI as output, can be implemented by the following gene network:<br />
{|align="center"<br />
|[[Image:pv_MUX.png|thumb|420px|left|Genetic Mux]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_MUXint.png|thumb|420px|left|Genetic Mux - interactions]]<br />
|}<br />
We want to supply a device that can be generalized to detect every kind of input and to express every kind of genes as output. So, input promoters and output protein generators are not part of the genetic Mux we want to build up. In this way, a hypothetical user can re-use our device assembling the desired input and output elements.<br />
<br><br />
Note that the output genes are two, but, as explained in "OR" section, if the genes are identical, we can say that there is only one output; in fact, the real output is a protein synthesis and so it is not important which of the two identical genes is expressed.<br />
<br><br />
However, it is possible to assemble two different genes downstream of Plux and Plas, for example two reporters. In this way, debugging process becomes easy, because we can discriminate lux system and las system activities.<br />
<br><br />
Here we report the truth table of this network:<br />
{|align="center"<br />
|[[Image:pv_gmux_truth.png|thumb|420px|left|Genetic Mux - truth table]]<br />
|}<br />
<br><br />
Now we report two examples of genetic Mux behavior, in response to generic inputs.<br />
<br><br />
<br><br />
====Genetic Mux behavior - Example 1====<br />
{|<br />
|[[Image:pv_genmux_example1.png|thumb|420px|left|First example of genetic Mux behavior]]<br />
|}<br />
According to Mux truth table, if "A" is not present and "B" and "S" are present, we expect to have GOI synthesis.<br />
<br><br />
"A" molecule is not present, so PA promoter is not active and luxI gene is not expressed.<br />
<br><br />
"B" molecule is present, so PB promoter is active and lasI gene is expressed.<br />
<br><br />
"S" molecule is present, so PS promoter is active and lasR and cI genes are expressed.<br />
<br><br />
cI protein represses transcription of the gene downstream of Plambda promoter, which is luxR.<br />
<br><br />
lasI protein can synthesize 3-OC12-HSL. This molecule activates transcription factor lasR, which can activate Plas promoter. So, the copy of GOI gene under Plas regulation can be expressed.<br />
<br><br />
The other copy of GOI gene, which is under Plux promoter regulation, is not expressed. In fact Plux is not active, because both luxI and luxR proteins are not present.<br />
<br><br />
Only one of the two GOI genes is expressed, but this is sufficient to synthesize the output protein GOI.<br />
<br><br />
<br><br />
====Genetic Mux behavior - Example 2====<br />
{|<br />
|[[Image:pv_genmux_example2.png|thumb|420px|left|Second example of genetic Mux behavior]]<br />
|}<br />
We expect to have no GOI synthesis in response to this input combination.<br />
<br><br />
"A" molecule is not present, so PA promoter is not active and luxI gene is not expressed.<br />
<br><br />
"B" molecule is present, so PB promoter is active and lasI gene is expressed.<br />
<br><br />
"S" molecule is not present, so PS promoter can't transcribe cI and lasR genes.<br />
<br><br />
cI protein is not present, so Plambda promoter can transcribe luxR gene.<br />
<br><br />
None of the two logic AND systems is active, because lux system lacks of luxI and las system lacks of lasR. So, Plux and Plas promoters are unactive and can't express GOI genes. For this reason, GOI protein is not synthesized.<br />
<br><br />
<br><br />
These two examples show how "S" molecule can select the input to be conveyed into the single output channel: in fact its presence allows lasR expression and represses luxR expression, while "S" absence represses lasR expression and allows luxR expression.<br />
<br><br />
<br><br />
<br />
=== Genetic Demux ===<br />
Let PI and PS be two generic promoters that can be ACTIVATED respectively by the two exogenous molecules "I" and "S". A genetic Demux with input "I", selector "S" and the generic proteins GOI0 and GOI1 as outputs (OUT0 and OUT1 respectively), can be implemented by the following gene network:<br />
{|align="center"<br />
|[[Image:pv_DEMUX.png|thumb|420px|left|Genetic Demux]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_DEMUXint.png|thumb|420px|left|Genetic Demux - interactions]]<br />
|}<br />
As described for genetic Mux, we want to supply a device that can be generalized to detect every kind of inputs and to express every kind of genes as output. So, input promoters and output protein generators are not part of the genetic Demux. In this way, a hypothetical user can re-use our device assembling the desired input and output elements.<br />
<br><br />
Here we report the truth table of this network:<br />
{|align="center"<br />
|[[Image:pv_gdemux_truth.png|thumb|420px|left|Genetic Demux - truth table]]<br />
|}<br />
<br><br />
Now we report two examples of genetic Demux behavior, in response to generic inputs.<br />
<br><br />
<br><br />
====Genetic Demux behavior - Example 1====<br />
{|<br />
|[[Image:pv_gendemux_example1.png|thumb|420px|left|First example of genetic Demux behavior]]<br />
|}<br />
According to Demux truth table, if "I" molecule is present and "S" molecule is absent, we expect to have GOI0 protein synthesis and no GOI1 protein synthesis.<br />
<br><br />
"I" is present, so PI promoter is active and luxI and lasI genes are expressed.<br />
<br><br />
"S" is not present, so PS promoter can't transcribe lasR and cI genes.<br />
<br><br />
cI protein is not present, so Plambda promoter is not inhibited and luxR gene is expressed.<br />
<br><br />
luxI protein can synthesize 3-OC6-HSL lactone. This molecule activates luxR transcription factor which can activate Plux promoter. In this way, GOI0 gene, which is under Plux regulation, is expressed.<br />
<br><br />
On the other hand, lasI protein can synthesize 3-OC12-HSL, but lasR transcription factor is not present and so Plas promoter cannot be activated. In this way, GOI1 gene, which is under Plas regulation, is not expressed.<br />
<br><br />
So, we have GOI0 protein synthesis and no GOI1 protein synthesis.<br />
<br><br />
<br><br />
====Genetic Demux behavior - Example 2====<br />
{|<br />
|[[Image:pv_gendemux_example2.png|thumb|420px|left|Second example of genetic Demux behavior]]<br />
|}<br />
We expect to have GOI1 synthesis and no GOI0 synthesis in response to this input combination.<br />
<br><br />
"I" is present, so PI promoter is active and luxI and lasI genes are expressed.<br />
<br><br />
"S" is present, so PS promoter lasR and cI genes are expressed.<br />
<br><br />
cI protein is present, so Plambda promoter is inhibited and luxR gene is not expressed.<br />
<br><br />
lasI protein can synthesize 3-OC12-HSL lactone. This molecule activates lasR transcription factor which can activate Plas promoter. In this way, GOI1 gene, which is under Plas regulation, is expressed.<br />
<br><br />
On the other hand, luxI protein can synthesize 3-OC6-HSL, but luxR transcription factor is not present and so Plux promoter cannot be activated. In this way, GOI0 gene, which is under Plux regulation, is not expressed.<br />
<br><br />
So, we have GOI1 protein synthesis and no GOI0 protein synthesis.<br />
<br />
<br />
=== A complete genetic Mux===<br />
In this section a complete Mux gene network is described.<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_implementation.png|thumb|420px|left|Example of a complete genetic Mux]]<br />
|[[Image:pv_mux_gn_implementation.png|thumb|420px|left|Example of a complete genetic Mux - Gene network]]<br />
|}<br />
We want to build up a device in which Channel 0, Channel 1 and Selector are respectively sensitive to Tetracycline, IPTG and red light. The presence of each input corresponds to logic 1.<br />
We chose green fluorescence as Mux output: expression of GFP corresponds to logic 1, while absence of fluorescence corresponds to logic 0.<br />
<br><br />
Tetracycline and IPTG can be considered as "A" and "B" molecules, introduced in "Genetic Mux" section, because both Tetracycline and IPTG are indirect ACTIVATORS of Ptet and Plac promoters respectively.<br />
On the other hand, red light is quite different from "S" molecule, because red light is an indirect REPRESSOR of Pomp promoter and not an activator as required by the original schema. For this reason, if we want a device in which the presence of Tetracycline, IPTG and red light correspond to logic 1, red light input should be ''inverted''. The simplest way to do this, is to cross-exchange the two input channels. So, Tetracycline sensor (CH0) has to be assembled to las system and IPTG sensor (CH1) has to be assembled to lux system.<br />
<br />
<br />
====Complete genetic Mux - Example====<br />
{|<br />
|[[Image:pv_mux_example1.png|thumb|420px|left|Example of a complete genetic Mux behavior]]<br />
|}<br />
According to Mux truth table, if SEL=0, CH0=1, CH1=0, output channel must be logic 1.<br />
Red light is not present, so it can't dephosphorylate cph8-ho1-pcyA complex, which is constitutively expressed. cph8-ho1-pcyA complex activates endogenous ompR, which can activate Pomp promoter, so cI and lasR genes are transcribed.<br />
cI protein binds Plambda promoter and so luxR expression is inhibited.<br />
TetR is a repressor for Ptet promoter, but Tetracycline is present, so it can bind tetR protein, which is constitutively expressed, and can activate transcription of downstream gene: lasI.<br />
Simultaneous expression of lasI and lasR can activate GFP, which is under Plas promoter.<br />
IPTG is not present, so lacI protein binds Plac promoter and represses luxI transcription.<br />
<br />
<br />
=== A complete genetic Demux===<br />
In this section a complete Demux gene network is described.<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_demux_implementation.png|thumb|420px|left|Example of a complete genetic Demux]]<br />
|[[Image:pv_demux_gn_implementation.png|thumb|420px|left|Example of a complete genetic Demux - Gene network]]<br />
|}<br />
<br />
We want to build up a device in which Input and Selector are respectively IPTG and Tetracycline.<br><br />
In Demux we have two output channels: red fluorescence corresponds to logic 1 at Channel 0, while green fluorescence corresponds to logic 1 at Channel 1.<br><br />
Absence of reporters expression corresponds to logic 0 at Channel 0 and Channel 1.<br><br />
There isn't any input combination that corresponds to logic 1 at Channel 0 and Channel 1 together.<br />
<br />
====Complete genetic Demux - Example====<br />
{|<br />
|[[Image:pv_demux_example1.png|thumb|420px|left|Example of a complete genetic Demux behavior]]<br />
|}<br />
According to Demux truth table, if IN=1 and SEL=1, output channel 0 is logic 0 and output channel 1 is logic 1.<br><br />
IPTG is present, so Plac promoter is active because IPTG binds lacI protein. This allows lasI and luxI transcription.<br><br />
Tetracycline is present, so Ptet promoter is active because Tetracycline binds tetR protein. This allows cI and lasR transcription.<br><br />
cI protein binds Plambda promoter and so luxR expression is inhibited.<br><br />
Simultaneous expression of lasI and lasR can activate GFP, which is under Plas promoter.<br><br />
There is no simultaneous expression of luxI and luxR, so RFP (which is under Plux regulation) cannot be expressed.<br />
<br />
== Final devices==<br />
BioBrick standard parts for genetic Mux and Demux are summarized in the following schemas:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_final.png|thumb|420px|left|Three standard parts for mux]]<br />
|[[Image:pv_demux_final.png|thumb|450px|left|Two standard parts for demux]]<br />
|}<br />
<br />
A hypothetical user of Mux or Demux has to ligate our standard parts with desired inputs and outputs, as shown in the pictures above.<br />
<br><br />
The structure of our devices show that Mux and Demux systems both conform to the PoPS device boundary standard.<br />
<br><br />
<br />
==Applications==<br />
Mux and Demux are two fundamental devices in electronics. They are used in several applications, for example in communication devices, in Arithmetic Logic Units (ALUs), or in applications that involve channel sharing.<br />
<br><br />
Analogously, they could play a crucial role in the building of complex genetic circuits. In fact, both of them can be used as controlled genetic switches.<br />
<br><br />
Because our devices had been designed to be general, their application field is very wide. For example, genetic Mux can be used to integrate signals from the environment in a two-inputs and one-output biosensor; once detected, the selector controls which of the two inputs must be transferred in output. In this way, two sensing devices can be integrated in only one circuit that can compute multiplexing logic function.<br />
On the other hand, genetic Demux can be used for controlled protein productions, where the choice of the protein to produce is made by the selector. In this case, we can imagine an industrial process where two consequent enzyme reactions are necessary to build a final product. Using a Demux, we can consider the two enzymes as system outputs and then we can easily switch on and off their production modulating selection signal.<br />
<br><br />
Selector in Mux and Demux can be controlled manually, but also automatically. In fact, it is possible to use feedback control to pilot the selector in order, according with Demux example, to switch enzyme production when a specific condition (for example a pH threshold) is reached.<br />
<br><br />
Switching activity in these two devices is a very powerful tool to manage multi-input and multi-output systems.<br />
<br />
<br />
== Experiments and results ==<br />
=== Assemblies ===<br />
We successfully amplified the following BioBrick standard parts from Spring 2008 DNA Distribution:<br />
{|align="center"<br />
|[[Image:pv_resusp.png|thumb|600px|left|Successful amplifications]]<br />
|}<br />
<br><br />
while we couldn't amplify correctly the following parts:<br />
{|align="center"<br />
|[[Image:pv_notresusp.png|thumb|600px|left|Unsuccessful amplifications]]<br />
|}<br />
<br><br />
To build up our designed devices, we followed and completed this assembly tree schema:<br />
{|align="center"<br />
|[[Image:pv_assemblyschema.png|thumb|600px|left|Assembly tree schema]]<br />
|}<br />
<br />
=== Functional tests ===<br />
We performed these boolean (on/off) fluorescence tests:<br />
<br><br />
<hr><br />
*'''TEST 1'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test1.png|thumb|250px|left|GFP protein generator under Plambda]]<br />
|}<br />
'''Description:''' we assembled an available GFP protein generator (E0240) under Plambda promoter, keeping pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' cI protein was not present, so Plambda should work like a constitutive promoter. A reporter gene downstream of this promoter allows us to validate Plambda costitutive activity. That can be very useful to validate our NOT logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of R0051 and E0240, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test1.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that GFP was expressed in positive transformants, so Plambda activity was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 2'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test2.png|thumb|250px|left|GFP protein generator under Ptet]]<br />
|}<br />
'''Description:''' we assembled E0240 under Ptet promoter, keeping pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' tetR protein was not present, so Ptet should work like a constitutive promoter. A reporter gene downstream of this promoter allows us to validate Ptet costitutive activity. Ptet is useful for specific input building.<br />
<br><br />
'''Methods:''' after ligation reaction of R0040 and E0240, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test2.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that GFP was expressed in positive transformants, so Ptet activity was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 3'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test3.png|thumb|250px|left|RFP protein generator under constitutive promoter]]<br />
|}<br />
'''Description:''' we didn't perform any assembly for this experiment, because the promoter we wanted to test was contained into BBa_J61002 vector, which places a RFP protein generator between SpeI and PstI restriction sites.<br />
<br><br />
'''Motivation:''' J23100, which we call Pcon, should have a strong constitutive activity. A reporter gene downstream of this promoter allows us to validate this activity. Pcon is useful to build our inputs because some sensors like IPTG and Tetracycline sensors need the constitutive production of specific proteins, in this case lacI and tetR respectively.<br />
<br><br />
'''Methods''' we transformed J23100 using Invitrogen TOP10 and plated transformed bacteria. We expected to observe red fluorescence (using transluminator) in all the colonies. We also picked up a colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through TRITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' all the grown colonies glowed under UV rays and red fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test3.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that RFP was expressed in transformed bacteria, so Pcon functionality was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 4'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test4.png|thumb|250px|left|Our GFP protein generator (K081012) under a constitutive promoter]]<br />
|}<br />
'''Description:''' we assembled a constitutive promoter family member (J23100) upstream of K081012, a GFP protein generator we built. We decided to excide J23100 from its plasmid (E-S) and to open K081012 plasmid (pSB1AK3) (E-X).<br />
<br><br />
'''Motivation:''' we already validated qualitatively J23100 constitutive activity, so this promoter can be used to test the GFP protein generator we built. We wanted to check if K081012 actually generates GFP.<br />
<br><br />
'''Methods''' after ligation reaction of J23100 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays (see figure) and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test4bis.jpg|thumb|400px|left|TOP10 colonies under UV]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test4.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that our GFP protein generator actually generates GFP.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 5'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test5.png|thumb|250px|left|Our RFP protein generator (K081014) under a constitutive promoter]]<br />
|}<br />
'''Description:''' we assembled a constitutive promoter family member (J23100) upstream of K081014, a RFP protein generator we built. We decided to excide J23100 from its plasmid (E-S) and to open K081014 plasmid (pSB1AK3) (E-X).<br />
<br><br />
'''Motivation:''' we already validated qualitatively J23100 constitutive activity, so this promoter can be used to test the RFP protein generator we built. We wanted to check if K081014 actually generates RFP.<br />
<br><br />
'''Methods''' after ligation reaction of J23100 and K081014, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through TRITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays (see figure) and red fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test5bis.jpg|thumb|400px|left|TOP10 colonies under UV]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test5.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that our RFP protein generator actually generates RFP.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 6'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test6.png|thumb|350px|left|GFP protein generator under Plux]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081000. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we didn't assemble any input to K081000 device, so we could study only the promoter basic activity. We expected to find a weak basic activity. Plux is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of K081000 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we performed a screening on 5 colonies through colony PCR to search for correctly ligated plasmid. We infected 9 ml of LB + Amp with the chosen colony and incubated the culture at 37°C, 220 rpm for 15 hours. Then we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test6.png|thumb|400px|left|No fluorescent TOP10 cells can be seen at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' the picture above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria. Increasing exposition time, green fluorescence could be observed (picture not reported). So, we can say that GFP was expressed very weakly. This experiment confirmed that Plux promoter has a weak activity without luxI and luxR proteins.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 7'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test7.png|thumb|350px|left|GFP protein generator under Plas]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plas promoter that was contained into K081001. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we didn't assemble any input to K081001 device, so we could study only the promoter basic activity. We expected to find a weak basic activity. Plas is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of K081001 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we performed a screening on 5 colonies through colony PCR to search for correctly ligated plasmid. We infected 9 ml of LB + Amp with the chosen colony and incubated the culture at 37°C, 220 rpm for 15 hours. Then we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test7.png|thumb|400px|left|No fluorescent TOP10 cells can be seen at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' the picture above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria. Increasing exposition time, green fluorescence could be observed (picture not reported). So, we can say that GFP was expressed very weakly. This experiment confirmed that Plas promoter has a weak activity without lasI and lasR proteins.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 8'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test8.png|thumb|600px|left|GFP protein generator under Plux and constitutive expression of luxI and luxR]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081000. We also assembled K081011 upstream of K081000. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' Plux promoter activity was studied in response of a constitutive expression of luxI and luxR. Plambda promoter without cI repressor guaranteed the constitutive expression of these genes. We expected to find a strong activity because Plux is turned on. lux system is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' we ligated K081012 under K081000, transformed ligation, plated transformed bacteria, performed PCR screening on some colonies and extracted correctly ligated plasmids. We repeated these steps to assemble K081011 upstream of K081000-K081012, but we didn't perform PCR screening. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test8.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that Plux promoter can be activated by the contemporary presence of luxI and luxR. This is a crucial result for our AND logic gate.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 9'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test9.png|thumb|500px|left|GFP protein generator under Plux and constitutive expression of luxR]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081022. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' Plux promoter activity was studied in response to a constitutive expression of luxR. Plambda promoter without cI repressor guaranteed the constitutive expression of this gene. We expected to find a weak activity because luxR transcription factor is not active and so Plux cannot be turned on. We also expected to find a strong activity if we induce luxR activation using 3OC6-HSL (this is equivalent to TEST 8 conditions, because 3OC6-HSL is synthesized by luxI). lux system is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' we ligated K081012 downstream of K081022, transformed ligation, plated transformed bacteria and screened three colonies to insulate a colony containing correctly ligated plasmids. We inoculated the positive colony into 9 ml of LB + Amp and incubated the culture at 37°C, 220 rpm for 15 hours. Then we diluted 1:10 the culture in two falcon tubes (5 ml cultures). One of these cultures was induced with 3OC6-HSL 1 uM. We let the two cultures grow for 2 hours and then we watched 50 ul at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture.<br />
{|align="center"<br />
|[[Image:pv_fig_test9.png|thumb|400px|left|Non induced culture: TOP10 cells cannot be seen at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test9bis.png|thumb|400px|left|Induced culture: fluorescent TOP10 cells at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test9superexp.png|thumb|400px|left|Same frames as above, but superexposed: non induced (left) and induced (right) cultures]]<br />
|}<br />
<br><br />
'''Comments:''' the pictures above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria for the non induced culture, while we can see green fluorescent TOP10 in the induced culture. As we wrote for TEST 6 and TEST 7, increasing exposition time green fluorescence could be observed even for the non induced culture (last picture), confirming the weak activity of Plux promoter in response of unactive luxR. This experiment confirmed that Plux promoter has a weak activity without luxI (or 3OC6-HSL) and luxR protein is not sufficient to induce a strong transcription. Adding 3OC6-HSL, luxR becomes active and so Plux is turned on.<br />
<br><br />
NOTE: K081022 has a point mutation in position 349 of C0062 coding sequence. This mutation changes the aminoacid, but luxR seems to work as expected.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 10'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test10.png|thumb|600px|left|RFP protein generator under Plux and constitutive expression of luxR]]<br />
|}<br />
'''Description:''' this test is equivalent to TEST 9: we assembled K081014 (our RFP) under the Plux promoter that was contained into K081004. We kept pSB1AK3 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we wanted to repeat TEST 9 experiment using a longer construct and a different reporter gene.<br />
<br><br />
'''Methods:''' the same as TEST 9, but using K081004 instead of K081022 and using RFP (K081014) instead of GFP (K081012).<br />
<br><br />
'''Results:''' red fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture.<br />
{|align="center"<br />
|[[Image:pv_fig_test10.png|thumb|400px|left|Non induced culture: TOP10 cells cannot be seen at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test10bis.png|thumb|400px|left|Induced culture: fluorescent TOP10 cells at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test10superexp.png|thumb|400px|left|Same frames as above, but superexposed: non induced (left) and induced (right) cultures]]<br />
|}<br />
<br><br />
'''Comments:''' the same as TEST 9.<br />
<br><br />
NOTE: C0062 has the same mutation described in TEST 9. We also performed this test with an old version of K081004 carrying another point mutation (C->T at position 704 of C0062 coding sequence) that changed an aminoacid. Even in this case K081004 seemed to work as expected (results not shown).<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 11'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test11.png|thumb|600px|left|Terminator efficiency test]]<br />
|}<br />
'''Description:''' we assembled K081014 (our RFP) under the artificial 39 bp terminator (B1006) that is at the end of K081022-K081012 (composite part used for TEST 9). We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we wanted to test qualitatively if our terminator actually stops transcription.<br />
<br><br />
'''Methods:''' we assembled K081014 downstream of K081022, transformed ligation, plated transformed bacteria and insulated a colony containing correctly ligated plasmid. We inoculated the colony into 9 ml of LB + Amp and incubated the culture at 37°C, 220 rpm for 15 hours. Then we diluted 1:10 the culture in two falcon tubes (5 ml cultures). One of these cultures was induced with 3OC6-HSL 1 uM. We let the two cultures grow for 2 hours and then we watched 50 ul at microscope through FITC channel (positive control), TRITC channel and DAPI channel (negative control).<br />
<br><br />
'''Results:''' soon<br />
<br><br />
'''Comments:''' soon<br />
<br><br />
<br><br />
<hr><br />
<br><br />
We also performed these quantitative fluorescence tests:<br />
<hr><br />
*'''TEST 1'''<br />
<br><br />
'''Description:''' this test is an extension of TEST 9. We induced six cultures with 3OC6-HSL at different concentrations to study quantitatively HSL-GFP static transfer function.<br />
<br><br />
'''Motivation:''' we want to know cutoff point of this device.<br />
<br><br />
'''Methods:''' the same as TEST 9, but we diluted the overnight 9 ml culture 1:10 in six falcon tubes (5 ml cultures). We induced the six cultures with 3OC6-HSL 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM and 1 uM respectively. We incubated the cultures at 37°C, 220 rpm for 2 hours and then we watched 50 ul at microscope through FITC channel and DAPI channel for negative control. We prepared two 50 ul glasses for each culture. We acquired three frames at 2.5 s (maximum exposition time) and 10 ms exposition time for every sample. We used ImageJ software to count automatically the number of cells for every frame. Then we computed n10/n2.5 (where n is the number of cells) to calculate the percentage of cells that glowed in the 10 ms acquisition, assuming that in the 2.5 s acquisition we can see the total number of cells. For each HSL concentration, we calculated the mean value of the 6 statistics (3 frames for each of the 2 glasses).<br />
<br><br />
'''Results:'''<br />
{|align="center"<br />
|[[Image:pv_fig_test1q.png|thumb|600px|left|GFP (arbitrary units) vs 3OC6-HSL concentration: 1=0nM, 2=0.1nM, 3=1nM, 4=10nM, 5=100nM, 6=1uM]]<br />
|}<br />
<br><br />
'''Comments:''' we computed the statistic n10/n2.5 because we know that when fluorescence is weak, cells cannot be seen at low exposition times. So, we calculate the ratio between the cells we can see at a low exposition time and the total number of cells in the frame. We chose 10 ms because the previous experiments with GFP had been performed using this parameter. We know that this is not an exact statistic, in fact we have to consider the count errors of ImageJ software, especially when cells are superimposed. Further quantitative experiments will have to be performed using standard measurement, in order to characterize parts.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 2'''<br />
<br><br />
'''Description:''' this test is an extension of TEST 10. We induced seven cultures with 3OC6-HSL at different concentrations to study quantitatively HSL-RFP static transfer function.<br />
<br><br />
'''Motivation:''' we want to know cutoff point of this device.<br />
<br><br />
'''Methods:''' the same as TEST 10, but we diluted the overnight 9 ml culture 1:10 in seven falcon tubes (5 ml cultures). We induced the seven cultures with 3OC6-HSL 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 uM and 10 uM respectively. We incubated the cultures at 37°C, 220 rpm for 2 hours and then we watched 50 ul at microscope through TRITC channel and DAPI channel for negative control. We prepared two 50 ul glasses for each culture. We acquired three frames at 2.5 s (maximum exposition time) and 90 ms exposition time for every sample. We used ImageJ software to count automatically the number of cells for every acquisition. Then we computed n90/n2.5 (where n is the number of cells) to calculate the percentage of cells that glowed in the 90 ms acquisition, assuming that in the 2.5 s acquisition we can see the total number of cells. For each HSL concentration, we calculated the mean value of the 6 statistics (3 frames for each of the 2 glasses).<br />
<br><br />
'''Results:'''<br />
{|align="center"<br />
|[[Image:pv_fig_test2q.png|thumb|600px|left|RFP (arbitrary units) vs 3OC6-HSL concentration: 1=0nM, 2=0.1nM, 3=1nM, 4=10nM, 5=100nM, 6=1uM, 7=10uM]]<br />
|}<br />
<br><br />
'''Comments:''' we computed the statistic n90/n2.5 because we know that when fluorescence is weak, cells cannot be seen at low exposition times. So, we calculate the ratio between the cells we can see at a low exposition time and the total number of cells in the frame. We chose 90 ms because the previous experiments with RFP had been performed using this parameter. We know that this is not an exact statistic, in fact we have to consider the count errors of ImageJ software, especially when cells are superimposed. As we wrote for TEST 9, further quantitative experiments will have to be performed using standard measurement, in order to characterize parts.<br />
<br><br />
<hr><br />
<br />
=== Conclusions ===<br />
All the experiments we performed were consistent with our expectations.<br />
<br><br />
Considering the elementary logic gates of our final devices (AND, OR, NOT), we validated some truth table rows:<br />
{|align="center"<br />
|[[Image:pv_summary.png|thumb|600px|left|Biological truth tables]]<br />
|}<br />
<br />
In particular:<br />
*'''TEST 1 validated the first row of NOT logic gate.'''<br />
*'''TEST 6 validated the first row of AND (lux) logic gate.'''<br />
*'''TEST 7 validated the first row of AND (las) logic gate.'''<br />
*'''TEST 8, TEST 9 (with induction) and TEST 10 (with induction) validated the fourth row of AND (lux) logic gate.'''<br />
*'''TEST 9 (without induction) and TEST 10 (without induction) validated the third row of AND (lux) logic gate.'''</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/ProjectTeam:UNIPV-Pavia/Project2008-10-27T00:13:05Z<p>Magni: </p>
<hr />
<div>{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
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|-<br />
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|}<br />
<br />
<br><br />
<br />
== '''Overall project''' ==<br />
<br />
We are trying to mimic Multiplexer (Mux) and Demultiplexer (Demux) logic functions in E. coli.<br />
<br><br />
In the following paragraphs project details will be described from both digital electronic and genetic points of view.<br />
<br><br />
<br><br />
<br />
== '''Electronic Implementation''' ==<br />
<br><br />
=== '''What kind of components are Mux and Demux?''' ===<br />
'''Mux''' is a component which conveys one of the two input channels values into a single output channel. The choice of the input channel is made by a selector.<br />
<br><br />
'''Demux''' is a component which conveys the only input channel value into one of the two output channels. The choice of the output channel is made by a selector.<br />
<br><br />
<br><br />
The following pictures show data flow in Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_dataflow0.png|thumb|300px|left|Data flow in Multiplexer - SELECTOR=0]]<br />
|[[Image:pv_mux_dataflow1.png|thumb|300px|left|Data flow in Multiplexer - SELECTOR=1]]<br />
|-<br />
|[[Image:pv_demux_dataflow0.png|thumb|300px|left|Data flow in Demultiplexer - SELECTOR=0]]<br />
|[[Image:pv_demux_dataflow1.png|thumb|300px|left|Data flow in Demultiplexer - SELECTOR=1]]<br />
|}<br />
<br><br />
<br />
=== '''What kind of signals do we process?''' ===<br />
In this project we consider Boolean logic signals, thus every input/output value can assume only the values 0 and 1. A function that processes Boolean values is called logic function.<br />
<br><br />
Mux and Demux can be considered by now as black boxes which implement a logic function that can process input signals to output signals. Here you can see examples of Boolean data flow in Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_bool.png|thumb|300px|left|Example: Mux Boolean data flow]]<br />
|[[Image:pv_demux_bool.png|thumb|300px|left|Example: Demux Boolean data flow]]<br />
|}<br />
In the following documentation we will see what is inside these black boxes.<br />
<br><br />
<br><br />
=== How can we formalize Mux and Demux logic behavior? ===<br />
Logic functions can be formalized writing a truth table; a truth table is a mathematical table in which every row represents a combination of input values and its respective output values. The table has to be filled with every input combination.<br />
<br><br />
<br><br />
Here you can see Mux and Demux truth tables (output columns are gray):<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_truth.png|thumb|300px|left|Mux truth table]]<br />
|[[Image:pv_demux_truth.png|thumb|300px|left|Demux truth table]]<br />
|}<br />
<br><br />
<br />
=== Building a logic circuit from a truth table ===<br />
Our goal in this section is to project two logic gates networks which behave like Mux and Demux truth tables. A very useful tool to transform a truth table into a logic network is Karnaugh map.<br />
<br><br />
It is possible to read about Karnaugh maps at: [http://en.wikipedia.org/wiki/Karnaugh_map]<br />
<br><br />
<br><br />
Following Karnaugh maps method, we can write these two logic networks for Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux.png|thumb|340px|left|Mux - logic circuit]]<br />
|[[Image:pv_mux_example.png|thumb|340px|left|Mux - Example]]<br />
|-<br />
|[[Image:pv_demux.png|thumb|340px|left|Demux - logic circuit]]<br />
|[[Image:pv_demux_example.png|thumb|340px|left|Demux - Example]]<br />
|}<br />
<br />
<br><br />
<br />
== '''Genetic Implementation''' ==<br />
Our goal is to mimic Mux and Demux logic networks in a biological device, such as E. coli. To perform this, we use protein/DNA and protein/protein interactions to build up biological logic gates.<br />
Mux and Demux logic circuits are composed by three fundamental logic gates, AND, OR, NOT: in the next paragraphs genetic implementation of these logic gates will be provided.<br />
<br><br />
<br><br />
=== AND ===<br />
To mimic an AND gate, we need a biological function, such as a promoter activation, which is directly turned on by the interaction between two upstream genes. In our synthetic devices, we use the luxR/luxI system: luxR can activate Plux promoter only upon 3-oxo-hexanoyl-homoserine lactone (HSL) binding; luxI generates HSL; so, only the contemporary expression of LuxR and luxI proteins can activate the downstream Plux-dependent gene expression. Another AND gate we use is the lasR/lasI system, which works in a very similar way but through another chemical intermediate, N-(3-oxododecanoyl) homoserine lactone (PAI-1).<br />
{|<br />
|[[Image:pv_proj_AND.png|thumb|340px|left|Genetic AND: Plux can be turned on only when the two proteins luxI and luxR are present.]]<br />
|}<br />
=== OR ===<br />
To mimic an OR gate in Mux, we need a biological function which can be activated alternatively by two independent upstream signals or by both. Thus, we combine the outputs of the upstream AND gates to assemble directly an OR reporter function, by simply repeating the reporter gene (GFP) under two different promoters (Plux and Plas). It’s sufficient to activate one of the two promoters (or both) to recover the GFP signal from engineered bacteria.<br />
There should not be an over-expression problem for GFP, in fact, in Mux device, only one promoter can be active, either Plux or Plas. Here we considered GFP output, but OR device can be generalized for every output gene.<br />
{|<br />
|[[Image:pv_proj_OR.png|thumb|340px|left|Genetic OR: it is sufficient that one of the two input promoters is active to obtain GFP expression.]]<br />
|}<br />
=== NOT ===<br />
To mimic a NOT gate, we need an efficient and regulated repressor of a specific downstream promoter: in this case, we choose cI repression on Plambda, which should be specific and, upon cI inactivation, quick and efficient.<br />
{|<br />
|[[Image:pv_proj_NOT.png|thumb|340px|left|Genetic NOT: Plambda can be turned on only when cI protein is not present.]]<br />
|}<br />
<br />
According to what above stated, genetic implementation of Mux and Demux can be obtained connecting these basic logic gates and can be summarized in this way:<br />
<br><br />
<br><br />
<br><br />
<br />
=== Genetic Mux ===<br />
Let PA, PB and PS be three generic promoters that can be ACTIVATED respectively by the three exogenous molecules "A", "B" and "S". A genetic Mux with inputs "A" (CH0) and "B" (CH1), selector "S" and the generic protein GOI as output, can be implemented by the following gene network:<br />
{|align="center"<br />
|[[Image:pv_MUX.png|thumb|420px|left|Genetic Mux]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_MUXint.png|thumb|420px|left|Genetic Mux - interactions]]<br />
|}<br />
We want to supply a device that can be generalized to detect every kind of input and to express every kind of genes as output. So, input promoters and output protein generators are not part of the genetic Mux we want to build up. In this way, a hypothetical user can re-use our device assembling the desired input and output elements.<br />
<br><br />
Note that the output genes are two, but, as explained in "OR" section, if the genes are identical, we can say that there is only one output; in fact, the real output is a protein synthesis and so it is not important which of the two identical genes is expressed.<br />
<br><br />
However, it is possible to assemble two different genes downstream of Plux and Plas, for example two reporters. In this way, debugging process becomes easy, because we can discriminate lux system and las system activities.<br />
<br><br />
Here we report the truth table of this network:<br />
{|align="center"<br />
|[[Image:pv_gmux_truth.png|thumb|420px|left|Genetic Mux - truth table]]<br />
|}<br />
<br><br />
Now we report two examples of genetic Mux behavior, in response to generic inputs.<br />
<br><br />
<br><br />
====Genetic Mux behavior - Example 1====<br />
{|<br />
|[[Image:pv_genmux_example1.png|thumb|420px|left|First example of genetic Mux behavior]]<br />
|}<br />
According to Mux truth table, if "A" is not present and "B" and "S" are present, we expect to have GOI synthesis.<br />
<br><br />
"A" molecule is not present, so PA promoter is not active and luxI gene is not expressed.<br />
<br><br />
"B" molecule is present, so PB promoter is active and lasI gene is expressed.<br />
<br><br />
"S" molecule is present, so PS promoter is active and lasR and cI genes are expressed.<br />
<br><br />
cI protein represses transcription of the gene downstream of Plambda promoter, which is luxR.<br />
<br><br />
lasI protein can synthesize 3-OC12-HSL. This molecule activates transcription factor lasR, which can activate Plas promoter. So, the copy of GOI gene under Plas regulation can be expressed.<br />
<br><br />
The other copy of GOI gene, which is under Plux promoter regulation, is not expressed. In fact Plux is not active, because both luxI and luxR proteins are not present.<br />
<br><br />
Only one of the two GOI genes is expressed, but this is sufficient to synthesize the output protein GOI.<br />
<br><br />
<br><br />
====Genetic Mux behavior - Example 2====<br />
{|<br />
|[[Image:pv_genmux_example2.png|thumb|420px|left|Second example of genetic Mux behavior]]<br />
|}<br />
We expect to have no GOI synthesis in response to this input combination.<br />
<br><br />
"A" molecule is not present, so PA promoter is not active and luxI gene is not expressed.<br />
<br><br />
"B" molecule is present, so PB promoter is active and lasI gene is expressed.<br />
<br><br />
"S" molecule is not present, so PS promoter can't transcribe cI and lasR genes.<br />
<br><br />
cI protein is not present, so Plambda promoter can transcribe luxR gene.<br />
<br><br />
None of the two logic AND systems is active, because lux system lacks of luxI and las system lacks of lasR. So, Plux and Plas promoters are unactive and can't express GOI genes. For this reason, GOI protein is not synthesized.<br />
<br><br />
<br><br />
These two examples show how "S" molecule can select the input to be conveyed into the single output channel: in fact its presence allows lasR expression and represses luxR expression, while "S" absence represses lasR expression and allows luxR expression.<br />
<br><br />
<br><br />
<br />
=== Genetic Demux ===<br />
Let PI and PS be two generic promoters that can be ACTIVATED respectively by the two exogenous molecules "I" and "S". A genetic Demux with input "I", selector "S" and the generic proteins GOI0 and GOI1 as outputs (OUT0 and OUT1 respectively), can be implemented by the following gene network:<br />
{|align="center"<br />
|[[Image:pv_DEMUX.png|thumb|420px|left|Genetic Demux]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_DEMUXint.png|thumb|420px|left|Genetic Demux - interactions]]<br />
|}<br />
As described for genetic Mux, we want to supply a device that can be generalized to detect every kind of inputs and to express every kind of genes as output. So, input promoters and output protein generators are not part of the genetic Demux. In this way, a hypothetical user can re-use our device assembling the desired input and output elements.<br />
<br><br />
Here we report the truth table of this network:<br />
{|align="center"<br />
|[[Image:pv_gdemux_truth.png|thumb|420px|left|Genetic Demux - truth table]]<br />
|}<br />
<br><br />
Now we report two examples of genetic Demux behavior, in response to generic inputs.<br />
<br><br />
<br><br />
====Genetic Demux behavior - Example 1====<br />
{|<br />
|[[Image:pv_gendemux_example1.png|thumb|420px|left|First example of genetic Demux behavior]]<br />
|}<br />
According to Demux truth table, if "I" molecule is present and "S" molecule is absent, we expect to have GOI0 protein synthesis and no GOI1 protein synthesis.<br />
<br><br />
"I" is present, so PI promoter is active and luxI and lasI genes are expressed.<br />
<br><br />
"S" is not present, so PS promoter can't transcribe lasR and cI genes.<br />
<br><br />
cI protein is not present, so Plambda promoter is not inhibited and luxR gene is expressed.<br />
<br><br />
luxI protein can synthesize 3-OC6-HSL lactone. This molecule activates luxR transcription factor which can activate Plux promoter. In this way, GOI0 gene, which is under Plux regulation, is expressed.<br />
<br><br />
On the other hand, lasI protein can synthesize 3-OC12-HSL, but lasR transcription factor is not present and so Plas promoter cannot be activated. In this way, GOI1 gene, which is under Plas regulation, is not expressed.<br />
<br><br />
So, we have GOI0 protein synthesis and no GOI1 protein synthesis.<br />
<br><br />
<br><br />
====Genetic Demux behavior - Example 2====<br />
{|<br />
|[[Image:pv_gendemux_example2.png|thumb|420px|left|Second example of genetic Demux behavior]]<br />
|}<br />
We expect to have GOI1 synthesis and no GOI0 synthesis in response to this input combination.<br />
<br><br />
"I" is present, so PI promoter is active and luxI and lasI genes are expressed.<br />
<br><br />
"S" is present, so PS promoter lasR and cI genes are expressed.<br />
<br><br />
cI protein is present, so Plambda promoter is inhibited and luxR gene is not expressed.<br />
<br><br />
lasI protein can synthesize 3-OC12-HSL lactone. This molecule activates lasR transcription factor which can activate Plas promoter. In this way, GOI1 gene, which is under Plas regulation, is expressed.<br />
<br><br />
On the other hand, luxI protein can synthesize 3-OC6-HSL, but luxR transcription factor is not present and so Plux promoter cannot be activated. In this way, GOI0 gene, which is under Plux regulation, is not expressed.<br />
<br><br />
So, we have GOI1 protein synthesis and no GOI0 protein synthesis.<br />
<br />
<br />
=== A complete genetic Mux===<br />
In this section a complete Mux gene network is described.<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_implementation.png|thumb|420px|left|Example of a complete genetic Mux]]<br />
|[[Image:pv_mux_gn_implementation.png|thumb|420px|left|Example of a complete genetic Mux - Gene network]]<br />
|}<br />
We want to build up a device in which Channel 0, Channel 1 and Selector are respectively sensitive to Tetracycline, IPTG and red light. The presence of each input corresponds to logic 1.<br />
We chose green fluorescence as Mux output: expression of GFP corresponds to logic 1, while absence of fluorescence corresponds to logic 0.<br />
<br><br />
Tetracycline and IPTG can be considered as "A" and "B" molecules, introduced in "Genetic Mux" section, because both Tetracycline and IPTG are indirect ACTIVATORS of Ptet and Plac promoters respectively.<br />
On the other hand, red light is quite different from "S" molecule, because red light is an indirect REPRESSOR of Pomp promoter and not an activator as required by the original schema. For this reason, if we want a device in which the presence of Tetracycline, IPTG and red light correspond to logic 1, red light input should be ''inverted''. The simplest way to do this, is to cross-exchange the two input channels. So, Tetracycline sensor (CH0) has to be assembled to las system and IPTG sensor (CH1) has to be assembled to lux system.<br />
<br />
<br />
====Complete genetic Mux - Example====<br />
{|<br />
|[[Image:pv_mux_example1.png|thumb|420px|left|Example of a complete genetic Mux behavior]]<br />
|}<br />
According to Mux truth table, if SEL=0, CH0=1, CH1=0, output channel must be logic 1.<br />
Red light is not present, so it can't dephosphorylate cph8-ho1-pcyA complex, which is constitutively expressed. cph8-ho1-pcyA complex activates endogenous ompR, which can activate Pomp promoter, so cI and lasR genes are transcribed.<br />
cI protein binds Plambda promoter and so luxR expression is inhibited.<br />
TetR is a repressor for Ptet promoter, but Tetracycline is present, so it can bind tetR protein, which is constitutively expressed, and can activate transcription of downstream gene: lasI.<br />
Simultaneous expression of lasI and lasR can activate GFP, which is under Plas promoter.<br />
IPTG is not present, so lacI protein binds Plac promoter and represses luxI transcription.<br />
<br />
<br />
=== A complete genetic Demux===<br />
In this section a complete Demux gene network is described.<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_demux_implementation.png|thumb|420px|left|Example of a complete genetic Demux]]<br />
|[[Image:pv_demux_gn_implementation.png|thumb|420px|left|Example of a complete genetic Demux - Gene network]]<br />
|}<br />
<br />
We want to build up a device in which Input and Selector are respectively IPTG and Tetracycline.<br><br />
In Demux we have two output channels: red fluorescence corresponds to logic 1 at Channel 0, while green fluorescence corresponds to logic 1 at Channel 1.<br><br />
Absence of reporters expression corresponds to logic 0 at Channel 0 and Channel 1.<br><br />
There isn't any input combination that corresponds to logic 1 at Channel 0 and Channel 1 together.<br />
<br />
====Complete genetic Demux - Example====<br />
{|<br />
|[[Image:pv_demux_example1.png|thumb|420px|left|Example of a complete genetic Demux behavior]]<br />
|}<br />
According to Demux truth table, if IN=1 and SEL=1, output channel 0 is logic 0 and output channel 1 is logic 1.<br><br />
IPTG is present, so Plac promoter is active because IPTG binds lacI protein. This allows lasI and luxI transcription.<br><br />
Tetracycline is present, so Ptet promoter is active because Tetracycline binds tetR protein. This allows cI and lasR transcription.<br><br />
cI protein binds Plambda promoter and so luxR expression is inhibited.<br><br />
Simultaneous expression of lasI and lasR can activate GFP, which is under Plas promoter.<br><br />
There is no simultaneous expression of luxI and luxR, so RFP (which is under Plux regulation) cannot be expressed.<br />
<br />
== Final devices==<br />
BioBrick standard parts for genetic Mux and Demux are summarized in the following schemas:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_final.png|thumb|420px|left|Three standard parts for mux]]<br />
|[[Image:pv_demux_final.png|thumb|450px|left|Two standard parts for demux]]<br />
|}<br />
<br />
A hypothetical user of Mux or Demux has to ligate our standard parts with desired inputs and outputs, as shown in the pictures above.<br />
<br><br />
The structure of our devices show that Mux and Demux systems both conform to the PoPS device boundary standard.<br />
<br><br />
<br />
==Applications==<br />
Mux and Demux are two fundamental devices in electronics. They are used in several applications, for example in communication devices, in Arithmetic Logic Units (ALUs), or in applications that involve channel sharing.<br />
<br><br />
Analogously, they could play a crucial role in the building of complex genetic circuits. In fact, both of them can be used as controlled genetic switches.<br />
<br><br />
Because our devices had been designed to be general, their application field is very wide. For example, genetic Mux can be used to integrate signals from the environment in a two-inputs and one-output biosensor; once detected, the selector controls which of the two inputs must be transferred in output. In this way, two sensing devices can be integrated in only one circuit that can compute multiplexing logic function.<br />
On the other hand, genetic Demux can be used for controlled protein productions, where the choice of the protein to produce is made by the selector. In this case, we can imagine an industrial process where two consequent enzyme reactions are necessary to build a final product. Using a Demux, we can consider the two enzymes as system outputs and then we can easily switch on and off their production modulating selection signal.<br />
<br><br />
Selector in Mux and Demux can be controlled manually, but also automatically. In fact, it is possible to use feedback control to pilot the selector in order, according with Demux example, to switch enzyme production when a specific condition (for example a pH threshold) is reached.<br />
<br><br />
Switching activity in these two devices is a very powerful tool to manage multi-input and multi-output systems.<br />
<br />
<br />
== Experiments and results ==<br />
=== Assemblies ===<br />
We successfully amplified the following BioBrick standard parts from Spring 2008 DNA Distribution:<br />
{|align="center"<br />
|[[Image:pv_resusp.png|thumb|600px|left|Successful amplifications]]<br />
|}<br />
<br><br />
while we couldn't amplify correctly the following parts:<br />
{|align="center"<br />
|[[Image:pv_notresusp.png|thumb|600px|left|Unsuccessful amplifications]]<br />
|}<br />
<br><br />
To build up our designed devices, we followed and completed this assembly tree schema:<br />
{|align="center"<br />
|[[Image:pv_assemblyschema.png|thumb|600px|left|Assembly tree schema]]<br />
|}<br />
<br />
=== Functional tests ===<br />
We performed these boolean (on/off) fluorescence tests:<br />
<br><br />
<hr><br />
*'''TEST 1'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test1.png|thumb|250px|left|GFP protein generator under Plambda]]<br />
|}<br />
'''Description:''' we assembled an available GFP protein generator (E0240) under Plambda promoter, keeping pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' cI protein was not present, so Plambda should work like a constitutive promoter. A reporter gene downstream of this promoter allows us to validate Plambda costitutive activity. That can be very useful to validate our NOT logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of R0051 and E0240, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test1.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that GFP was expressed in positive transformants, so Plambda activity was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 2'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test2.png|thumb|250px|left|GFP protein generator under Ptet]]<br />
|}<br />
'''Description:''' we assembled E0240 under Ptet promoter, keeping pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' tetR protein was not present, so Ptet should work like a constitutive promoter. A reporter gene downstream of this promoter allows us to validate Ptet costitutive activity. Ptet is useful for specific input building.<br />
<br><br />
'''Methods:''' after ligation reaction of R0040 and E0240, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test2.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that GFP was expressed in positive transformants, so Ptet activity was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 3'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test3.png|thumb|250px|left|RFP protein generator under constitutive promoter]]<br />
|}<br />
'''Description:''' we didn't perform any assembly for this experiment, because the promoter we wanted to test was contained into BBa_J61002 vector, which places a RFP protein generator between SpeI and PstI restriction sites.<br />
<br><br />
'''Motivation:''' J23100, which we call Pcon, should have a strong constitutive activity. A reporter gene downstream of this promoter allows us to validate this activity. Pcon is useful to build our inputs because some sensors like IPTG and Tetracycline sensors need the constitutive production of specific proteins, in this case lacI and tetR respectively.<br />
<br><br />
'''Methods''' we transformed J23100 using Invitrogen TOP10 and plated transformed bacteria. We expected to observe red fluorescence (using transluminator) in all the colonies. We also picked up a colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through TRITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' all the grown colonies glowed under UV rays and red fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test3.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that RFP was expressed in transformed bacteria, so Pcon functionality was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 4'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test4.png|thumb|250px|left|Our GFP protein generator (K081012) under a constitutive promoter]]<br />
|}<br />
'''Description:''' we assembled a constitutive promoter family member (J23100) upstream of K081012, a GFP protein generator we built. We decided to excide J23100 from its plasmid (E-S) and to open K081012 plasmid (pSB1AK3) (E-X).<br />
<br><br />
'''Motivation:''' we already validated qualitatively J23100 constitutive activity, so this promoter can be used to test the GFP protein generator we built. We wanted to check if K081012 actually generates GFP.<br />
<br><br />
'''Methods''' after ligation reaction of J23100 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays (see figure) and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test4bis.jpg|thumb|400px|left|TOP10 colonies under UV]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test4.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that our GFP protein generator actually generates GFP.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 5'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test5.png|thumb|250px|left|Our RFP protein generator (K081014) under a constitutive promoter]]<br />
|}<br />
'''Description:''' we assembled a constitutive promoter family member (J23100) upstream of K081014, a RFP protein generator we built. We decided to excide J23100 from its plasmid (E-S) and to open K081014 plasmid (pSB1AK3) (E-X).<br />
<br><br />
'''Motivation:''' we already validated qualitatively J23100 constitutive activity, so this promoter can be used to test the RFP protein generator we built. We wanted to check if K081014 actually generates RFP.<br />
<br><br />
'''Methods''' after ligation reaction of J23100 and K081014, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through TRITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays (see figure) and red fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test5bis.jpg|thumb|400px|left|TOP10 colonies under UV]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test5.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that our RFP protein generator actually generates RFP.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 6'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test6.png|thumb|350px|left|GFP protein generator under Plux]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081000. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we didn't assemble any input to K081000 device, so we could study only the promoter basic activity. We expected to find a weak basic activity. Plux is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of K081000 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we performed a screening on 5 colonies through colony PCR to search for correctly ligated plasmid. We infected 9 ml of LB + Amp with the chosen colony and incubated the culture at 37°C, 220 rpm for 15 hours. Then we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test6.png|thumb|400px|left|No fluorescent TOP10 cells can be seen at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' the picture above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria. Increasing exposition time, green fluorescence could be observed (picture not reported). So, we can say that GFP was expressed very weakly. This experiment confirmed that Plux promoter has a weak activity without luxI and luxR proteins.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 7'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test7.png|thumb|350px|left|GFP protein generator under Plas]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plas promoter that was contained into K081001. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we didn't assemble any input to K081001 device, so we could study only the promoter basic activity. We expected to find a weak basic activity. Plas is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of K081001 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we performed a screening on 5 colonies through colony PCR to search for correctly ligated plasmid. We infected 9 ml of LB + Amp with the chosen colony and incubated the culture at 37°C, 220 rpm for 15 hours. Then we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test7.png|thumb|400px|left|No fluorescent TOP10 cells can be seen at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' the picture above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria. Increasing exposition time, green fluorescence could be observed (picture not reported). So, we can say that GFP was expressed very weakly. This experiment confirmed that Plas promoter has a weak activity without lasI and lasR proteins.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 8'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test8.png|thumb|600px|left|GFP protein generator under Plux and constitutive expression of luxI and luxR]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081000. We also assembled K081011 upstream of K081000. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' Plux promoter activity was studied in response of a constitutive expression of luxI and luxR. Plambda promoter without cI repressor guaranteed the constitutive expression of these genes. We expected to find a strong activity because Plux is turned on. lux system is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' we ligated K081012 under K081000, transformed ligation, plated transformed bacteria, performed PCR screening on some colonies and extracted correctly ligated plasmids. We repeated these steps to assemble K081011 upstream of K081000-K081012, but we didn't perform PCR screening. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test8.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that Plux promoter can be activated by the contemporary presence of luxI and luxR. This is a crucial result for our AND logic gate.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 9'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test9.png|thumb|500px|left|GFP protein generator under Plux and constitutive expression of luxR]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081022. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' Plux promoter activity was studied in response to a constitutive expression of luxR. Plambda promoter without cI repressor guaranteed the constitutive expression of this gene. We expected to find a weak activity because luxR transcription factor is not active and so Plux cannot be turned on. We also expected to find a strong activity if we induce luxR activation using 3OC6-HSL (this is equivalent to TEST 8 conditions, because 3OC6-HSL is synthesized by luxI). lux system is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' we ligated K081012 downstream of K081022, transformed ligation, plated transformed bacteria and screened three colonies to insulate a colony containing correctly ligated plasmids. We inoculated the positive colony into 9 ml of LB + Amp and incubated the culture at 37°C, 220 rpm for 15 hours. Then we diluted 1:10 the culture in two falcon tubes (5 ml cultures). One of these cultures was induced with 3OC6-HSL 1 uM. We let the two cultures grow for 2 hours and then we watched 50 ul at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture.<br />
{|align="center"<br />
|[[Image:pv_fig_test9.png|thumb|400px|left|Non induced culture: TOP10 cells cannot be seen at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test9bis.png|thumb|400px|left|Induced culture: fluorescent TOP10 cells at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test9superexp.png|thumb|400px|left|Same frames as above, but superexposed: non induced (left) and induced (right) cultures]]<br />
|}<br />
<br><br />
'''Comments:''' the pictures above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria for the non induced culture, while we can see green fluorescent TOP10 in the induced culture. As we wrote for TEST 6 and TEST 7, increasing exposition time green fluorescence could be observed even for the non induced culture (last picture), confirming the weak activity of Plux promoter in response of unactive luxR. This experiment confirmed that Plux promoter has a weak activity without luxI (or 3OC6-HSL) and luxR protein is not sufficient to induce a strong transcription. Adding 3OC6-HSL, luxR becomes active and so Plux is turned on.<br />
<br><br />
NOTE: K081022 has a point mutation in position 349 of C0062 coding sequence. This mutation changes the aminoacid, but luxR seems to work as expected.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 10'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test10.png|thumb|600px|left|RFP protein generator under Plux and constitutive expression of luxR]]<br />
|}<br />
'''Description:''' this test is equivalent to TEST 9: we assembled K081014 (our RFP) under the Plux promoter that was contained into K081004. We kept pSB1AK3 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we wanted to repeat TEST 9 experiment using a longer construct and a different reporter gene.<br />
<br><br />
'''Methods:''' the same as TEST 9, but using K081004 instead of K081022 and using RFP (K081014) instead of GFP (K081012).<br />
<br><br />
'''Results:''' red fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture.<br />
{|align="center"<br />
|[[Image:pv_fig_test10.png|thumb|400px|left|Non induced culture: TOP10 cells cannot be seen at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test10bis.png|thumb|400px|left|Induced culture: fluorescent TOP10 cells at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test10superexp.png|thumb|400px|left|Same frames as above, but superexposed: non induced (left) and induced (right) cultures]]<br />
|}<br />
<br><br />
'''Comments:''' the same as TEST 9.<br />
<br><br />
NOTE: C0062 has the same mutation described in TEST 9. We also performed this test with an old version of K081004 carrying another point mutation (C->T at position 704 of C0062 coding sequence) that changed an aminoacid. Even in this case K081004 seemed to work as expected (results not shown).<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 11'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test11.png|thumb|600px|left|Terminator efficiency test]]<br />
|}<br />
'''Description:''' we assembled K081014 (our RFP) under the artificial 39 bp terminator (B1006) that is at the end of K081022-K081012 (composite part used for TEST 9). We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we wanted to test qualitatively if our terminator actually stops transcription.<br />
<br><br />
'''Methods:''' we assembled K081014 downstream of K081022, transformed ligation, plated transformed bacteria and insulated a colony containing correctly ligated plasmid. We inoculated the colony into 9 ml of LB + Amp and incubated the culture at 37°C, 220 rpm for 15 hours. Then we diluted 1:10 the culture in two falcon tubes (5 ml cultures). One of these cultures was induced with 3OC6-HSL 1 uM. We let the two cultures grow for 2 hours and then we watched 50 ul at microscope through FITC channel (positive control), TRITC channel and DAPI channel (negative control).<br />
<br><br />
'''Results:''' soon<br />
<br><br />
'''Comments:''' soon<br />
<br><br />
<br><br />
<hr><br />
<br><br />
We also performed these quantitative fluorescence tests:<br />
<hr><br />
*'''TEST 1'''<br />
<br><br />
'''Description:''' this test is an extension of TEST 9. We induced six cultures with 3OC6-HSL at different concentrations to study quantitatively HSL-GFP static transfer function.<br />
<br><br />
'''Motivation:''' we want to know cutoff point of this device.<br />
<br><br />
'''Methods:''' the same as TEST 9, but diluted the overnight 9 ml culture 1:10 in six falcon tubes (5 ml cultures). We induced the six cultures with 3OC6-HSL 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM and 1 uM respectively. We incubated the cultures at 37°C, 220 rpm for 2 hours and then we watched 50 ul at microscope through FITC channel and DAPI channel for negative control. We prepared two 50 ul glasses for each culture. We acquired three frames at 2.5 s (maximum exposition time) and 10 ms exposition time for every sample. We used ImageJ software to count automatically the number of cells for every frame. Then we computed n10/n2.5 (where n is the number of cells) to calculate the percentage of cells that glowed in the 10 ms acquisition, assuming that in the 2.5 s acquisition we can see the total number of cells. For each HSL concentration, we calculated the mean value of the 6 statistics (3 frames for each of the 2 glasses).<br />
<br><br />
'''Results:'''<br />
{|align="center"<br />
|[[Image:pv_fig_test1q.png|thumb|600px|left|GFP (arbitrary units) vs 3OC6-HSL concentration: 1=0nM, 2=0.1nM, 3=1nM, 4=10nM, 5=100nM, 6=1uM]]<br />
|}<br />
<br><br />
'''Comments:''' we computed the statistic n10/n2.5 because we know that when fluorescence is weak, cells cannot be seen at low exposition times. So, we calculate the ratio between the cells we can see at a low exposition time and the total number of cells in the frame. We chose 10 ms because the previous experiments with GFP had been performed using this parameter. We know that this is not an exact statistic, in fact we have to consider the count errors of ImageJ software, especially when cells are superimposed. Further quantitative experiments will have to be performed using standard measurement, in order to characterize parts.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 2'''<br />
<br><br />
'''Description:''' this test is an extension of TEST 10. We induced seven cultures with 3OC6-HSL at different concentrations to study quantitatively HSL-RFP static transfer function.<br />
<br><br />
'''Motivation:''' we want to know cutoff point of this device.<br />
<br><br />
'''Methods:''' the same as TEST 10, but diluted the overnight 9 ml culture 1:10 in seven falcon tubes (5 ml cultures). We induced the seven cultures with 3OC6-HSL 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 uM and 10 uM respectively. We incubated the cultures at 37°C, 220 rpm for 2 hours and then we watched 50 ul at microscope through TRITC channel and DAPI channel for negative control. We prepared two 50 ul glasses for each culture. We acquired three frames at 2.5 s (maximum exposition time) and 90 ms exposition time for every sample. We used ImageJ software to count automatically the number of cells for every acquisition. Then we computed n90/n2.5 (where n is the number of cells) to calculate the percentage of cells that glowed in the 90 ms acquisition, assuming that in the 2.5 s acquisition we can see the total number of cells. For each HSL concentration, we calculated the mean value of the 6 statistics (3 frames for each of the 2 glasses).<br />
<br><br />
'''Results:'''<br />
{|align="center"<br />
|[[Image:pv_fig_test2q.png|thumb|600px|left|RFP (arbitrary units) vs 3OC6-HSL concentration: 1=0nM, 2=0.1nM, 3=1nM, 4=10nM, 5=100nM, 6=1uM, 7=10uM]]<br />
|}<br />
<br><br />
'''Comments:''' we computed the statistic n90/n2.5 because we know that when fluorescence is weak, cells cannot be seen at low exposition times. So, we calculate the ratio between the cells we can see at a low exposition time and the total number of cells in the frame. We chose 90 ms because the previous experiments with RFP had been performed using this parameter. We know that this is not an exact statistic, in fact we have to consider the count errors of ImageJ software, especially when cells are superimposed. As we wrote for TEST 9, further quantitative experiments will have to be performed using standard measurement, in order to characterize parts.<br />
<br><br />
<hr><br />
<br />
=== Conclusions ===<br />
All the experiments we performed were consistent with our expectations.<br />
<br><br />
Considering the elementary logic gates of our final devices (AND, OR, NOT), we validated some truth table rows:<br />
{|align="center"<br />
|[[Image:pv_summary.png|thumb|600px|left|Biological truth tables]]<br />
|}<br />
<br />
In particular:<br />
*'''TEST 1 validated the first row of NOT logic gate.'''<br />
*'''TEST 6 validated the first row of AND (lux) logic gate.'''<br />
*'''TEST 7 validated the first row of AND (las) logic gate.'''<br />
*'''TEST 8, TEST 9 (with induction) and TEST 10 (with induction) validated the fourth row of AND (lux) logic gate.'''<br />
*'''TEST 9 (without induction) and TEST 10 (without induction) validated the third row of AND (lux) logic gate.'''</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/ProjectTeam:UNIPV-Pavia/Project2008-10-26T23:41:31Z<p>Magni: </p>
<hr />
<div>{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
== '''Overall project''' ==<br />
<br />
We are trying to mimic Multiplexer (Mux) and Demultiplexer (Demux) logic functions in E. coli.<br />
<br><br />
In the following paragraphs project details will be described from both digital electronic and genetic points of view.<br />
<br><br />
<br><br />
<br />
== '''Electronic Implementation''' ==<br />
<br><br />
=== '''What kind of components are Mux and Demux?''' ===<br />
'''Mux''' is a component which conveys one of the two input channels values into a single output channel. The choice of the input channel is made by a selector.<br />
<br><br />
'''Demux''' is a component which conveys the only input channel value into one of the two output channels. The choice of the output channel is made by a selector.<br />
<br><br />
<br><br />
The following pictures show data flow in Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_dataflow0.png|thumb|300px|left|Data flow in Multiplexer - SELECTOR=0]]<br />
|[[Image:pv_mux_dataflow1.png|thumb|300px|left|Data flow in Multiplexer - SELECTOR=1]]<br />
|-<br />
|[[Image:pv_demux_dataflow0.png|thumb|300px|left|Data flow in Demultiplexer - SELECTOR=0]]<br />
|[[Image:pv_demux_dataflow1.png|thumb|300px|left|Data flow in Demultiplexer - SELECTOR=1]]<br />
|}<br />
<br><br />
<br />
=== '''What kind of signals do we process?''' ===<br />
In this project we consider Boolean logic signals, thus every input/output value can assume only the values 0 and 1. A function that processes Boolean values is called logic function.<br />
<br><br />
Mux and Demux can be considered by now as black boxes which implement a logic function that can process input signals to output signals. Here you can see examples of Boolean data flow in Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_bool.png|thumb|300px|left|Example: Mux Boolean data flow]]<br />
|[[Image:pv_demux_bool.png|thumb|300px|left|Example: Demux Boolean data flow]]<br />
|}<br />
In the following documentation we will see what is inside these black boxes.<br />
<br><br />
<br><br />
=== How can we formalize Mux and Demux logic behavior? ===<br />
Logic functions can be formalized writing a truth table; a truth table is a mathematical table in which every row represents a combination of input values and its respective output values. The table has to be filled with every input combination.<br />
<br><br />
<br><br />
Here you can see Mux and Demux truth tables (output columns are gray):<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux_truth.png|thumb|300px|left|Mux truth table]]<br />
|[[Image:pv_demux_truth.png|thumb|300px|left|Demux truth table]]<br />
|}<br />
<br><br />
<br />
=== Building a logic circuit from a truth table ===<br />
Our goal in this section is to project two logic gates networks which behave like Mux and Demux truth tables. A very useful tool to transform a truth table into a logic network is Karnaugh map.<br />
<br><br />
It is possible to read about Karnaugh maps at: [http://en.wikipedia.org/wiki/Karnaugh_map]<br />
<br><br />
<br><br />
Following Karnaugh maps method, we can write these two logic networks for Mux and Demux:<br />
<br><br />
{|cellpadding="12px" align="center"<br />
|[[Image:pv_mux.png|thumb|340px|left|Mux - logic circuit]]<br />
|[[Image:pv_mux_example.png|thumb|340px|left|Mux - Example]]<br />
|-<br />
|[[Image:pv_demux.png|thumb|340px|left|Demux - logic circuit]]<br />
|[[Image:pv_demux_example.png|thumb|340px|left|Demux - Example]]<br />
|}<br />
<br />
<br><br />
<br />
== '''Genetic Implementation''' ==<br />
Our goal is to mimic Mux and Demux logic networks in a biological device, such as E. coli. To perform this, we use protein/DNA and protein/protein interactions to build up biological logic gates.<br />
Mux and Demux logic circuits are composed by three fundamental logic gates, AND, OR, NOT: in the next paragraphs genetic implementation of these logic gates will be provided.<br />
<br><br />
<br><br />
=== AND ===<br />
To mimic an AND gate, we need a biological function, such as a promoter activation, which is directly turned on by the interaction between two upstream genes. In our synthetic devices, we use the luxR/luxI system: luxR can activate Plux promoter only upon 3-oxo-hexanoyl-homoserine lactone (HSL) binding; luxI generates HSL; so, only the contemporary expression of LuxR and luxI proteins can activate the downstream Plux-dependent gene expression. Another AND gate we use is the lasR/lasI system, which works in a very similar way but through another chemical intermediate, N-(3-oxododecanoyl) homoserine lactone (PAI-1).<br />
{|<br />
|[[Image:pv_proj_AND.png|thumb|340px|left|Genetic AND: Plux can be turned on only when the two proteins luxI and luxR are present.]]<br />
|}<br />
=== OR ===<br />
To mimic an OR gate in Mux, we need a biological function which can be activated alternatively by two independent upstream signals or by both. Thus, we combine the outputs of the upstream AND gates to assemble directly an OR reporter function, by simply repeating the reporter gene (GFP) under two different promoters (Plux and Plas). It’s sufficient to activate one of the two promoters (or both) to recover the GFP signal from engineered bacteria.<br />
There should not be an over-expression problem for GFP, in fact, in Mux device, only one promoter can be active, either Plux or Plas. Here we considered GFP output, but OR device can be generalized for every output gene.<br />
{|<br />
|[[Image:pv_proj_OR.png|thumb|340px|left|Genetic OR: it is sufficient that one of the two input promoters is active to obtain GFP expression.]]<br />
|}<br />
=== NOT ===<br />
To mimic a NOT gate, we need an efficient and regulated repressor of a specific downstream promoter: in this case, we choose cI repression on Plambda, which should be specific and, upon cI inactivation, quick and efficient.<br />
{|<br />
|[[Image:pv_proj_NOT.png|thumb|340px|left|Genetic NOT: Plambda can be turned on only when cI protein is not present.]]<br />
|}<br />
<br />
According to what above stated, genetic implementation of Mux and Demux can be obtained connecting these basic logic gates and can be summarized in this way:<br />
<br><br />
<br><br />
<br><br />
<br />
=== Genetic Mux ===<br />
Let PA, PB and PS be three generic promoters that can be ACTIVATED respectively by the three exogenous molecules "A", "B" and "S". A genetic Mux with inputs "A" (CH0) and "B" (CH1), selector "S" and the generic protein GOI as output, can be implemented by the following gene network:<br />
{|align="center"<br />
|[[Image:pv_MUX.png|thumb|420px|left|Genetic Mux]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_MUXint.png|thumb|420px|left|Genetic Mux - interactions]]<br />
|}<br />
We want to supply a device that can be generalized to detect every kind of input and to express every kind of genes as output. So, input promoters and output protein generators are not part of the genetic Mux we want to build up. In this way, a hypothetical user can re-use our device assembling the desired input and output elements.<br />
<br><br />
Note that the output genes are two, but, as explained in "OR" section, if the genes are identical, we can say that there is only one output; in fact, the real output is a protein synthesis and so it is not important which of the two identical genes is expressed.<br />
<br><br />
However, it is possible to assemble two different genes downstream of Plux and Plas, for example two reporters. In this way, debugging process becomes easy, because we can discriminate lux system and las system activities.<br />
<br><br />
Here we report the truth table of this network:<br />
{|align="center"<br />
|[[Image:pv_gmux_truth.png|thumb|420px|left|Genetic Mux - truth table]]<br />
|}<br />
<br><br />
Now we report two examples of genetic Mux behavior, in response to generic inputs.<br />
<br><br />
<br><br />
====Genetic Mux behavior - Example 1====<br />
{|<br />
|[[Image:pv_genmux_example1.png|thumb|420px|left|First example of genetic Mux behavior]]<br />
|}<br />
According to Mux truth table, if "A" is not present and "B" and "S" are present, we expect to have GOI synthesis.<br />
<br><br />
"A" molecule is not present, so PA promoter is not active and luxI gene is not expressed.<br />
<br><br />
"B" molecule is present, so PB promoter is active and lasI gene is expressed.<br />
<br><br />
"S" molecule is present, so PS promoter is active and lasR and cI genes are expressed.<br />
<br><br />
cI protein represses transcription of the gene downstream of Plambda promoter, which is luxR.<br />
<br><br />
lasI protein can synthesize 3-OC12-HSL. This molecule activates transcription factor lasR, which can activate Plas promoter. So, the copy of GOI gene under Plas regulation can be expressed.<br />
<br><br />
The other copy of GOI gene, which is under Plux promoter regulation, is not expressed. In fact Plux is not active, because both luxI and luxR proteins are not present.<br />
<br><br />
Only one of the two GOI genes is expressed, but this is sufficient to synthesize the output protein GOI.<br />
<br><br />
<br><br />
====Genetic Mux behavior - Example 2====<br />
{|<br />
|[[Image:pv_genmux_example2.png|thumb|420px|left|Second example of genetic Mux behavior]]<br />
|}<br />
We expect to have no GOI synthesis in response to this input combination.<br />
<br><br />
"A" molecule is not present, so PA promoter is not active and luxI gene is not expressed.<br />
<br><br />
"B" molecule is present, so PB promoter is active and lasI gene is expressed.<br />
<br><br />
"S" molecule is not present, so PS promoter can't transcribe cI and lasR genes.<br />
<br><br />
cI protein is not present, so Plambda promoter can transcribe luxR gene.<br />
<br><br />
None of the two logic AND systems is active, because lux system lacks of luxI and las system lacks of lasR. So, Plux and Plas promoters are unactive and can't express GOI genes. For this reason, GOI protein is not synthesized.<br />
<br><br />
<br><br />
These two examples show how "S" molecule can select the input to be conveyed into the single output channel: in fact its presence allows lasR expression and represses luxR expression, while "S" absence represses lasR expression and allows luxR expression.<br />
<br><br />
<br><br />
<br />
=== Genetic Demux ===<br />
Let PI and PS be two generic promoters that can be ACTIVATED respectively by the two exogenous molecules "I" and "S". A genetic Demux with input "I", selector "S" and the generic proteins GOI0 and GOI1 as outputs (OUT0 and OUT1 respectively), can be implemented by the following gene network:<br />
{|align="center"<br />
|[[Image:pv_DEMUX.png|thumb|420px|left|Genetic Demux]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_DEMUXint.png|thumb|420px|left|Genetic Demux - interactions]]<br />
|}<br />
As described for genetic Mux, we want to supply a device that can be generalized to detect every kind of inputs and to express every kind of genes as output. So, input promoters and output protein generators are not part of the genetic Demux. In this way, a hypothetical user can re-use our device assembling the desired input and output elements.<br />
<br><br />
Here we report the truth table of this network:<br />
{|align="center"<br />
|[[Image:pv_gdemux_truth.png|thumb|420px|left|Genetic Demux - truth table]]<br />
|}<br />
<br><br />
Now we report two examples of genetic Demux behavior, in response to generic inputs.<br />
<br><br />
<br><br />
====Genetic Demux behavior - Example 1====<br />
{|<br />
|[[Image:pv_gendemux_example1.png|thumb|420px|left|First example of genetic Demux behavior]]<br />
|}<br />
According to Demux truth table, if "I" molecule is present and "S" molecule is absent, we expect to have GOI0 protein synthesis and no GOI1 protein synthesis.<br />
<br><br />
"I" is present, so PI promoter is active and luxI and lasI genes are expressed.<br />
<br><br />
"S" is not present, so PS promoter can't transcribe lasR and cI genes.<br />
<br><br />
cI protein is not present, so Plambda promoter is not inhibited and luxR gene is expressed.<br />
<br><br />
luxI protein can synthesize 3-OC6-HSL lactone. This molecule activates luxR transcription factor which can activate Plux promoter. In this way, GOI0 gene, which is under Plux regulation, is expressed.<br />
<br><br />
On the other hand, lasI protein can synthesize 3-OC12-HSL, but lasR transcription factor is not present and so Plas promoter cannot be activated. In this way, GOI1 gene, which is under Plas regulation, is not expressed.<br />
<br><br />
So, we have GOI0 protein synthesis and no GOI1 protein synthesis.<br />
<br><br />
<br><br />
====Genetic Demux behavior - Example 2====<br />
{|<br />
|[[Image:pv_gendemux_example2.png|thumb|420px|left|Second example of genetic Demux behavior]]<br />
|}<br />
We expect to have GOI1 synthesis and no GOI0 synthesis in response to this input combination.<br />
<br><br />
"I" is present, so PI promoter is active and luxI and lasI genes are expressed.<br />
<br><br />
"S" is present, so PS promoter lasR and cI genes are expressed.<br />
<br><br />
cI protein is present, so Plambda promoter is inhibited and luxR gene is not expressed.<br />
<br><br />
lasI protein can synthesize 3-OC12-HSL lactone. This molecule activates lasR transcription factor which can activate Plas promoter. In this way, GOI1 gene, which is under Plas regulation, is expressed.<br />
<br><br />
On the other hand, luxI protein can synthesize 3-OC6-HSL, but luxR transcription factor is not present and so Plux promoter cannot be activated. In this way, GOI0 gene, which is under Plux regulation, is not expressed.<br />
<br><br />
So, we have GOI1 protein synthesis and no GOI0 protein synthesis.<br />
<br />
<br />
=== A complete genetic Mux===<br />
In this section a complete Mux gene network is described.<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_implementation.png|thumb|420px|left|Example of a complete genetic Mux]]<br />
|[[Image:pv_mux_gn_implementation.png|thumb|420px|left|Example of a complete genetic Mux - Gene network]]<br />
|}<br />
We want to build up a device in which Channel 0, Channel 1 and Selector are respectively sensitive to Tetracycline, IPTG and red light. The presence of each input corresponds to logic 1.<br />
We chose green fluorescence as Mux output: expression of GFP corresponds to logic 1, while absence of fluorescence corresponds to logic 0.<br />
<br><br />
Tetracycline and IPTG can be considered as "A" and "B" molecules, introduced in "Genetic Mux" section, because both Tetracycline and IPTG are indirect ACTIVATORS of Ptet and Plac promoters respectively.<br />
On the other hand, red light is quite different from "S" molecule, because red light is an indirect REPRESSOR of Pomp promoter and not an activator as required by the original schema. For this reason, if we want a device in which the presence of Tetracycline, IPTG and red light correspond to logic 1, red light input should be ''inverted''. The simplest way to do this, is to cross-exchange the two input channels. So, Tetracycline sensor (CH0) has to be assembled to las system and IPTG sensor (CH1) has to be assembled to lux system.<br />
<br />
<br />
====Complete genetic Mux - Example====<br />
{|<br />
|[[Image:pv_mux_example1.png|thumb|420px|left|Example of a complete genetic Mux behavior]]<br />
|}<br />
According to Mux truth table, if SEL=0, CH0=1, CH1=0, output channel must be logic 1.<br />
Red light is not present, so it can't dephosphorylate cph8-ho1-pcyA complex, which is constitutively expressed. cph8-ho1-pcyA complex activates endogenous ompR, which can activate Pomp promoter, so cI and lasR genes are transcribed.<br />
cI protein binds Plambda promoter and so luxR expression is inhibited.<br />
TetR is a repressor for Ptet promoter, but Tetracycline is present, so it can bind tetR protein, which is constitutively expressed, and can activate transcription of downstream gene: lasI.<br />
Simultaneous expression of lasI and lasR can activate GFP, which is under Plas promoter.<br />
IPTG is not present, so lacI protein binds Plac promoter and represses luxI transcription.<br />
<br />
<br />
=== A complete genetic Demux===<br />
In this section a complete Demux gene network is described.<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_demux_implementation.png|thumb|420px|left|Example of a complete genetic Demux]]<br />
|[[Image:pv_demux_gn_implementation.png|thumb|420px|left|Example of a complete genetic Demux - Gene network]]<br />
|}<br />
<br />
We want to build up a device in which Input and Selector are respectively IPTG and Tetracycline.<br><br />
In Demux we have two output channels: red fluorescence corresponds to logic 1 at Channel 0, while green fluorescence corresponds to logic 1 at Channel 1.<br><br />
Absence of reporters expression corresponds to logic 0 at Channel 0 and Channel 1.<br><br />
There isn't any input combination that corresponds to logic 1 at Channel 0 and Channel 1 together.<br />
<br />
====Complete genetic Demux - Example====<br />
{|<br />
|[[Image:pv_demux_example1.png|thumb|420px|left|Example of a complete genetic Demux behavior]]<br />
|}<br />
According to Demux truth table, if IN=1 and SEL=1, output channel 0 is logic 0 and output channel 1 is logic 1.<br><br />
IPTG is present, so Plac promoter is active because IPTG binds lacI protein. This allows lasI and luxI transcription.<br><br />
Tetracycline is present, so Ptet promoter is active because Tetracycline binds tetR protein. This allows cI and lasR transcription.<br><br />
cI protein binds Plambda promoter and so luxR expression is inhibited.<br><br />
Simultaneous expression of lasI and lasR can activate GFP, which is under Plas promoter.<br><br />
There is no simultaneous expression of luxI and luxR, so RFP (which is under Plux regulation) cannot be expressed.<br />
<br />
== Final devices==<br />
BioBrick standard parts for genetic Mux and Demux are summarized in the following schemas:<br />
{|cellpadding="1px" align="center"<br />
|[[Image:pv_mux_final.png|thumb|420px|left|Three standard parts for mux]]<br />
|[[Image:pv_demux_final.png|thumb|450px|left|Two standard parts for demux]]<br />
|}<br />
<br />
A hypothetical user of Mux or Demux has to ligate our standard parts with desired inputs and outputs, as shown in the pictures above.<br />
<br><br />
The structure of our devices show that Mux and Demux systems both conform to the PoPS device boundary standard.<br />
<br><br />
<br />
==Applications==<br />
Mux and Demux are two fundamental devices in electronics. They are used in several applications, for example in communication devices, in Arithmetic Logic Units (ALUs), or in applications that involve channel sharing.<br />
<br><br />
Analogously, they should play a crucial role in building complex genetic circuits. In fact, both of them can be used as controlled genetic switches.<br />
<br />
== Experiments and results ==<br />
=== Assemblies ===<br />
We successfully amplified the following BioBrick standard parts from Spring 2008 DNA Distribution:<br />
{|align="center"<br />
|[[Image:pv_resusp.png|thumb|600px|left|Successful amplifications]]<br />
|}<br />
<br><br />
while we couldn't amplify correctly the following parts:<br />
{|align="center"<br />
|[[Image:pv_notresusp.png|thumb|600px|left|Unsuccessful amplifications]]<br />
|}<br />
<br><br />
To build up our designed devices, we followed and completed this assembly tree schema:<br />
{|align="center"<br />
|[[Image:pv_assemblyschema.png|thumb|600px|left|Assembly tree schema]]<br />
|}<br />
<br />
=== Functional tests ===<br />
We performed these boolean (on/off) fluorescence tests:<br />
<br><br />
<hr><br />
*'''TEST 1'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test1.png|thumb|250px|left|GFP protein generator under Plambda]]<br />
|}<br />
'''Description:''' we assembled an available GFP protein generator (E0240) under Plambda promoter, keeping pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' cI protein was not present, so Plambda should work like a constitutive promoter. A reporter gene downstream of this promoter allows us to validate Plambda costitutive activity. That can be very useful to validate our NOT logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of R0051 and E0240, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test1.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that GFP was expressed in positive transformants, so Plambda activity was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 2'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test2.png|thumb|250px|left|GFP protein generator under Ptet]]<br />
|}<br />
'''Description:''' we assembled E0240 under Ptet promoter, keeping pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' tetR protein was not present, so Ptet should work like a constitutive promoter. A reporter gene downstream of this promoter allows us to validate Ptet costitutive activity. Ptet is useful for specific input building.<br />
<br><br />
'''Methods:''' after ligation reaction of R0040 and E0240, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test2.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that GFP was expressed in positive transformants, so Ptet activity was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 3'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test3.png|thumb|250px|left|RFP protein generator under constitutive promoter]]<br />
|}<br />
'''Description:''' we didn't perform any assembly for this experiment, because the promoter we wanted to test was contained into BBa_J61002 vector, which places a RFP protein generator between SpeI and PstI restriction sites.<br />
<br><br />
'''Motivation:''' J23100, which we call Pcon, should have a strong constitutive activity. A reporter gene downstream of this promoter allows us to validate this activity. Pcon is useful to build our inputs because some sensors like IPTG and Tetracycline sensors need the constitutive production of specific proteins, in this case lacI and tetR respectively.<br />
<br><br />
'''Methods''' we transformed J23100 using Invitrogen TOP10 and plated transformed bacteria. We expected to observe red fluorescence (using transluminator) in all the colonies. We also picked up a colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through TRITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' all the grown colonies glowed under UV rays and red fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test3.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that RFP was expressed in transformed bacteria, so Pcon functionality was qualitatively validated.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 4'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test4.png|thumb|250px|left|Our GFP protein generator (K081012) under a constitutive promoter]]<br />
|}<br />
'''Description:''' we assembled a constitutive promoter family member (J23100) upstream of K081012, a GFP protein generator we built. We decided to excide J23100 from its plasmid (E-S) and to open K081012 plasmid (pSB1AK3) (E-X).<br />
<br><br />
'''Motivation:''' we already validated qualitatively J23100 constitutive activity, so this promoter can be used to test the GFP protein generator we built. We wanted to check if K081012 actually generates GFP.<br />
<br><br />
'''Methods''' after ligation reaction of J23100 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays (see figure) and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test4bis.jpg|thumb|400px|left|TOP10 colonies under UV]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test4.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that our GFP protein generator actually generates GFP.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 5'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test5.png|thumb|250px|left|Our RFP protein generator (K081014) under a constitutive promoter]]<br />
|}<br />
'''Description:''' we assembled a constitutive promoter family member (J23100) upstream of K081014, a RFP protein generator we built. We decided to excide J23100 from its plasmid (E-S) and to open K081014 plasmid (pSB1AK3) (E-X).<br />
<br><br />
'''Motivation:''' we already validated qualitatively J23100 constitutive activity, so this promoter can be used to test the RFP protein generator we built. We wanted to check if K081014 actually generates RFP.<br />
<br><br />
'''Methods''' after ligation reaction of J23100 and K081014, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through TRITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays (see figure) and red fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test5bis.jpg|thumb|400px|left|TOP10 colonies under UV]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test5.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that our RFP protein generator actually generates RFP.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 6'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test6.png|thumb|350px|left|GFP protein generator under Plux]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081000. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we didn't assemble any input to K081000 device, so we could study only the promoter basic activity. We expected to find a weak basic activity. Plux is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of K081000 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we performed a screening on 5 colonies through colony PCR to search for correctly ligated plasmid. We infected 9 ml of LB + Amp with the chosen colony and incubated the culture at 37°C, 220 rpm for 15 hours. Then we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test6.png|thumb|400px|left|No fluorescent TOP10 cells can be seen at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' the picture above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria. Increasing exposition time, green fluorescence could be observed (picture not reported). So, we can say that GFP was expressed very weakly. This experiment confirmed that Plux promoter has a weak activity without luxI and luxR proteins.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 7'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test7.png|thumb|350px|left|GFP protein generator under Plas]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plas promoter that was contained into K081001. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we didn't assemble any input to K081001 device, so we could study only the promoter basic activity. We expected to find a weak basic activity. Plas is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' after ligation reaction of K081001 and K081012, we transformed ligation product using Invitrogen TOP10 cells and plated transformed bacteria. Then we performed a screening on 5 colonies through colony PCR to search for correctly ligated plasmid. We infected 9 ml of LB + Amp with the chosen colony and incubated the culture at 37°C, 220 rpm for 15 hours. Then we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test7.png|thumb|400px|left|No fluorescent TOP10 cells can be seen at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' the picture above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria. Increasing exposition time, green fluorescence could be observed (picture not reported). So, we can say that GFP was expressed very weakly. This experiment confirmed that Plas promoter has a weak activity without lasI and lasR proteins.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 8'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test8.png|thumb|600px|left|GFP protein generator under Plux and constitutive expression of luxI and luxR]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081000. We also assembled K081011 upstream of K081000. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' Plux promoter activity was studied in response of a constitutive expression of luxI and luxR. Plambda promoter without cI repressor guaranteed the constitutive expression of these genes. We expected to find a strong activity because Plux is turned on. lux system is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' we ligated K081012 under K081000, transformed ligation, plated transformed bacteria, performed PCR screening on some colonies and extracted correctly ligated plasmids. We repeated these steps to assemble K081011 upstream of K081000-K081012, but we didn't perform PCR screening. Then we watched the plate on the transluminator to check if positive transformants glowed under UV rays. We also picked up a fluorescent colony with a tip and infected 1 ml of LB + Amp. After 3 hours at 37°C, 220 rpm we watched 50 ul of the culture at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' positive transformants glowed under UV rays and green fluorescent cells could be observed at microscope (see figure).<br />
{|align="center"<br />
|[[Image:pv_fig_test8.png|thumb|400px|left|TOP10 cells at microscope]]<br />
|}<br />
<br><br />
'''Comments:''' this experiment confirmed that Plux promoter can be activated by the contemporary presence of luxI and luxR. This is a crucial result for our AND logic gate.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 9'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test9.png|thumb|500px|left|GFP protein generator under Plux and constitutive expression of luxR]]<br />
|}<br />
'''Description:''' we assembled K081012 (our GFP) under the Plux promoter that was contained into K081022. We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' Plux promoter activity was studied in response to a constitutive expression of luxR. Plambda promoter without cI repressor guaranteed the constitutive expression of this gene. We expected to find a weak activity because luxR transcription factor is not active and so Plux cannot be turned on. We also expected to find a strong activity if we induce luxR activation using 3OC6-HSL (this is equivalent to TEST 8 conditions, because 3OC6-HSL is synthesized by luxI). lux system is useful for our AND logic gate.<br />
<br><br />
'''Methods:''' we ligated K081012 downstream of K081022, transformed ligation, plated transformed bacteria and screened three colonies to insulate a colony containing correctly ligated plasmids. We inoculated the positive colony into 9 ml of LB + Amp and incubated the culture at 37°C, 220 rpm for 15 hours. Then we diluted 1:10 the culture in two falcon tubes (5 ml cultures). One of these cultures was induced with 3OC6-HSL 1 uM. We let the two cultures grow for 2 hours and then we watched 50 ul at microscope through FITC channel and DAPI channel for negative control.<br />
<br><br />
'''Results:''' green fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture.<br />
{|align="center"<br />
|[[Image:pv_fig_test9.png|thumb|400px|left|Non induced culture: TOP10 cells cannot be seen at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test9bis.png|thumb|400px|left|Induced culture: fluorescent TOP10 cells at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test9superexp.png|thumb|400px|left|Same frames as above, but superexposed: non induced (left) and induced (right) cultures]]<br />
|}<br />
<br><br />
'''Comments:''' the pictures above was taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria for the non induced culture, while we can see green fluorescent TOP10 in the induced culture. As we wrote for TEST 6 and TEST 7, increasing exposition time green fluorescence could be observed even for the non induced culture (last picture), confirming the weak activity of Plux promoter in response of unactive luxR. This experiment confirmed that Plux promoter has a weak activity without luxI (or 3OC6-HSL) and luxR protein is not sufficient to induce a strong transcription. Adding 3OC6-HSL, luxR becomes active and so Plux is turned on.<br />
<br><br />
NOTE: K081022 has a point mutation in position 349 of C0062 coding sequence. This mutation changes the aminoacid, but luxR seems to work as expected.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 10'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test10.png|thumb|600px|left|RFP protein generator under Plux and constitutive expression of luxR]]<br />
|}<br />
'''Description:''' this test is equivalent to TEST 9: we assembled K081014 (our RFP) under the Plux promoter that was contained into K081004. We kept pSB1AK3 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we wanted to repeat TEST 9 experiment using a longer construct and a different reporter gene.<br />
<br><br />
'''Methods:''' the same as TEST 9, but using K081004 instead of K081022 and using RFP (K081014) instead of GFP (K081012).<br />
<br><br />
'''Results:''' red fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture.<br />
{|align="center"<br />
|[[Image:pv_fig_test10.png|thumb|400px|left|Non induced culture: TOP10 cells cannot be seen at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test10bis.png|thumb|400px|left|Induced culture: fluorescent TOP10 cells at microscope]]<br />
|}<br />
{|align="center"<br />
|[[Image:pv_fig_test10superexp.png|thumb|400px|left|Same frames as above, but superexposed: non induced (left) and induced (right) cultures]]<br />
|}<br />
<br><br />
'''Comments:''' the same as TEST 9.<br />
<br><br />
NOTE: C0062 has the same mutation described in TEST 9. We also performed this test with an old version of K081004 carrying another point mutation (C->T at position 704 of C0062 coding sequence) that changed an aminoacid. Even in this case K081004 seemed to work as expected (results not shown).<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 11'''<br />
<br><br />
{|align="center"<br />
|[[Image:pv_test11.png|thumb|600px|left|Terminator efficiency test]]<br />
|}<br />
'''Description:''' we assembled K081014 (our RFP) under the artificial 39 bp terminator (B1006) that is at the end of K081022-K081012 (composite part used for TEST 9). We kept pSB1A2 as the scaffold vector.<br />
<br><br />
'''Motivation:''' we wanted to test qualitatively if our terminator actually stops transcription.<br />
<br><br />
'''Methods:''' we assembled K081014 downstream of K081022, transformed ligation, plated transformed bacteria and insulated a colony containing correctly ligated plasmid. We inoculated the colony into 9 ml of LB + Amp and incubated the culture at 37°C, 220 rpm for 15 hours. Then we diluted 1:10 the culture in two falcon tubes (5 ml cultures). One of these cultures was induced with 3OC6-HSL 1 uM. We let the two cultures grow for 2 hours and then we watched 50 ul at microscope through FITC channel (positive control), TRITC channel and DAPI channel (negative control).<br />
<br><br />
'''Results:''' soon<br />
<br><br />
'''Comments:''' soon<br />
<br><br />
<br><br />
<hr><br />
<br><br />
We also performed these quantitative fluorescence tests:<br />
<hr><br />
*'''TEST 1'''<br />
<br><br />
'''Description:''' this test is an extension of TEST 9. We induced six cultures with 3OC6-HSL at different concentrations to study quantitatively HSL-GFP static transfer function.<br />
<br><br />
'''Motivation:''' we want to know cutoff point of this device.<br />
<br><br />
'''Methods:''' the same as TEST 9, but diluted the overnight 9 ml culture 1:10 in six falcon tubes (5 ml cultures). We induced the six cultures with 3OC6-HSL 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM and 1 uM respectively. We incubated the cultures at 37°C, 220 rpm for 2 hours and then we watched 50 ul at microscope through FITC channel and DAPI channel for negative control. We prepared two 50 ul glasses for each culture. We acquired three frames at 2.5 s (maximum exposition time) and 10 ms exposition time for every sample. We used ImageJ software to count automatically the number of cells for every frame. Then we computed n10/n2.5 (where n is the number of cells) to calculate the percentage of cells that glowed in the 10 ms acquisition, assuming that in the 2.5 s acquisition we can see the total number of cells. For each HSL concentration, we calculated the mean value of the 6 statistics (3 frames for each of the 2 glasses).<br />
<br><br />
'''Results:'''<br />
{|align="center"<br />
|[[Image:pv_fig_test1q.png|thumb|600px|left|GFP (arbitrary units) vs 3OC6-HSL concentration: 1=0nM, 2=0.1nM, 3=1nM, 4=10nM, 5=100nM, 6=1uM]]<br />
|}<br />
<br><br />
'''Comments:''' we computed the statistic n10/n2.5 because we know that when fluorescence is weak, cells cannot be seen at low exposition times. So, we calculate the ratio between the cells we can see at a low exposition time and the total number of cells in the frame. We chose 10 ms because the previous experiments with GFP had been performed using this parameter. We know that this is not an exact statistic, in fact we have to consider the count errors of ImageJ software, especially when cells are superimposed. Further quantitative experiments will have to be performed using standard measurement, in order to characterize parts.<br />
<br><br />
<hr><br />
<hr><br />
*'''TEST 2'''<br />
<br><br />
'''Description:''' this test is an extension of TEST 10. We induced seven cultures with 3OC6-HSL at different concentrations to study quantitatively HSL-RFP static transfer function.<br />
<br><br />
'''Motivation:''' we want to know cutoff point of this device.<br />
<br><br />
'''Methods:''' the same as TEST 10, but diluted the overnight 9 ml culture 1:10 in seven falcon tubes (5 ml cultures). We induced the seven cultures with 3OC6-HSL 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 uM and 10 uM respectively. We incubated the cultures at 37°C, 220 rpm for 2 hours and then we watched 50 ul at microscope through TRITC channel and DAPI channel for negative control. We prepared two 50 ul glasses for each culture. We acquired three frames at 2.5 s (maximum exposition time) and 90 ms exposition time for every sample. We used ImageJ software to count automatically the number of cells for every acquisition. Then we computed n90/n2.5 (where n is the number of cells) to calculate the percentage of cells that glowed in the 90 ms acquisition, assuming that in the 2.5 s acquisition we can see the total number of cells. For each HSL concentration, we calculated the mean value of the 6 statistics (3 frames for each of the 2 glasses).<br />
<br><br />
'''Results:'''<br />
{|align="center"<br />
|[[Image:pv_fig_test2q.png|thumb|600px|left|RFP (arbitrary units) vs 3OC6-HSL concentration: 1=0nM, 2=0.1nM, 3=1nM, 4=10nM, 5=100nM, 6=1uM, 7=10uM]]<br />
|}<br />
<br><br />
'''Comments:''' we computed the statistic n90/n2.5 because we know that when fluorescence is weak, cells cannot be seen at low exposition times. So, we calculate the ratio between the cells we can see at a low exposition time and the total number of cells in the frame. We chose 90 ms because the previous experiments with RFP had been performed using this parameter. We know that this is not an exact statistic, in fact we have to consider the count errors of ImageJ software, especially when cells are superimposed. As we wrote for TEST 9, further quantitative experiments will have to be performed using standard measurement, in order to characterize parts.<br />
<br><br />
<hr><br />
<br />
=== Conclusions ===<br />
All the experiments we performed were consistent with our expectations.<br />
<br><br />
Considering the elementary logic gates of our final devices (AND, OR, NOT), we validated some truth table rows:<br />
{|align="center"<br />
|[[Image:pv_summary.png|thumb|600px|left|Biological truth tables]]<br />
|}<br />
<br />
In particular:<br />
*'''TEST 1 validated the first row of NOT logic gate.'''<br />
*'''TEST 6 validated the first row of AND (lux) logic gate.'''<br />
*'''TEST 7 validated the first row of AND (las) logic gate.'''<br />
*'''TEST 8, TEST 9 (with induction) and TEST 10 (with induction) validated the fourth row of AND (lux) logic gate.'''<br />
*'''TEST 9 (without induction) and TEST 10 (without induction) validated the third row of AND (lux) logic gate.'''</div>Magnihttp://2008.igem.org/File:Pv_miniprep27.jpgFile:Pv miniprep27.jpg2008-10-26T21:55:24Z<p>Magni: uploaded a new version of "Image:Pv miniprep27.jpg": 27 minipreps</p>
<hr />
<div>27 miniprep</div>Magnihttp://2008.igem.org/File:Pv_miniprep27.jpgFile:Pv miniprep27.jpg2008-10-26T21:54:57Z<p>Magni: 27 miniprep</p>
<hr />
<div>27 miniprep</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week23Team:UNIPV-Pavia/Notebook/Week232008-10-26T21:54:33Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 19: 10/20/08 - 10/24/08==<br />
<br />
Preparation of miniprepped DNA for the 27 parts we want to submit to the Registry:<br />
*9 ml overnight cultures from glycerol stocks.<br />
*Miniprep.<br />
*8-tubes strip preparation.<br />
*DNA shipment with DHL.<br />
*Online documentation: Registry & online submission form.<br />
<br />
{|<br />
|[[Image:pv_miniprep27.jpg|thumb|370px|left|27 bacterial pellets to begin miniprep!]]<br />
|}</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week24Team:UNIPV-Pavia/Notebook/Week242008-10-26T21:53:11Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 19: 10/27/08 - 10/29/08==<br />
<br />
'''10/29/08'''<br />
<br><br />
Deadline for wiki!</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week24Team:UNIPV-Pavia/Notebook/Week242008-10-26T21:52:58Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 19: 10/27/08 - 10/29/08==<br />
<br />
'''10/29/08'''<br />
<br><br />
Deadline for Wiki!</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week24Team:UNIPV-Pavia/Notebook/Week242008-10-26T21:52:13Z<p>Magni: New page: <!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --> {| style="color:#000000;background-color:#...</p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 19: 10/27/08 - 10/29/08==</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week23Team:UNIPV-Pavia/Notebook/Week232008-10-26T21:51:17Z<p>Magni: New page: <!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --> {| style="color:#000000;background-color:#...</p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 19: 10/20/08 - 10/24/08==<br />
<br />
Preparation of miniprepped DNA for the 27 parts we want to submit to the Registry:<br />
*9 ml overnight cultures from glycerol stocks.<br />
*Miniprep.<br />
*8-tubes strip preparation.<br />
*DNA shipment with DHL.<br />
*Online documentation: Registry & online submission form.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week22Team:UNIPV-Pavia/Notebook/Week222008-10-26T21:42:53Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 19: 10/13/08 - 10/16/08==<br />
<br />
'''10/13/08'''<br />
<br><br />
*Lorenzo graduated in Biomedical Engineering with the first Synthetic Biology master thesis at the University of Pavia.<br />
<br />
'''10/14/08'''<br />
<br><br />
*Lorenzo is still at work...:) Wiki updating.<br />
<br />
'''10/15/08'''<br />
<br><br />
*Meeting for activity planning.<br />
<br />
'''10/16/08'''<br />
<br><br />
*Wiki updating.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week22Team:UNIPV-Pavia/Notebook/Week222008-10-26T21:30:21Z<p>Magni: New page: <!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --> {| style="color:#000000;background-color:#...</p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 19: 10/13/08 - 10/17/08==<br />
<br />
*10/1</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week21Team:UNIPV-Pavia/Notebook/Week212008-10-26T21:28:57Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 19: 10/6/08 - 10/10/08==<br />
<br />
Mathematical modeling using Matlab and Simulink. Our aim was to build a scaffold for future quantitative standard characterization.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week20Team:UNIPV-Pavia/Notebook/Week202008-10-26T21:28:44Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 20: 09/29/08 - 10/3/08==<br />
<br />
'''09/29/08'''<br />
<br><br />
*Ligations:<br />
**Lig.34-Lig.15 (=TR)(our RFP protein generator downstream, to check Lig.34 functionality)<br />
**T5-Lig.15 (=TT)(our RFP protein generator after our GFP protein generator, to check terminator efficiency)<br />
*We incubated ligations at 16°C overnight.<br />
<br />
'''09/30/08'''<br />
<br><br />
*We transformed the two overnight ligations and plated transformed bacteria. We incubated the two plates at 37°C overnight.<br />
<br />
'''10/1/08'''<br />
<br><br />
*We received sequencing results for Lig.31 and Lig.36. Sequences were OK!!! Notice that both of the two parts are long. So, sequencing could only confirm partially the nucleotides composing these two parts.<br />
<br />
*Plates were ok!<br />
<br />
*We performed colony PCR on the two plates. Medium and large gel could not be ran because instrumentation was buisy. We could ran two small gels (8 wells), so the maximum number of colonies was 13 (considering two markers and one blank). Whe chose to screen 6 colonies for TT and 7 colonies for TR.<br />
<br />
*Gel results:<br />
**TT - 2nd colony<br />
**TR - 1st colony (gel picture not available...sorry!)<br />
<br />
{|<br />
|[[Image:pv_colonypcr_TT.jpg|thumb|370px|left|Marker 1Kb, blank, R0051-B0030-C0062-B1006-R0062-B0030-E0040-B1006-B0030-E1010-B1006 (6 colonies)]]<br />
|}<br />
<br />
*We incubated the chosen colonies at 37°C, 220 rpm overnight.<br />
<br />
'''10/2/08'''<br />
<br><br />
*Glycerol stocks for TT-2 and TR-1.<br />
<br />
*TR fluorescence test. Experiment details and results are reported in The Project section, Experiments.<br />
<br />
*Data processing.<br />
<br />
'''10/3/08'''<br />
<br><br />
*We prepared 0.5 l of LB + Amp for liquid cultures because our LB was looked turbid.<br />
<br />
*Wiki updating.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week19Team:UNIPV-Pavia/Notebook/Week192008-10-26T21:28:34Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 19: 09/22/08 - 09/26/08==<br />
<br />
'''09/22/08'''<br />
<br><br />
*Sequencing results:<br />
*Lig.30 - we had another mutation in luxR coding sequence...no comments...(position 349 of C0062, G->C, aminoacid changes...)<br />
*Lig.34-1 - OK<br />
*Lig.34-3 - OK<br />
*Lig.34-4 - OK<br />
*Lig.34dil-1 - OK<br />
*Lig.34dil-2 - OK<br />
*Lig.35-3 - OK<br />
*Lig.35-4 - OK<br />
*Lig.35dil-1 - OK<br />
*Lig.35dil-4 - OK<br />
<br />
*Comments: Lig.34 and Lig.35 sequences were correct, but we have to notice that they are long parts, so their sequencing could not be extended to all the nucleotides. In particular, the mutated nucleotide of Lig.30 was not included in Lig.34 and Lig.35 sequencing. For this reason, we expect to find in Lig.34 the same G->C mutation as Lig.30.<br />
<br />
*We decided to test qualitatively and quantitatively the mutated construct:<br />
**T5 (R0051-B0030-C0062-B1006-R0062-B0030-E0040-B1006, that carries the G->C mutation)<br />
*So, we infected 9 ml of LB + Amp with 30 ul of T5 glycerol stock. We incubated the culture at 37°C, 220 rpm overnight.<br />
<br />
'''09/23/08'''<br />
<br><br />
*We performed the experiment described in TEST 9 and quantitativeTEST 1 (The Project section, Experiments).<br />
<br />
*We infected 9 ml of LB + Amp with 30 ul of Lig.31 and Lig.36 glycerol stocks. We incubated the cultures at 37°C, 220 rpm overnight.<br />
<br />
'''09/24/08'''<br />
<br><br />
*Data processing for 09/23/08 experiment.<br />
<br />
*Glycerol stocks/miniprep for Lig.31 and Lig.36.<br />
<br />
*We sent purified plasmids to Primm for sequencing.<br />
<br />
'''09/24/08'''<br />
<br><br />
*We infected 9 ml of LB + Amp with 30 ul of:<br />
**Lig.34-1<br />
**T5 (R0051-B0030-C0062-B1006-R0062-B0030-E0040-B1006)<br />
**Lig.15<br />
*glycerol stocks. We incubated the three cultures at 37°C, 220 rpm overnight.<br />
<br />
*Using these parts we are going to perform our last ligations...cross the fingers!:)<br />
<br />
'''09/25/08'''<br />
<br><br />
*Glycerol stocks/miniprep for Lig.34-1, T5 and Lig.15.<br />
<br />
*Plasmid digestions:<br />
**Lig.34-1 (S-P)<br />
**T5 (S-P)<br />
**Lig.15 (X-P)<br />
<br />
*Gel run/cut. Gel extraction for the three parts.<br />
<br />
*We put purified and digested parts at -20°C. Next week we will perform two ligations!</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week18Team:UNIPV-Pavia/Notebook/Week182008-10-26T21:28:22Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 18: 09/15/08 - 09/19/08==<br />
<br />
'''09/15-19/08'''<br />
<br><br />
*National School for Bioengineering (Brixen, Italy).</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week17Team:UNIPV-Pavia/Notebook/Week172008-10-26T21:28:10Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 17: 09/8/08 - 09/12/08==<br />
<br />
'''09/8/08'''<br />
<br><br />
*We prepared 0.5 l of LB + Amp for liquid cultures.<br />
<br />
*We infected 9 ml of LB + Amp with 30 µl of Lig.30(3), Lig.31, Lig.22, Lig.13 and two falcon tubes for Lig.a.<br />
<br />
*We received 3OC6HSL from Sigma! we resuspended it in ddH2O, we prepared some stocks (2 mM) and stored them at -20°C.<br />
<br />
'''09/9/08'''<br />
<br><br />
*Glycerol stocks/miniprep for Lig.30(3), Lig.31, Lig.22 and Lig.13.<br />
<br />
*Plasmid digestion for:<br />
**Lig.30 (S-P)<br />
**Lig.31 (S-P)<br />
**Lig.30 (X-P)<br />
**Lig.22 (X-P)<br />
**13 (X-P)<br />
<br />
*Run/gel extraction.<br />
<br />
*Ligations:<br />
**Lig.31-Lig.30 (="Lig.34")<br />
**Lig.31-Lig.22 (="Lig.35")<br />
**Lig.30-Lig.13 (="Lig.T5" for green fluorescence test)<br />
<br />
*We induced one of the two Lig.a overnight culture with 3OC6HSL 1 µM. We incubated the two cultures for 1 hour and then watched TRITC channel at microscope. (We didn't synchronize the two cultures, but performed a qualitative test for luxR mutated protein integrity evaluation).<br />
<br />
*Fluorescence test results: HSL induced Lig.a show RFP expression, while Lig.a without HSL showed a weak red fluorescence. Result pictures are not available at the moment.<br />
<br />
'''09/10/08'''<br />
<br><br />
*We transformed/plated ligations. We decided to perform two transformations for each ligation:<br />
**one normal transformation (1 µl of ligation);<br />
**one diluted ligation (1:10)<br />
*We decided to try diluted transformations to have less colonies in the plate and to avoid streaking single colonies plates when colonies are not insulated.<br />
<br />
'''09/11/08'''<br />
<br><br />
*Plates grew correctly and diluted transformation showed less colonies.<br />
<br />
*Colony PCR for Lig.34, Lig.35 and Lig.T5 (4 colonies for normal transformation plates and 4 colonies for diluted transformation plates).<br />
<br />
*Gel results (gel picture not available, we're sorry...!):<br />
**Lig.34 (1st,3rd,4th colonies for normal transformation plate; 1st,2nd colonies for diluted transformation plate)<br />
**Lig.35 (3rd,4th colonies for normal transformation plate; 1st,4th colonies for diluted transformation plate) (this time there were not unexpected contaminants!)<br />
**Lig.T5 (1st colony)<br />
<br />
*Comments: we chose MANY colonies because Lig.34 and Lig.35 are our final assemblies and we want to be sure that the parts were correct. We decided to extract plasmids from all these colonies and to sequence all of them.<br />
<br />
*We incubated the chosen colonies at 37°C, 220 rpm overnight.<br />
<br />
'''09/12/08'''<br />
<br><br />
*Glycerol stocks/miniprep for our ten overnight cultures.<br />
<br />
*We sent:<br />
**Lig.34-1<br />
**Lig.34-3<br />
**Lig.34-4<br />
**Lig.34dil-1<br />
**Lig.34dil-2<br />
**Lig.35-3<br />
**Lig.35-4<br />
**Lig.35dil-1<br />
**Lig.35dil-4<br />
*to Primm for sequencing.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week16Team:UNIPV-Pavia/Notebook/Week162008-10-26T21:27:59Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 16: 09/1/08 - 09/5/08==<br />
<br />
'''09/1/08'''<br />
<br><br />
*We had to perform these ligations:<br />
**Lig.22-R0062<br />
**Lig.31-Lig.22<br />
*but we didn't have enough Lig.22 and Lig.31 plasmids...<br />
<br />
*We infected 9 ml of LB + Amp with 30 µl of Lig.22 and Lig.31 glycerol stocks.<br />
<br />
'''09/2/08'''<br />
<br><br />
*Glycerol stocks/miniprep for Lig.22 and Lig.31.<br />
<br />
*Plasmid digestion for:<br />
**Lig.22 (E-S)<br />
**Lig.22 (X-P)<br />
**Lig.31 (S-P)<br />
**R0062 (E-X)<br />
<br />
*Run/gel extraction.<br />
<br />
*Ligations:<br />
**Lig.31-Lig.22 (=Lig.35)<br />
**Lig.22-R0062 (=Lig.30)<br />
*We incubated ligations at 16°C overnight.<br />
<br />
'''09/3/08'''<br />
<br><br />
*We transformed/plated ligations.<br />
<br />
'''09/4/08'''<br />
<br><br />
*Colony PCR for the two ligation plates<br />
<br />
{|<br />
|[[Image:pv_colonypcr_30_35again.jpg|thumb|370px|left|Marker 1Kb, Lig.30 (5 colonies), Marker 1Kb, Lig.35 (5 colonies)]]<br />
|}<br />
<br />
*Gel results:<br />
**Lig.30 (3rd colony)<br />
**Lig.35 (gel showed an unexpected contaminant...we decided to re-perform this ligation)<br />
<br />
'''09/5/08'''<br />
<br><br />
*Glycerol stock/miniprep for Lig.30.<br />
<br />
*We sent Lig.30 (3rd col) to Primm for sequencing.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week15Team:UNIPV-Pavia/Notebook/Week152008-10-26T21:27:47Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 15: 08/25/08 - 08/29/08==<br />
<br />
'''08/25/08'''<br />
<br><br />
*Plasmid digestion for:<br />
**R0051 (S-P)<br />
**R0040 (S-P)<br />
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (E-X) (=Lig.b (E-X))<br />
**B1006 (E-X)<br />
**Lig.12 (E-S)<br />
<br />
*Run/gel extraction.<br />
<br />
*Ligations:<br />
**BBa_R0051 (S-P) - BBa_E0240 (X-P) (for promoter test)<br />
**BBa_R0040 (S-P) - BBa_E0240 (X-P) (for promoter test)<br />
**Lig.12 (E-S) - B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (E-X) (for AND logic gate test)<br />
**Lig.12 (E-S) - '''BBa_B1006 (E-X)''' (to re-perform mutated assemblies)<br />
*We incubated ligations at 16°C overnight.<br />
<br />
*We ordered 3OC6HSL (Sigma).<br />
<br />
'''08/26/08'''<br />
<br><br />
*We transformed/plated ligations.<br />
<br />
'''08/27/08'''<br />
<br><br />
*Plates grew correctly. We checked colony fluorescence of three plates under UV rays:<br />
**'''R0051'''-E0240 glowed<br />
**'''R0040'''-E0240 glowed<br />
**Lig.12-Lig.b (R0051-B0030-C0062-B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006) glowed<br />
<br />
*We picked up fluorescent colonies from these three plates to infect three 15 ml falcon tubes containing 1 ml of LB + Amp. We let the culture grow at 37°C, 220 rpm for 3 hours, then we prepared three 50 ul samples and watch them at microscope. Results are shown in The Project section (Experiments).<br />
<br />
*Colony PCR for 5 colonies of Lig.12-'''B1006''' (called Lig.22).<br />
<br />
{|<br />
|[[Image:pv_colonypcr_22again.jpg|thumb|370px|left|Lig.22 (5 colonies), Marker 1Kb]]<br />
|}<br />
<br />
*Gel results: OK! we chose 1st colony to grow a 9 ml overnight culture.<br />
<br />
'''08/28/08'''<br />
<br><br />
*Glycerol stocks/miniprep for Lig.22.<br />
<br />
*We sent purified plasmids to Primm for sequencing.<br />
<br />
*We infected 9 ml of LB + Amp with 30 µl of R0062 glycerol stock.<br />
<br />
'''08/29/08'''<br />
<br><br />
*Glycerol stock/miniprep for R0062.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week14Team:UNIPV-Pavia/Notebook/Week142008-10-26T21:27:35Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 11: 08/21/08 - 08/22/08==<br />
<br />
'''08/21/08'''<br />
<br><br />
*Sequencing results:<br />
**J23100-B0030-C0012-B1006-'''R0010''' - completely wrong sequence<br />
**C0062 - OK<br />
**Lig.12 - OK<br />
**Lig.22 - point mutation C->T<br />
**Lig.30 - point mutation C->T<br />
*We decided to restart from Lig.12, whose sequence was correct, to perform ligations Lig.22, Lig.30, Lig.34 and Lig.35.<br />
<br />
*We prepared 0.5 l of LB + Amp for plates.<br />
<br />
*We infected 9 ml LB + Amp with 30 µl of B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 and Lig.12 glycerol stocks.<br />
<br />
'''08/22/08'''<br />
<br><br />
*Glycerol stocks/miniprep for B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 and Lig.12.<br />
<br />
*We planned experiments for qualitative and quantitative tests.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week13Team:UNIPV-Pavia/Notebook/Week132008-10-26T21:27:24Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 13: 08/10/08 - 08/15/08==<br />
<br />
'''08/10-15/08'''<br />
<br><br />
Holiday:)</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week12Team:UNIPV-Pavia/Notebook/Week122008-10-26T21:27:13Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 11: 08/4/08 - 08/7/08==<br />
<br />
'''08/4/08'''<br />
<br><br />
*Plasmid digestion for:<br />
{|cellpadding="20px"<br />
|J23100-B0030-C0040-'''B1006''' (E-S)<br />
|R0040 (E-X)<br />
|}<br />
<br />
*Gel run/cut/gel extraction.<br />
<br />
*Ligation: J23100-B0030-C0040-B1006-'''R0040'''. We incubated ligation at 16°C overnight.<br />
<br />
*We had 5 plates to screen with colony PCR:<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006 (that we call "a")<br />
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (that we call "b")<br />
**B0030-C0078-B1006-'''R0079'''-B0030-E0040-B1006 (that we call "c")<br />
**B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006 (that we call "d")<br />
**J23100-B0030-C0012-B1006-'''R0010'''<br />
<br />
*Last week J23100-B0030-C0012-B1006-'''R0010''' colony PCR gave a bad result. For this reason, we decided to perform colony PCR only for:<br />
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (7 colonies)<br />
**J23100-B0030-C0012-B1006-'''R0010''' (6 colonies)<br />
<br />
{|<br />
|[[Image:pv_colonypcr_27_b.jpg|thumb|370px|left|Marker 1Kb, blank, J23100-B0030-C0012-B1006-'''R0010''' (6 colonies), B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (7 colonies)]]<br />
|}<br />
<br />
*Gel result:<br />
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (1st colony, but it was not pure. We decided to prepare single colonies plate for it)<br />
**J23100-B0030-C0012-B1006-'''R0010''' (2nd colony)<br />
<br />
'''08/4/08'''<br />
<br><br />
*We transformed J23100-B0030-C0040-B1006-'''R0040''' overnight ligation. We plated transformed bacteria and incubated plate at 37°C overnight.<br />
<br />
*We infected 9 ml of LB + Amp with 30 µl of C0062, Lig.12, Lig.22, Lig.30, Lig.27(2nd col), B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (1st col) (see "Parts" section for our nomenclature).<br />
<br />
*Single colonies plates for:<br />
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 ("Lig.b")<br />
**B0030-C0078-B1006-'''R0079'''-B0030-E0040-B1006 ("Lig.c")<br />
**B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006 ("Lig.d")<br />
<br />
'''08/5/08'''<br />
<br><br />
*Glycerol stocks/miniprep for C0062, Lig.12, Lig.22, Lig.30, Lig.27(2nd col), B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006(1).<br />
<br />
*We sent C0062, Lig.12, Lig.22 and Lig.30 purified plasmids to Primm for sequencing: all these parts contain BBa_C0062.<br />
<br />
*We transformed/plated J23100-B0030-C0040-B1006-'''R0040''' overnight ligation.<br />
<br />
*Colony PCR for a (7 colonies), Lig.b(single colonies)(6 colonies), Lig.c(single colonies)(6 colonies), Lig.d(single colonies)(6 colonies).<br />
<br />
{|<br />
|[[Image:pv_colonypcr_abcd.jpg|thumb|370px|left|Marker 1Kb, blank, Lig.a (7 colonies), Lig.b (6 colonies), Marker 1Kb, Lig.c (6 colonies), Lig.d (6 colonies)]]<br />
|}<br />
<br />
*Gel results were not so clear: the length of some fragments was not expected and there were some contaminants. Maybe those parts were too long for our PCR reaction. We decided to grow 9 ml cultures for some of those colonies, to extract plasmids, to cut them and to check their length in a new run. We chose:<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006 (1, 4, 6, 7)<br />
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (no colony was chosen: we already had them and this run didn't show any 100% pure colony)<br />
**B0030-C0078-B1006-'''R0079'''-B0030-E0040-B1006 (5)<br />
**B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006 (2)<br />
<br />
'''08/6/08'''<br />
<br><br />
*Single colonies plate for J23100-B0030-C0040-B1006-'''R0040''', because where were too many bacteria on its plate.<br />
<br />
*Glycerol stocks/miniprep for:<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(1)<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(4)<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(6)<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(7)<br />
**B0030-C0078-B1006-'''R0079'''-B0030-E0040-B1006(5)<br />
**B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006(2)<br />
<br />
*We cut these 6 plasmids and Lig.b in (E-P) (5 µl of DNA in a final reaction volume of 20 µl).<br />
<br />
*Run for digested plasmids.<br />
<br />
{|<br />
|[[Image:pv_colonypcr_aaaabcd.jpg|thumb|370px|left|Marker 1Kb, Lig.a (1), Lig.a (4), Lig.a (6), Lig.a (7), Lig.b (1), Lig.c (5), Lig.d (2)]]<br />
|}<br />
<br />
*Gel results:<br />
**(Lig.a) B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(1) OK<br />
**(Lig.a) B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(4) False positive<br />
**(Lig.a) B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(6) OK<br />
**(Lig.a) B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(7) False positive<br />
**(Lig.b) B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006(1) OK<br />
**(Lig.c) B0030-C0078-B1006-'''R0079'''-B0030-E0040-B1006(5) OK<br />
**(Lig.d) B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006(2) OK<br />
<br />
*We 9 ml of LB + Amp with 30 ul of Lig.b(1) and Lig.c(5) to perform tests.<br />
<br />
'''08/7/08'''<br />
<br><br />
*Qualitative fluorescence tests for Lig.b and Lig.c. Results are shown in The Project section (Experiments).<br />
<br />
*Colony PCR for J23100-B0030-C0040-B1006-'''R0040''' single colonies plate (screening on 6 colonies).<br />
<br />
*Gel results: all screened colonies were negative...<br />
{|<br />
|[[Image:pv_colonypcr_28_allnegatives.jpg|thumb|300px|left|Marker 1Kb, blank, J23100-B0030-C0040-B1006-'''R0040''' (6 colonies)]]<br />
|}</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week11Team:UNIPV-Pavia/Notebook/Week112008-10-26T21:27:01Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 11: 07/28/08 - 08/1/08==<br />
<br />
'''07/28/08'''<br />
<br><br />
*Colony PCR for B0030-C0061-B0030-C0078-B1006-'''R0079''' plate: 6 colonies.<br />
<br />
{|<br />
|[[Image:pv_colonypcr_36.jpg|thumb|300px|left|B0030-C0061-B0030-C0078-B1006-'''R0079''']]<br />
|}<br />
<br />
*Gel results: all colonies were true positives! we decided to keep 1st colony to grow a 9 ml overnight culture.<br />
<br />
*Plasmid digestion for:<br />
{|cellpadding="20px"<br />
|J23100-B0030-C0012-'''B1006''' (E-S)<br />
|R0010 (E-X)<br />
|}<br />
<br />
*Gel run/cut/gel extraction.<br />
<br />
*Ligation: J23100-B0030-C0012-B1006-'''R0010'''<br />
<br />
*We incubated ligation at 16°C overnight.<br />
<br />
*We infected 9 ml LB + Amp with 30 µl of these glycerol stocks:<br />
{|cellpadding="20px"<br />
|B0030-E0040-'''B1006'''<br />
|B0030-C0061-B1006-'''R0062'''<br />
|-<br />
|B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (1)<br />
|B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (1)<br />
|-<br />
|B0030-E1010-'''B1006'''<br />
|B0030-C0078-B1006-'''R0079'''<br />
|-<br />
|B0030-C0061-B0030-C0079-B1006-'''R0079'''<br />
|}<br />
<br />
<br><br><br />
'''07/29/08'''<br />
<br><br />
*LB + Amp preparation.<br />
<br />
*Glycerol stocks/miniprep for the 7 overnight cultures. Unfortunately we had low yield...<br />
<br />
*DNA precipitation with sodium acetate for the 7 purified plasmids to concetrate DNA in a final volume of 10 µl.<br />
<br />
*We transformed J23100-B0030-C0012-B1006-'''R0010''' ligation. We plated transformed bacteria and incubated plate at 37°C overnight.<br />
<br />
*We received sequencing results for:<br />
**J23100-B0030-C0040-B1006-'''R0040''': sequence was not correct...we ligated the wrong part in the last ligation step of this composite part. We decided to re-ligate J23100-B0030-C0040-'''B1006''' and R0040.<br />
**B0030-C0078-B1006-'''R0079''': apart from an extra dna fragment at the end of C0078 (already noticed in previous sequencing), sequence was correct!<br />
<br />
<br><br><br />
'''07/30/08'''<br />
<br><br />
*Plasmid digestion for:<br />
{|cellpadding="20px"<br />
|B0030-BBa_E0040-'''BBa_B1006''' (X-P)<br />
|B0030-BBa_C0061-BBa_B1006-'''BBa_R0062''' (S-P)<br />
|-<br />
|B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (1) (S-P)<br />
|B0030-C0061-B0030-C0079-B1006-'''R0079''' (1) (S-P)<br />
|-<br />
|B0030-E1010-'''B1006''' (X-P)<br />
|B0030-C0078-B1006-'''R0079''' (S-P)<br />
|}<br />
<br />
*Gel run/cut/gel extraction. DNA quantification with NanoDrop showed that all cut parts were enough for the four ligations!<br />
<br />
*Ligations:<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006<br />
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006<br />
**B0030-C0078-B1006-'''R0079'''-B0030-E0040-B1006<br />
**B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006<br />
<br />
*We incubated ligations at 16°C overnight. These ligations are test assemblies, in fact reporter protein generators are assembled downstream of promoters.<br />
<br />
*We infected 9 ml LB + Amp with 30 µl of J23100-B0030-C0040-'''B1006''' and R0040 glycerol stocks. We incubated cultures at 37°C, 220 rpm overnight.<br />
<br />
<br><br><br />
'''07/31/08'''<br />
<br><br />
*We transformed overnight ligations. We plated transformed bacteria and incubated plates at 37°C overnight.<br />
<br />
*Glycerol stocks/miniprep for J23100-B0030-C0040-'''B1006 and R0040'''. Next week we will be ready to re-perform the unlucky ligation between these two parts;)<br />
<br />
*Colony PCR for J23100-B0030-C0012-B1006-'''R0010''' (the plate was stored at +4°C): we picked up 12 colonies.<br />
<br />
*Gel results: we couldn't see any band from gel...maybe we made a mistake in PCR mix...Next week we will re-perform this colony PCR.<br />
<br />
*Sequencing results: we had a point mutation (C->T) in C0062 coding sequence, in B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 composite parts...This mutation determines a Ser->Phe change in DNA binding domain of luxR gene. We decided to test mutated part anyway, to understand if protein function is actually compromised. In the meanwhile, we decided to sequence all parts with C0062 coding sequence to find the step in which mutation occurred and the re-perform ligations.<br />
<br />
<br><br><br />
'''08/1/08'''<br />
<br><br />
*We put:<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006<br />
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006<br />
**B0030-C0078-B1006-'''R0079'''-B0030-E0040-B1006<br />
**B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006<br />
<br />
*ligation plates at +4°C. Next week we will perform colony PCR on these plates.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week10Team:UNIPV-Pavia/Notebook/Week102008-10-26T21:26:50Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
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{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 10: 07/21/08 - 07/25/08==<br />
<br />
'''07/21/08'''<br />
<br><br />
*Colony PCR for B0030-C0078-B1006-'''R0079''': 6 colonies from single colonies plate.<br />
<br />
{|<br />
|[[Image:pv_colonypcr_25_single_col.jpg|thumb|300px|left|B0030-C0078-B1006-'''R0079''']]<br />
|}<br />
<br />
*Gel results: all the 6 picked colonies were true positive! we chose the 5th colony to grow a 9 ml overnight culture.<br />
<br />
*We also infected 9 ml LB + Amp with 30 µl of J23100-B0030-C0040-B1006-'''R0040''' and '''B0030'''-C0061 glycerol stocks to grow two overnight cultures.<br />
<br />
*Ligation:<br />
**J23100-B0030-C0012-'''B1006''' (1:2 ratio, 40 ng of vector)<br />
**B0030-I15009-B1006-'''R0082''' (30 ng of vector)<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006<br />
<br />
*We incubated ligations at 16°C overnight.<br />
<br />
<br><br><br />
'''07/22/08'''<br />
<br><br />
*Transformation for the 4 overnight ligations (1 µl). We plated transformed bacteria and incubated plates at 37°C overnight.<br />
<br />
*Glycerol stocks/miniprep for:<br />
{|cellpadding="20px"<br />
|J23100-B0030-C0040-B1006-'''R0040'''<br />
|'''B0030'''-C0061<br />
|-<br />
|B0030-C0078-B1006-'''R0079'''<br />
|}<br />
<br />
*We sent J23100-B0030-C0040-B1006-'''R0040''' and B0030-C0078-B1006-'''R0079''' purified plasmids to Primm for sequencing.<br />
<br />
*Plasmid digestion for:<br />
{|cellpadding="20px"<br />
|'''B0030'''-C0061 (E-S)<br />
|B0030-C0078-B1006-'''R0079''' (E-X)<br />
|}<br />
<br />
*Gel run/cut/gel extraction.<br />
<br />
<br><br><br />
'''07/23/08'''<br />
<br><br />
*Ligation plates showed colonies!We put ligation plates at +4°C.<br />
<br />
*Ligation for B0030-C0061-B0030-C0078-B1006-'''R0079'''.<br />
<br />
*We incubated ligation at 16°C overnight.<br />
<br />
<br><br><br />
'''07/24/08'''<br />
<br><br />
*Transformation (1 µl) for B0030-C0061-B0030-C0078-B1006-'''R0079''' ligation. We plated transformed bacteria and incubated plate at 37°C overnight.<br />
<br />
*Colony PCR for:<br />
**J23100-B0030-C0012-'''B1006'''<br />
**B0030-I15009-B1006-'''R0082'''<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006<br />
<br />
*(We performed one PCR for J23100-B0030-C0012-'''B1006''' and B0030-I15009-B1006-'''R0082''' with 2 min elongation and another PCR for the remaining two ligations with 3 min elongation).<br />
<br />
{|cellpadding="1px"<br />
|[[Image:pv_pcr_parallel1.jpg|thumb|300px|left|Thermal cycler working on J23100-B0030-C0012-'''B1006''' and B0030-I15009-B1006-'''R0082''' colonies: 2 min elongation]]<br />
|[[Image:pv_pcr_parallel2.jpg|thumb|300px|left|Thermal cycler working on B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 colonies: 3 min elongation]]<br />
|}<br />
<br />
*Electrophoresis (1.2% agarose) for J23100-B0030-C0012-'''B1006''' and B0030-I15009-B1006-'''R0082''':<br />
<br />
{|<br />
|[[Image:pv_colonypcr_19_32.jpg|thumb|300px|left|Marker 1Kb, blank, J23100-B0030-C0012-'''B1006''' (7 lanes), B0030-I15009-B1006-'''R0082''' (6 lanes)]]<br />
|}<br />
<br />
*Electrophoresis (1% agarose) for B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006:<br />
<br />
{|<br />
|[[Image:pv_colonypcr_34_35.jpg|thumb|300px|left|Marker 1Kb, B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (7 lanes), B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (7 lanes)]]<br />
|}<br />
<br />
*Gel results:<br />
**J23100-B0030-C0012-'''B1006''' 1st colony<br />
**B0030-I15009-B1006-'''R0082''' 1st colony<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 1st and 3rd colony<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 1st and 4th colony<br />
<br />
*We decided to keep two colonies for B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 ligations because they are important parts for our project and we wanted to be sure they were correct.<br />
<br />
*The fifth colony of B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 showed an unexpected length...(@_@?) We decided to ignore it. Sequencing results will tell us if we made mistakes in assemblies.<br />
<br />
*We infected 9 ml LB + Amp with chosen colonies to grow 6 overnight cultures.<br />
<br />
<br><br><br />
'''07/25/08'''<br />
<br><br />
*Glycerol stocks/miniprep for:<br />
**J23100-B0030-C0012-'''B1006''' (1)<br />
**B0030-I15009-B1006-'''R0082''' (1)<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (1)<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (3)<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (1)<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (4)<br />
<br />
*We sent these purified plasmids:<br />
**B0030-I15009-B1006-'''R0082''' (1)<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (1)<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (3)<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (1)<br />
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (4)<br />
<br />
*to Primm for sequencing.<br />
<br />
*We put B0030-C0061-B0030-C0078-B1006-'''R0079''' plate at +4°C.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week9Team:UNIPV-Pavia/Notebook/Week92008-10-26T21:26:34Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 9: 07/14/08 - 07/17/08==<br />
<br />
'''07/14/08'''<br />
<br><br />
*We received sequencing results for BBa_B0030-BBa_E0040-BBa_B1006 and BBa_B0030-BBa_E1010-BBa_B1006: sequences were correct!<br />
<br />
*We transformed 1 µl of the 6 ligations stored at -20°C. We plated transformed bacteria and incubated plates at 37°C overnight.<br />
<br />
*Plasmid digestion for:<br />
{|cellpadding="20px"<br />
|BBa_R0079 (E-X)<br />
|BBa_B0030-BBa_C0078-'''BBa_B1006''' (E-S)<br />
|}<br />
<br />
*Gel run/cut/gel extraction for the 2 parts.<br />
<br />
*Ligation: BBa_B0030-BBa_C0078-BBa_B1006-'''BBa_R0079'''.<br />
<br />
*We incubated ligation at 16°C overnight.<br />
<br />
<br><br><br />
'''07/15/08'''<br />
<br><br />
*We transformed 1 µl of BBa_B0030-BBa_C0078-BBa_B1006-'''BBa_R0079''' ligation and plated transformed bacteria. We incubated the plate at 37°C overnight.<br />
<br />
*All the 6 overnight plates showed colonies. We performed colony PCR for (we decided to pick up more colonies for the ligations with long inserts):<br />
#BBa_J23100-'''BBa_B0030''' (S-P) -BBa_C0012 (standard ligation) - 10 colonies<br />
#'''pSB1AK3'''-BBa_J23100-BBa_B0030-BBa_C0012 (double ligation) - 7 colonies<br />
#BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-'''BBa_R0040''' - 3 colonies<br />
#BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-'''BBa_R0062''' - 3 colonies<br />
#BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006''' - 10 colonies<br />
#BBa_B0030-BBa_I15009-'''BBa_B1006''' - 3 colonies<br />
<br />
{|<br />
|[[Image:pv_colonypcr_15_7_08.jpg|thumb|300px|left|Picked up colonies in LB + Amp]]<br />
|}<br />
<br />
{|<br />
|[[Image:pv_pcr_9s_9d_26_28_30_31.png|thumb|700px|left|BBa_J23100-'''BBa_B0030'''-BBa_C0012 (standard ligation), '''pSB1AK3'''-BBa_J23100-BBa_B0030-BBa_C0012 (double ligation), BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-'''BBa_R0040''', BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-'''BBa_R0062''', BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006''', BBa_B0030-BBa_I15009-'''BBa_B1006''']]<br />
|}<br />
<br />
*Gel results:<br />
#BBa_J23100-'''BBa_B0030'''-BBa_C0012 (standard ligation) - 9th colony<br />
#'''pSB1AK3'''-BBa_J23100-BBa_B0030-BBa_C0012 (double ligation) - no true positives<br />
#BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-'''BBa_R0040''' - no true positives<br />
#BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-'''BBa_R0062''' - 3rd colony<br />
#BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006''' - 7th colony<br />
#BBa_B0030-BBa_I15009-'''BBa_B1006''' - 1st colony<br />
<br />
*Comments:<br />
**double ligation apparently did not work, but this time we had our true positive colonies for BBa_J23100-'''BBa_B0030'''-BBa_C0012 with a standard ligation.<br />
**BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-'''BBa_R0040''' did not show colonies with ligated plasmid...Maybe we picked up too too few colonies (in addition, we probably made a mistake with the 2nd colony which was not dipped in PCR tube...). We will repeat colony PCR on this ligation soon.<br />
<br />
*We infected 9 ml LB + Amp with these glycerol stocks:<br />
{|cellpadding="20px"<br />
|BBa_R0082<br />
|BBa_R0051-BBa_B0030-BBa_C0062-'''BBa_B1006'''<br />
|}<br />
<br />
*We grew the 4 chosen colonies and the 2 glycerol stocks colonies overnight.<br />
<br />
<br><br><br />
'''07/16/08'''<br />
<br><br />
*BBa_B0030-BBa_C0078-BBa_B1006-'''BBa_R0079''' plate showed a carpet...We decided to streak the plate to grow a "single colonies" plate overnight. We also decided to perform "colony" PCR (we did not use colonies, but little streaks) to check if there were ligated plasmids.<br />
<br />
*So, we performed colony PCR for BBa_B0030-BBa_C0078-BBa_B1006-'''BBa_R0079''' and we repeated colony PCR for BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-'''BBa_R0040'''.<br />
<br />
{|<br />
|[[Image:pv_pcr_25_28_.jpg|thumb|300px|left|]]<br />
|}<br />
<br />
*Gel results:<br />
**All the 8 BBa_B0030-BBa_C0078-BBa_B1006-'''BBa_R0079''' streaks contained ligated plasmid! but gel run showed an extra band that could correspond to not ligated plasmid...We decided to perform colony PCR on single colonies plate. We also decided to grow a 9 ml culture overnight with the 4th colony (in which the extra band is very weak) to have a backup of this result.<br />
**BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-'''BBa_R0040''' showed some pure true positive colonies; we chose the 2nd colony to grow a 9 ml culture overnight.<br />
<br />
*Glycerol stocks for:<br />
{|cellpadding="20px"<br />
|BBa_R0082<br />
|BBa_R0051-BBa_B0030-BBa_C0062-'''BBa_B1006'''<br />
|-<br />
|BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-'''BBa_R0062''' (3)<br />
|BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006''' (7)<br />
|-<br />
|BBa_B0030-BBa_I15009-'''BBa_B1006''' (1)<br />
|BBa_J23100-'''BBa_B0030'''-BBa_C0012 (9)<br />
|}<br />
<br />
*Miniprep for these 6 parts.<br />
<br />
*Plasmid digestion for:<br />
{|cellpadding="20px"<br />
|BBa_R0082 (E-X)<br />
|BBa_R0051-BBa_B0030-BBa_C0062-'''BBa_B1006''' (X-P)<br />
|-<br />
|BBa_J23100-'''BBa_B0030'''-BBa_C0012 (9) (E-S)<br />
|BBa_B0030-BBa_I15009-'''BBa_B1006''' (1) (E-S)<br />
|-<br />
|BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-'''BBa_R0062''' (3) (X-P)<br />
|BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006''' (7) (S-P)<br />
|}<br />
<br />
*Gel run/cut/gel extraction for these 6 parts.<br />
<br />
<br><br><br />
'''07/17/08'''<br />
<br><br />
*Glycerol stocks for BBa_B0030-BBa_C0078-BBa_B1006-'''BBa_R0079''' (4) and BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-'''BBa_R0040''' (2).<br />
<br />
*We put BBa_B0030-BBa_C0078-BBa_B1006-'''BBa_R0079''' single colonies plate at +4°C.<br />
<br />
*We received sequencing results for:<br />
**BBa_B0030-BBa_C0061-BBa_B1006-BBa_R0062 - the sequence was correct!<br />
**BBa_I15010: - sequencing failed (according to DNA Repository Quality Control): we cannot use this part.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week8Team:UNIPV-Pavia/Notebook/Week82008-10-26T21:26:21Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 8: 07/7/08 - 07/12/08==<br />
<br />
'''07/7/08'''<br />
<br><br />
*Colony PCR (5 colonies for each plate) for:<br />
**BBa_J23100-'''BBa_B0030'''-BBa_C0012 (for the second time, we hoped to find true positive colonies)<br />
**BBa_J23100-'''BBa_B0030'''-BBa_I15010 (for the second time, we hoped to find true positive colonies)<br />
**BBa_B0030-BBa_E0040-'''BBa_B1006'''<br />
**BBa_B0030-BBa_C0051-'''BBa_B0030'''<br />
**BBa_B0030-BBa_E1010-'''BBa_B1006'''<br />
<br />
{|<br />
|[[Image:pv_pcr_09_11_13_14_15.jpg|thumb|650px|left|Colony PCR: Marker (1), empty (2) BBa_J23100-'''BBa_B0030'''-BBa_C0012, BBa_J23100-'''BBa_B0030'''-BBa_I15010, '''BBa_B0030'''-BBa_E0040-BBa_B1006, '''BBa_B0030'''-BBa_C0051-BBa_B0030, '''BBa_B0030'''-BBa_E1010-BBa_B1006, blank]]<br />
|}<br />
<br />
*Gel results:<br />
**No true positives for BBa_J23100-'''BBa_B0030'''-BBa_C0012<br />
**No true positives for BBa_J23100-'''BBa_B0030'''-BBa_I15010<br />
**Non pure true positives for BBa_B0030-BBa_E0040-'''BBa_B1006'''<br />
**Pure true positives for BBa_B0030-BBa_C0051-'''BBa_B0030'''<br />
**Non pure true positives for BBa_B0030-BBa_E1010-'''BBa_B1006'''<br />
<br />
*We chose to keep the first colony for all the 3 working ligation plates.<br />
<br />
*NOTE: BBa_B0030-BBa_E0040-'''BBa_B1006''' and BBa_B0030-BBa_E1010-'''BBa_B1006''' are final parts; we decided to sequence these 2 parts even if gel showed a weak contamination: sequencing results will tell us if there are false positive plasmids in those colonies or if the contamination was only a PCR contamination.<br />
<br />
*We infected 9 ml LB + suitable antibiotic with 30 µl of these glycerol stocks:<br />
{|cellpadding="20px"<br />
|BBa_B0030<br />
|BBa_I15010<br />
|BBa_B0030-BBa_E0040-'''BBa_B1006''' (1)<br />
|BBa_B0030-BBa_E1010-'''BBa_B1006''' (1)<br />
|-<br />
|BBa_C0012<br />
|BBa_R0062<br />
|BBa_B0030-BBa_C0051-'''BBa_B0030'''<br />
|BBa_J23100-'''BBa_B0030'''<br />
|}<br />
<br />
*We incubated the 8 cultures at 37°C, 220 rpm overnight.<br />
<br />
*Tomorrow we will be ready to perform NINE LIGATIONS...(@_@!)<br />
<br />
<br><br><br />
'''07/8/08'''<br />
<br><br />
*We received sequencing results for '''BBa_B0030'''-BBa_C0078: sequence showed an extra DNA fragment between BBa_C0078 and Suffix...We decided to ignore it because there was a stop codon before that fragment.<br />
<br />
*Glycerol stocks for:<br />
{|cellpadding="20px"<br />
|BBa_B0030<br />
|BBa_I15010<br />
|BBa_B0030-BBa_E0040-'''BBa_B1006''' (1)<br />
|-<br />
|BBa_C0012<br />
|BBa_R0062<br />
|BBa_B0030-BBa_C0051-'''BBa_B0030'''<br />
|-<br />
|BBa_J23100-'''BBa_B0030'''<br />
|BBa_B0030-BBa_E1010-'''BBa_B1006''' (1)<br />
|}<br />
<br />
*Miniprep for these parts.<br />
<br />
*Plasmid digestion for:<br />
{|cellpadding="20px"<br />
|BBa_B0030 (S-P)<br />
|BBa_B0030-BBa_E0040-'''BBa_B1006''' (1) (E-X)<br />
|BBa_B0030-BBa_E1010-'''BBa_B1006''' (1) (E-X)<br />
|-<br />
|BBa_C0012 (X-P)<br />
|BBa_B0030-BBa_C0051-'''BBa_B0030''' (S-P)<br />
|BBa_J23100-'''BBa_B0030''' (E-S)<br />
|-<br />
|BBa_I15010 (X-P)<br />
|BBa_J23100-'''BBa_B0030'''-BBa_C0040 (E-S)<br />
|'''BBa_R0051'''-BBa_B0030-BBa_C0062 (E-S)<br />
|-<br />
|BBa_R0062 (E-X)<br />
|BBa_B0030-BBa_C0061-'''BBa_B1006''' (E-S)<br />
|}<br />
<br />
*Gel run/cut/gel extraction for:<br />
{|cellpadding="20px"<br />
|BBa_I15010 (X-P)<br />
|BBa_J23100-'''BBa_B0030''' (E-S)<br />
|BBa_J23100-'''BBa_B0030'''-BBa_C0040 (E-S)<br />
|-<br />
|BBa_C0012 (X-P)<br />
|'''BBa_R0051'''-BBa_B0030-BBa_C0062 (E-S)<br />
|BBa_B0030-BBa_C0061-'''BBa_B1006''' (E-S)<br />
|}<br />
<br />
*DNA precipitation with sodium acetate for:<br />
{|cellpadding="20px"<br />
|BBa_B0030 (S-P)<br />
|BBa_B0030-BBa_E0040-'''BBa_B1006''' (1) (E-X)<br />
|BBa_B0030-BBa_E1010-'''BBa_B1006''' (1) (E-X)<br />
|-<br />
|BBa_R0062 (E-X)<br />
|BBa_B0030-BBa_C0051-'''BBa_B0030''' (S-P)<br />
|}<br />
<br />
*(We already had BBa_I15009(X-P) and BBa_B1006(E-X))<br />
<br />
*Ligations:<br />
#'''BBa_B0030'''-BBa_I15009<br />
#BBa_J23100-BBa_B0030-BBa_C0040-'''BBa_B1006'''<br />
#BBa_R0051-BBa_B0030-BBa_C0062-'''BBa_B1006'''<br />
#BBa_B0030-BBa_C0051-'''BBa_B0030'''-BBa_C0079<br />
#BBa_B0030-BBa_C0061-BBa_B1006-'''BBa_R0062'''<br />
#BBa_J23100-'''BBa_B0030'''-BBa_C0012 (again)<br />
#BBa_J23100-'''BBa_B0030'''-BBa_I15010 (again)<br />
#BBa_J23100-BBa_B0030-BBa_E0040-'''BBa_B1006''' (to test the part)<br />
#BBa_J23100-BBa_B0030-BBa_E1010-'''BBa_B1006''' (to test the part)<br />
<br />
*We incubated ligations at 16°C overnight.<br />
<br />
*We infected 9 ml LB + Amp with 30 µl of BBa_B1006 and '''BBa_B0030'''-BBa_C0078 glycerol stocks. We incubated the 2 cultures at 37°C, 220 rpm ovenight.<br />
<br />
<br><br><br />
'''07/9/08'''<br />
<br><br />
*We transformed the 9 ligations (2 µl) and plated transformed bacteria. We incubated plates at 37°C ovenight.<br />
<br />
*Glycerol stocks for BBa_B1006 and '''BBa_B0030'''-BBa_C0078.<br />
<br />
*Miniprep for BBa_B1006 and '''BBa_B0030'''-BBa_C0078.<br />
<br />
*Plasmid digestion:<br />
{|cellpadding="20px"<br />
|BBa_B1006 (E-X)<br />
|'''BBa_B0030'''-BBa_C0078 (E-S)<br />
|}<br />
<br />
*Gel run/cut/gel extraction for '''BBa_B0030'''-BBa_C0078 (E-S).<br />
<br />
*DNA precipitation with sodium acetate for BBa_B1006 (E-X).<br />
<br />
*Ligation: BBa_B0030-BBa_C0078-'''BBa_B1006'''<br />
<br />
*We incubated ligation at 16°C overnight.<br />
<br />
<br><br><br />
'''07/10/08'''<br />
<br><br />
*We transformed BBa_B0030-BBa_C0078-'''BBa_B1006''' ligation (2 µl) and plated transformed bacteria. We incubated the plate at 37°C ovenight.<br />
<br />
*All the ligation plates showed colonies! There were carpets, but we could pick up some single colonies for colony PCR.<br />
<br />
*Colony PCR for:<br />
#'''BBa_B0030'''-BBa_I15009<br />
#BBa_J23100-BBa_B0030-BBa_C0040-'''BBa_B1006'''<br />
#BBa_R0051-BBa_B0030-BBa_C0062-'''BBa_B1006'''<br />
#BBa_B0030-BBa_C0051-'''BBa_B0030'''-BBa_C0079<br />
#BBa_B0030-BBa_C0061-BBa_B1006-'''BBa_R0062'''<br />
#BBa_J23100-'''BBa_B0030'''-BBa_C0012<br />
#BBa_J23100-'''BBa_B0030'''-BBa_I15010<br />
<br />
{|<br />
|[[Image:pv_falcon_colonypcr_9_7_08.jpg|thumb|300px|left|1 ml of infected LB + antibiotic for all the colonies we picked up to perform colony PCR]]<br />
||[[Image:pv_pcr_9_7_08.jpg|thumb|300px|left|Colony PCR]]<br />
|}<br />
<br />
{|<br />
|[[Image:pv_pcr_9-11-18-20-22-23-24.jpg|thumb|850px|left|Colony PCR: Marker (1), BBa_J23100-'''BBa_B0030'''-BBa_C0012, BBa_J23100-'''BBa_B0030'''-BBa_I15010, '''BBa_B0030'''-BBa_I15009, BBa_J23100-BBa_B0030-BBa_C0040-'''BBa_B1006''', BBa_R0051-BBa_B0030-BBa_C0062-'''BBa_B1006''', BBa_B0030-BBa_C0051-'''BBa_B0030'''-BBa_C0079, BBa_B0030-BBa_C0061-BBa_B1006-'''BBa_R0062''']]<br />
|}<br />
<br />
*Gel results:<br />
#'''BBa_B0030'''-BBa_I15009 1st colony was chosen.<br />
#BBa_J23100-BBa_B0030-BBa_C0040-'''BBa_B1006''' 5th colony was chosen.<br />
#BBa_R0051-BBa_B0030-BBa_C0062-'''BBa_B1006''' 2nd colony was chosen.<br />
#BBa_B0030-BBa_C0051-'''BBa_B0030'''-BBa_C0079 4th colony was chosen.<br />
#BBa_B0030-BBa_C0061-BBa_B1006-'''BBa_R0062''' 3rd colont was chosen.<br />
#BBa_J23100-'''BBa_B0030'''-BBa_C0012 did not show true positive colonies.<br />
#BBa_J23100-'''BBa_B0030'''-BBa_I15010 did not show true positive colonies.<br />
<br />
*We streaked BBa_J23100-BBa_B0030-BBa_E0040-'''BBa_B1006''' and BBa_J23100-BBa_B0030-BBa_E1010-'''BBa_B1006''' with a top to test the parts (NOTE: we could see some red colonies on BBa_J23100-BBa_B0030-BBa_E1010-'''BBa_B1006''' plate!these colonies correspond to bacteria with correctly ligated plasmid, while normal color colonies correspond to bacteria with BBa_B0030-BBa_E1010-'''BBa_B1006''' plasmid, without constitutive promoter).<br />
**We infected 100 µl LB + Amp with the tips and incubated the culture at 37°C, 220 rpm for 2 hours.<br />
**Then we watched green, blue and red fluorescence channels at microscope (50 µl of each culture):<br />
<br />
{|<br />
|[[Image:pv_G_R_10_7_08.png|thumb|700px|left|3 acquisitions for red cells; 3 acquisition for green cells; one acquisition for DAPI channel (blue) for green cells to check for impurities (DAPI channel acquisition for red cells was not saved, but it didn't show any blue area)]]<br />
|}<br />
<br />
*LB preparation: 0.5 l LB + Amp for plates.<br />
<br />
{|<br />
|[[Image:pv_LB_tower_10_7_08.jpg|thumb|300px|left|Prepared LB plates tower]]<br />
|}<br />
<br />
*BBa_R0010 resuspension from Registry of Standard Parts.<br />
<br />
*We transformed (4 µl) BBa_R0010 and plated transformed bacteria. We incubated BBa_R0010 plate at 37°C overnight.<br />
<br />
*We infected 9 ml LB + suitable antibiotic with 30 µl of these glycerol stocks:<br />
{|cellpadding="20px"<br />
|BBa_C0012<br />
|'''BBa_B0030'''-BBa_I15009 (1)<br />
|-<br />
|BBa_J23100-BBa_B0030-BBa_C0040-'''BBa_B1006''' (5)<br />
|BBa_R0051-BBa_B0030-BBa_C0062-'''BBa_B1006''' (2)<br />
|-<br />
|BBa_J23100-'''BBa_B0030'''<br />
|BBa_B0030-BBa_C0051-'''BBa_B0030'''-BBa_C0079 (4)<br />
|-<br />
|BBa_B0030-BBa_C0061-BBa_B1006-'''BBa_R0062''' (3)<br />
|}<br />
<br />
*We also picked up one colony from BBa_I15010 plate with a tip and infected 9 ml LB + Kan. We incubated the 8 cultures at 37°C, 220 rpm overnight.<br />
<br />
<br><br><br />
'''07/11/08'''<br />
<br><br />
*BBa_R0010 plate showed 2 colonies. We picked up one colony and infected 9 ml LB + Amp to grow an overnight culture.<br />
<br />
*BBa_B0030-BBa_C0078-'''BBa_B1006''' plate showed many colonies. We used 7 colonies to perform colony PCR.<br />
<br />
{|<br />
|[[Image:pv_pcr_17.png|thumb|600px|left|7 colonies for BBa_B0030-BBa_C0078-'''BBa_B1006''']]<br />
|}<br />
<br />
*Gel results: all the 7 colonies contained ligated plasmid; we chose the 5th colony to grow a 9 ml overnigth culture because 5th it showed no impurities.<br />
<br />
*Glycerol stocks for:<br />
{|cellpadding="20px"<br />
|BBa_C0012<br />
|'''BBa_B0030'''-BBa_I15009 (1)<br />
|-<br />
|BBa_I15010<br />
|BBa_J23100-BBa_B0030-BBa_C0040-'''BBa_B1006''' (5)<br />
|-<br />
|BBa_J23100-'''BBa_B0030'''<br />
|BBa_R0051-BBa_B0030-BBa_C0062-'''BBa_B1006''' (2)<br />
|-<br />
|BBa_B0030-BBa_C0051-'''BBa_B0030'''-BBa_C0079 (4)<br />
|BBa_B0030-BBa_C0061-BBa_B1006-'''BBa_R0062''' (3)<br />
|}<br />
<br />
*Miniprep for these 8 parts.<br />
<br />
*We sent BBa_I15010 and BBa_B0030-BBa_C0061-BBa_B1006-'''BBa_R0062''' (3) to Primm for sequencing.<br />
<br />
*Plasmid digestion for:<br />
{|cellpadding="20px"<br />
|BBa_C0012 (X-P)<br />
|'''BBa_B0030'''-BBa_I15009 (1) (E-S)<br />
|BBa_B1006 (we already had this plasmid at -20°C) (E-P)<br />
|-<br />
|BBa_J23100-'''BBa_B0030''' (E-S)<br />
|BBa_J23100-BBa_B0030-BBa_C0040-'''BBa_B1006''' (5) (E-S)<br />
|BBa_R0051-BBa_B0030-BBa_C0062-'''BBa_B1006''' (2) (E-S)<br />
|-<br />
|BBa_J23100-'''BBa_B0030''' (S-P)<br />
|BBa_R0040 (we already had this plasmid at -20°C) (E-X)<br />
|BBa_B0030-BBa_C0051-'''BBa_B0030'''-BBa_C0079 (4) (E-S)<br />
|}<br />
<br />
*Gel run/cut/gel extraction for all these parts.<br />
<br />
*(We already had BBa_B1006 (E-X) and BBa_R0062 (E-X))<br />
<br />
*Ligation:<br />
#BBa_J23100-'''BBa_B0030''' (S-P) -BBa_C0012 (X-P) (1:2 ratio instead of 1:6)<br />
#'''BBa_B1006 (E-P)''' + BBa_J23100-BBa_B0030 (E-S, 1:4) -BBa_C0012 (X-P, 1:2) (we decided to try this double ligation)<br />
#BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-'''BBa_R0040'''<br />
#BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-'''BBa_R0062'''<br />
#BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006'''<br />
#BBa_B0030-BBa_I15009-'''BBa_B1006'''<br />
<br />
*We incubated ligations at 16°C overnight.<br />
<br />
*We infected 9 ml LB + Amp with 30 µl of BBa_R0079 glycerol stock.<br />
<br />
<br><br><br />
'''07/12/08'''<br />
<br><br />
*Glycerol stocks for:<br />
{|cellpadding="20px"<br />
|BBa_R0010<br />
|BBa_R0079<br />
|BBa_B0030-BBa_C0078-'''BBa_B1006'''<br />
|}<br />
<br />
*Miniprep for these 3 parts.<br />
<br />
*We put these 3 purified plasmids at -20°C.<br />
<br />
*We also put the 6 ovenight ligations at -20°C. Next week they will be transformed!<br />
<br />
<br></div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week7Team:UNIPV-Pavia/Notebook/Week72008-10-26T21:26:08Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 7: 06/30/08 - 06/5/08==<br />
<br />
'''06/30/08'''<br />
<br><br />
*We received sequencing results for '''BBa_B0030'''-BBa_C0061 (4th colony and 7th colony): sequences were correct! We decided to keep the 4th colony.<br />
<br />
*Colony PCR for '''BBa_J23100'''-BBa_E0240 and '''BBa_B0030'''-BBa_C0061: 5 colonies for every plate. Gel showed many working colonies: we chose first colonies for the two ligations.<br />
<br />
{|<br />
|[[Image:pv_pcr_01_08.jpg|thumb|470px|left|Colony PCR for '''BBa_J23100'''-BBa_E0240, '''BBa_B0030'''-BBa_E0061: Marker and 5 colonies for each ligation]]<br />
|}<br />
<br />
*We infected 9 ml LB + Amp with 30 µl of:<br />
{|cellpadding="20px"<br />
|BBa_C0012<br />
|BBa_B1006<br />
|'''BBa_R0051'''-BBa_B0030<br />
|'''BBa_B0030'''-BBa_C0061<br />
|-<br />
|BBa_C0062<br />
|'''BBa_J23100'''-BBa_E0240 (1)<br />
|'''BBa_B0030'''-BBa_C0078 (1)<br />
|BBa_J23100-'''BBa_B0030'''<br />
|-<br />
|BBa_C0040<br />
|BBa_I15010<br />
|}<br />
<br />
*Tomorrow we will be ready to perform 5 ligations!<br />
<br />
<br><br><br />
'''07/1/08'''<br />
<br><br />
*Glycerol stocks for:<br />
{|cellpadding="20px"<br />
|BBa_C0012<br />
|BBa_B1006<br />
|'''BBa_R0051'''-BBa_B0030<br />
|'''BBa_B0030'''-BBa_C0061<br />
|-<br />
|BBa_C0062<br />
|'''BBa_J23100'''-BBa_E0240 (1)<br />
|'''BBa_B0030'''-BBa_C0078 (1)<br />
|BBa_J23100-'''BBa_B0030'''<br />
|-<br />
|BBa_C0040<br />
|BBa_I15010<br />
|}<br />
<br />
*Miniprep for all these parts.<br />
<br />
*We sent '''BBa_J23100'''-BBa_E0240 (1) and '''BBa_B0030'''-BBa_C0078 (1) to Primm for sequencing.<br />
<br />
*We performed digestion for:<br />
{|cellpadding="20px"<br />
|BBa_C0012 (X-P)<br />
|BBa_B1006 (E-X)<br />
|'''BBa_R0051'''-BBa_B0030 (S-P)<br />
|'''BBa_B0030'''-BBa_C0061 (E-S)<br />
|-<br />
|BBa_C0062 (X-P)<br />
|BBa_C0040 (X-P)<br />
|BBa_I15010 (X-P)<br />
|BBa_J23100-'''BBa_B0030''' (S-P)<br />
|}<br />
<br />
*Gel run/cut for:<br />
{|cellpadding="20px"<br />
|BBa_C0012 (X-P)<br />
|BBa_C0062 (X-P)<br />
|'''BBa_B0030'''-BBa_C0061 (E-S)<br />
|-<br />
|BBa_C0040 (X-P)<br />
|BBa_I15010 (X-P)<br />
|}<br />
<br />
*Gel extraction for these 5 parts.<br />
<br />
*DNA precipitation with sodium acetate for:<br />
{|cellpadding="20px"<br />
|BBa_B1006 (E-X)<br />
|'''BBa_R0051'''-BBa_B0030 (S-P)<br />
|BBa_J23100-'''BBa_B0030''' (S-P)<br />
|}<br />
<br />
*Ligation:<br />
**BBa_J23100-'''BBa_B0030'''-BBa_C0012<br />
**BBa_J23100-'''BBa_B0030'''-BBa_C0040<br />
**BBa_J23100-'''BBa_B0030'''-BBa_I15010<br />
**'''BBa_R0051'''-BBa_B0030-BBa_C0062<br />
**'''BBa_B0030'''-BBa_C0061-BBa_B1006<br />
<br />
*We incubated ligation reaction at 16°C overnight.<br />
<br />
<br><br><br />
'''07/2/08'''<br />
<br><br />
*We transformed ligations (5 µl) and plated transformed bacteria.<br />
<br />
*We also transformed BBa_B1006(E-X), BBa_J23100-'''BBa_B0030'''(S-P) and '''BBa_R0051'''-BBa_B0030 (1 µl) and plated transformed bacteria to estimate background noise.<br />
<br />
*We received sequencing results for:<br />
**'''BBa_B0030'''-BBa_E0040<br />
**'''BBa_B0030'''-BBa_C0051<br />
**'''BBa_B0030'''-BBa_E1010<br />
*All the sequences were correct!<br />
<br />
*We infected 9 ml LB + Amp with 30 µl of:<br />
{|cellpadding="20px"<br />
|BBa_B0030<br />
|'''BBa_B0030'''-BBa_E0040<br />
|'''BBa_B0030'''-BBa_C0051<br />
|-<br />
|BBa_B1006<br />
|'''BBa_B0030'''-BBa_E1010<br />
|}<br />
*glycerol stocks.<br />
<br />
<br><br><br />
'''07/3/08'''<br />
<br><br />
*All the ligation plates showed carpets, while negative control plates showed a weak background noise.<br />
<br />
*Single colonies plates for ligation plates.<br />
<br />
*Glycerol stocks for:<br />
{|cellpadding="20px"<br />
|BBa_B0030<br />
|'''BBa_B0030'''-BBa_E0040<br />
|'''BBa_B0030'''-BBa_C0051<br />
|-<br />
|BBa_B1006<br />
|'''BBa_B0030'''-BBa_E1010<br />
|}<br />
<br />
*Miniprep for these parts.<br />
<br />
*We performed digestion:<br />
{|cellpadding="20px"<br />
|BBa_B0030 (E-X)<br />
|'''BBa_B0030'''-BBa_E0040 (E-S)<br />
|'''BBa_B0030'''-BBa_C0051 (E-S)<br />
|-<br />
|BBa_B1006 (E-X)<br />
|'''BBa_B0030'''-BBa_E1010 (E-S)<br />
|}<br />
<br />
*Gel run/cut for:<br />
{|cellpadding="20px"<br />
|'''BBa_B0030'''-BBa_E0040 (E-S)<br />
|'''BBa_B0030'''-BBa_C0051 (E-S)<br />
|'''BBa_B0030'''-BBa_E1010 (E-S)<br />
|}<br />
<br />
*Gel extraction.<br />
<br />
*DNA precipitation with sodium acetate for:<br />
{|cellpadding="20px"<br />
|BBa_B0030 (E-X)<br />
|BBa_B1006 (E-X)<br />
|}<br />
<br />
Ligation:<br />
**BBa_B0030-BBa_E0040-'''BBa_B1006'''<br />
**BBa_B0030-BBa_C0051-'''BBa_B0030'''<br />
**BBa_B0030-BBa_E1010-'''BBa_B1006'''<br />
<br />
*We incubated ligation reactions at 16°C overnight.<br />
<br />
<br><br><br />
'''07/4/08'''<br />
<br><br />
*We transformed the 3 ligations (2 µl) and plated transformed bacteria.<br />
<br />
*Colony PCR for single colonies plates (3 colonies for each plate):<br />
{|cellpadding="20px"<br />
|BBa_J23100-'''BBa_B0030'''-BBa_C0012<br />
|BBa_J23100-'''BBa_B0030'''-BBa_C0040<br />
|BBa_J23100-'''BBa_B0030'''-BBa_I15010<br />
|-<br />
|'''BBa_R0051'''-BBa_B0030-BBa_C0062<br />
|'''BBa_B0030'''-BBa_C0061-BBa_B1006<br />
|}<br />
<br />
*Electrophoresis for PCR result: unfortunately, BBa_J23100-'''BBa_B0030'''-BBa_C0012 and BBa_J23100-'''BBa_B0030'''-BBa_I15010 did not show any true positive colony. We decided to re-perform colony PCR for these two parts next week. We chose to keep the first colonies for the other 3 ligations to grow 9 ml cultures overnight.<br />
<br />
{|<br />
|[[Image:pv_pcr_09_10_11_12_16.jpg|thumb|450px|left|Colony PCR: Marker (1), BBa_J23100-'''BBa_B0030'''-BBa_C0012 (2-4), BBa_J23100-'''BBa_B0030'''-BBa_C0040 (5-7), BBa_J23100-'''BBa_B0030'''-BBa_I15010 (8-10), '''BBa_R0051'''-BBa_B0030-BBa_C0062 (11-13), '''BBa_B0030'''-BBa_C0061-BBa_B1006 (14-16), blank (17)]]<br />
|}<br />
<br />
<br><br><br />
'''07/5/08'''<br />
<br><br />
*All the ligation plates showed colonies! Next week we will perform colony PCR to find true positive colonies.<br />
<br />
*Glycerol stocks and Miniprep for: BBa_J23100-'''BBa_B0030'''-BBa_C0040 (1), '''BBa_R0051'''-BBa_B0030-BBa_C0062 (1), '''BBa_B0030'''-BBa_C0061-BBa_B1006 (1).</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week6Team:UNIPV-Pavia/Notebook/Week62008-10-26T21:25:57Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 6: 06/23/08 - 06/27/08==<br />
<br />
'''06/23/08'''<br />
<br><br />
*Colony PCR for '''BBa_B0030'''-BBa_C0061 and '''BBa_B0030 (no Ant.Phosph.)'''-BBa_C0061: 10 colonies for every plate.<br />
<br />
*We ran PCR results: some colonies showed both ligated plasmid (heavier band) and false positive plasmid, because it was difficult to pick up single colonies from bacteria carpet. Ligation with Antarctic Phosphatase showed non-pure colonies and 3 false positives. Ligation without Antarctic Phosphatase showed 3 non-pure colonies and 7 pure colonies.<br />
<br />
{|<br />
|[[Image:pv_pcr_07.jpg|thumb|300px|left|Colony PCR for '''BBa_B0030'''-BBa_C0061: Antarctic Phosphatase results (top) and non-Antarctic Phosphatase results (bottom)]]<br />
|}<br />
<br />
*We decided to avoid Antarctic Phosphatase treatment in next ligations to save time.<br />
<br />
*We also decided to cut up to 1 µg of vector, to reduce background noise.<br />
<br />
*We choose 4th and 7th colonies to grow 9 ml cultures overnight.<br />
<br />
*We also infected 9 ml LB + Amp with 30 µl of:<br />
{|cellpadding="20px"<br />
|BBa_E0240<br />
|BBa_C0078<br />
|BBa_B0030<br />
|BBa_J23100<br />
|}<br />
*glycerol stocks. We incubated all the 6 cultures at 37°C, 220 rpm.<br />
<br />
*We transformed 10 µl of the remaining ligations:<br />
**'''BBa_B0030'''-BBa_E0040<br />
**'''BBa_B0030'''-BBa_E1010<br />
**'''BBa_B0030'''-BBa_C0051<br />
<br />
*Wiki updating: Project section.<br />
<br />
<br><br><br />
'''06/24/08'''<br />
<br><br />
*All the 3 ligation plates showed carpets. We decided to streak plates to produce single colonies plates. We incubated the 3 single colonies plates at 37°C overnight.<br />
<br />
*Glycerol stocks for:<br />
{|cellpadding="20px"<br />
|BBa_E0240<br />
|BBa_C0078<br />
|'''BBa_B0030'''-BBa_C0061(4)<br />
|-<br />
|BBa_J23100<br />
|BBa_B0030<br />
|'''BBa_B0030'''-BBa_C0061(7)<br />
|}<br />
<br />
*Miniprep for these 6 plasmids.<br />
<br />
*Plasmid digestion:<br />
{|cellpadding="20px"<br />
|BBa_E0240 (X-P)<br />
|BBa_C0078 (X-P)<br />
|-<br />
|BBa_J23100 (S-P)<br />
|BBa_B0030 (S-P)<br />
|}<br />
<br />
*DNA precipitation with sodium acetate for BBa_J23100 (S-P) and BBa_B0030 (S-P).<br />
<br />
*Gel run for BBa_E0240 (X-P) and BBa_C0078 (X-P)<br />
<br />
*Gel extraction.<br />
<br />
*Ligation:<br />
**'''BBa_J23100'''-BBa_E0240<br />
**'''BBa_B0030'''-BBa_C0078<br />
<br />
<br><br><br />
'''06/25/08'''<br />
<br><br />
*We transformed 5 µl of the two ligations.<br />
<br />
*We also transformed 0.5 µl of BBa_B0030(S-P) and BBa_J23100(S-P) plasmids to estimate background noise.<br />
<br />
*Colony PCR (6 colonies for each plate) for:<br />
**'''BBa_B0030'''-BBa_E0040<br />
**'''BBa_B0030'''-BBa_E1010<br />
**'''BBa_B0030'''-BBa_C0051<br />
<br />
{|<br />
|[[Image:pv_pcr_04_05_06.jpg|thumb|300px|left|Colony PCR for '''BBa_B0030'''-BBa_E0040, '''BBa_B0030'''-BBa_E0051 and '''BBa_B0030'''-BBa_E1010: Marker and 6 colonies for each ligation]]<br />
|}<br />
<br />
*The gel showed many working ligations! we choose the 1st colony for '''BBa_B0030'''-BBa_E0040, the 6th colony for '''BBa_B0030'''-BBa_C0051 and the 2nd colony for '''BBa_B0030'''-BBa_E1010 to grow 9 ml LB + Amp overnight cultures.<br />
<br />
<br><br><br />
'''06/26/08'''<br />
<br><br />
*Glycerol stocks for:<br />
**'''BBa_B0030'''-BBa_E0040 (1)<br />
**'''BBa_B0030'''-BBa_E1010 (6)<br />
**'''BBa_B0030'''-BBa_C0051 (2)<br />
<br />
*Miniprep for these 3 ligations. We sent purified plasmids to Primm for sequencing.<br />
<br />
*'''BBa_J23100'''-BBa_E0240 and '''BBa_B0030'''-BBa_C0078 plates showed a carpet.<br />
<br />
*We prepared single colonies plates for '''BBa_J23100'''-BBa_E0240 and '''BBa_B0030'''-BBa_C0078.<br />
<br />
*NOTE: '''BBa_J23100'''-BBa_E0240 plate showed red colonies (false positives) and normal color colonies (ligations).<br />
<br />
*We tested fluorescence for '''BBa_J23100'''-BBa_E0240: we streaked the plate and infected 100 µl of LB + Amp. We incubated this culture for 2 hours at 37°C, 220 rpm. Then we watched green, blue and red fluorescence channels at microscope. Some cells expressed GFP (cells with ligated plasmid) and some cells expressed RFP (false positives).<br />
<br />
{|align="center"<br />
|[[Image:pv_test_gfp.jpg|thumb|300px|left|Cells with ligated plasmid]]<br />
|[[Image:pv_test_rfp.jpg|thumb|300px|left|False positive cells]]<br />
|-<br />
|[[Image:pv_test_bluech.jpg|thumb|300px|left|Blue channel (to be sure that our glowing cells were not impurities)]]<br />
|[[Image:pv_test_merge.jpg|thumb|300px|left|Merge: green, red and blue channels]]<br />
|}<br />
<br />
<br><br><br />
'''06/27/08'''<br />
<br><br />
We put '''BBa_J23100'''-BBa_E0240 and '''BBa_B0030'''-BBa_C0078 single colonies plates at +4°C. Next week we will perform colony PCR for these two ligations.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week5Team:UNIPV-Pavia/Notebook/Week52008-10-26T21:25:44Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 5: 06/16/08 - 06/20/08==<br />
<br />
'''06/16/08'''<br />
<br><br />
*We picked up the only colony in '''BBa_B0030'''-BBa_C0078 plate to grow a 9 ml culture of transformed bacteria overnight.<br />
<br />
*We also infected 9 ml of LB + suitable antibiotic with 30 µl of:<br />
{|cellpadding="20px"<br />
|BBa_B0030<br />
|BBa_E1010<br />
|BBa_E0040<br />
|-<br />
|BBa_C0061<br />
|BBa_C0051<br />
|}<br />
*glycerol stocks. We incubated the 9 ml culture overnight at 37°C, 220 rpm.<br />
<br />
<br><br><br />
'''06/17/08'''<br />
<br><br />
*Glycerol stocks for:<br />
{|cellpadding="20px"<br />
|BBa_B0030<br />
|BBa_E1010<br />
|BBa_E0040<br />
|-<br />
|BBa_C0061<br />
|BBa_C0051<br />
|'''BBa_B0030'''-BBa_C0078<br />
|}<br />
<br />
*Miniprep for all these parts.<br />
<br />
*Digestion for:<br />
{|cellpadding="20px"<br />
|BBa_B0030 (S-P)<br />
|BBa_E1010 (X-P)<br />
|BBa_E0040 (X-P)<br />
|-<br />
|BBa_C0061 (X-P)<br />
|BBa_C0051 (X-P)<br />
|'''BBa_B0030'''-BBa_C0078 (X-S)<br />
|}<br />
<br />
*Gel run for '''BBa_B0030'''-BBa_C0078 to check for insert length: unfortunately, there was not a band where we expected...the only colony was a false positive. We'll try to ligate it in the next days.<br />
<br />
*Gel run for:<br />
{|cellpadding="20px"<br />
|BBa_B0030 (S-P)<br />
|BBa_E1010 (X-P)<br />
|BBa_E0040 (X-P)<br />
|-<br />
|BBa_C0061 (X-P)<br />
|BBa_C0051 (X-P)<br />
|}<br />
<br />
*Gel cut and DNA extraction.<br />
<br />
*We put DNA at -20°C. The next day we will perform some ligation reaction in different conditions, looking for the best protocol.<br />
<br />
<br><br><br />
'''06/18/08'''<br />
<br><br />
*We planned the following ligation experiments:<br />
**Transformation with BBa_B0030(S-P), to check background noise (we will know the amount of not digested vector);<br />
**Transformation with this ligation: BBa_B0030(S-P) after Antarctic Phosphatase treatment and no insert;<br />
**Transformation with this ligation: BBa_B0030(S-P) without Antarctic Phosphatase treatment and no insert;<br />
**Transformation with this ligation: BBa_B0030(S-P) without Antarctic Phosphatase treatment and BBa_C0061 insert;<br />
**Transformation with this ligation: BBa_B0030(S-P) after Antarctic Phosphatase treatment and BBa_C0061 insert;<br />
**3 Transformation with these ligations: BBa_B0030(S-P) after Antarctic Phosphatase treatment and BBa_C0051, BBa_E1010, BBa_E0040 inserts;<br />
<br />
*Antarctic Phosphatase for half of BBa_B0030 (S-P) volume.<br />
<br />
*We transformed 60 µl of TOP10 with 1 µl of BBa_B0030 (S-P)<br />
<br />
*We plated transformed bacteria and incubated them at 37°C overnight.<br />
<br />
*Antarctic Phosphatase for half a BBa_B0030 (S-P) volume.<br />
<br />
*Ligation (10 µl final volume):<br />
**BBa_B0030 alone<br />
**BBa_B0030 (no Ant.Phosph.) alone<br />
**'''BBa_B0030(no Ant.Phosph.)'''-BBa_C0061<br />
**'''BBa_B0030'''-BBa_C0061<br />
**'''BBa_B0030'''-BBa_C0078<br />
**'''BBa_B0030'''-BBa_E0040<br />
**'''BBa_B0030'''-BBa_E1010<br />
<br />
*We gave a lecture about Synthetic Biology and our current work at DIS (Department of Informatics and System Science).<br />
<br />
<br><br><br />
'''06/19/08'''<br />
<br><br />
*We received sequencing results for:<br />
**BBa_J23100-'''BBa_E0240''' (4 samples from 4 different colonies): all the 4 colonies were false positives<br />
**BBa_J23100-'''BBa_B0030''': the sequence was correct!<br />
**'''BBa_R0051'''-BBa_B0030: the sequence was correct!<br />
<br />
*BBa_B0030(S-P) plate showed many colonies. We expect to find at least this amount of colonies in ligation plates.<br />
<br />
*We heated ligation at 65°C for 10 min to inactivate T4 Ligase.<br />
<br />
*We transformed 10 µl of the following ligations:<br />
**BBa_B0030 alone<br />
**BBa_B0030(no Ant.Phosph.) alone<br />
**'''BBa_B0030 (no Ant.Phosph.)'''-BBa_C0061<br />
**'''BBa_B0030'''-BBa_C0061<br />
<br />
*We didn't transform the other 3 ligations because we wanted to check plated transformation the next day, to save 3 agar plates if the experiment doesn't work.<br />
<br />
<br><br><br />
'''06/20/08'''<br />
<br><br />
*Transformation results:<br />
**BBa_B0030 alone showed many colonies (less than BBa_B0030(S-P) seen the previous day)<br />
**BBa_B0030(no Ant.Phosph.) alone showed many colonies (the same quantity of BBa_B0030(S-P) seen the previous day)<br />
**'''BBa_B0030 (no Ant.Phosph.)'''-BBa_C0061 showed a carpet<br />
**'''BBa_B0030'''-BBa_C0061 showed a carpet<br />
<br />
*Now we are happy with these plates! Next week we will check insert length by colony PCR/electrophoresis.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week4Team:UNIPV-Pavia/Notebook/Week42008-10-26T21:25:33Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 4: 06/09/08 - 06/14/08==<br />
<br />
'''06/09/08'''<br />
<br><br />
*We prepared 4 glycerol stocks taking 800 µl from 9 ml cultures containing:<br />
<br />
{|cellpadding="20"<br />
|BBa_B0030 (one of the two cultures)<br />
|BBa_R0051<br />
|BBa_E0240<br />
|-<br />
|BBa_J23100-'''BBa_B0030'''<br />
|}<br />
<br />
<table width="100%" cellspacing="0" cellpadding="0"><br />
<tr><br />
<td><br />
*Miniprep for the 5 cultures.<br />
<br />
*We performed PCR on extracted BBa_J23100-'''BBa_B0030'''.<br />
<br />
*We performed electrophoresis on PCR result to check for contaminating plasmids: the plasmid was correct.<br />
<br />
*We sent purified BBa_J23100-'''BBa_B0030''' to Primm for sequencing.<br />
</td><br />
<td align="center"><br />
{|<br />
|[[Image:pv_02pcr.jpg|thumb|300px|right|PCR results for BBa_J23100-'''BBa_B0030''' ligation: plasmid length is correct]]<br />
|}<br />
</td><br />
</tr><br />
</table><br />
<br />
*We performed digestion protocol to open plasmids:<br />
{|cellpadding="20"<br />
|BBa_B0030 (1st culture) (E-X)<br />
|BBa_R0051 (S-P)<br />
|BBa_E0240 (E-X)<br />
|-<br />
|BBa_B0030 (2nd culture) (S-P)<br />
|}<br />
<br />
*We ran a gel with these parts.<br />
<br />
*We performed gel extraction.<br />
<br />
*Antarctic Phosphatase for these 4 parts.<br />
<br />
*Ligation (30 µl final volume):<br />
#BBa_J23100-'''BBa_E0240''' &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; (Pcon(E-S)-'''assGFP(E-X)''')<br />
#<br />
#'''BBa_R0051'''-BBa_B0030 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''Plam(S-P)'''-RBS(X-P))<br />
#'''BBa_B0030'''-BBa_E0040 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''RBS(S-P)'''-GFP(X-P))<br />
#'''BBa_B0030'''-BBa_C0051 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''RBS(S-P)'''-cI(X-P))<br />
#'''BBa_B0030'''-BBa_E1010 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''RBS(S-P)'''-RFP(X-P))<br />
#'''BBa_B0030'''-BBa_C0061 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''RBS(S-P)'''-luxI_LVA(X-P))<br />
#'''BBa_B0030'''-BBa_C0078 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''RBS(S-P)'''-lasI(X-P))<br />
<br />
*We incubated ligation overnight at 16°C.<br />
<br />
<br><br><br />
'''06/10/08'''<br />
<br><br />
*We transformed the whole volume of the ligations.<br />
<br />
*We plated the 7 ligations.<br />
<br />
<br><br><br />
'''06/11/08'''<br />
<br><br />
*BBa_J23100-'''BBa_E0240''' and '''BBa_R0051'''-BBa_B0030 plates showed respectively 4 and 1 colonies, while the other plates didn't show any colony.<br />
<br />
*We picked up one colony from the two working plates to grow 9 ml cultures of transformed bacteria overnight.<br />
<br />
*We also infected 9 ml of LB + suitable antibiotic with 30 µl of:<br />
{|cellpadding="20"<br />
|BBa_E0240<br />
|BBa_B0030<br />
|BBa_E0040<br />
|BBa_E1010<br />
|-<br />
|BBa_C0051<br />
|BBa_C0061<br />
|BBa_C0078<br />
|}<br />
<br />
*glycerol stocks. We incubated all the 9 ml cultures overnight at 37°C.<br />
<br />
*We contacted Francesca Ceroni from Bologna team for suggestions about ligation reaction. We decided to reduce reaction volume from 30 to 20 µl, increment insert:vector molar ratio from 1:2 to 1:3.<br />
<br />
<br><br><br />
'''06/12/08'''<br />
<br><br />
*We prepared 9 glycerol stocks taking 800 µl from 9 ml cultures containing:<br />
{|cellpadding="20"<br />
|BBa_E0240<br />
|BBa_B0030<br />
|BBa_E0040<br />
|'''BBa_R0051'''-BBa_B0030<br />
|-<br />
|BBa_C0051<br />
|BBa_C0061<br />
|BBa_C0078<br />
|BBa_J23100-'''BBa_E0240'''<br />
|-<br />
|BBa_E1010<br />
|}<br />
<br />
*Miniprep for the 9 parts.<br />
<br />
*We tested our microscope and BBa_J23100-'''BBa_E0240''' fluorescence:<br />
**We infected 150 µl of LB + Amp with 30 µl of BBa_J23100 glycerol stock (positive control)<br />
**We took 30 µl of BBa_J23100-'''BBa_E0240''' 9 ml culture and infected 150 µl of LB + Amp.<br />
*We incubated these two cultures at 37°C, 220 rpm for 3 hours.<br />
<br />
*Then, we took the 180 µl cultures and tried to watch them respectively through red and green fluorescence channel. BBa_J23100-'''BBa_E0240''' didn't glow, while our positive control, BBa_J23100, glowed correctly through red channel.<br />
<br />
<table width="100%" border="0"><br />
<tr><br />
<td valign="top"><br />
<table border="0"><br />
<tr><br />
<td valign="top"><br />
<div style="padding:20px;">[[Image:pv_rfp_test_J23100.jpg|thumb|300px|left|Positive control for fluorescence test: RFP in cells with BBa_J23100 plasmid]]</div><br />
</td><br />
<tr><br />
<td><br />
*We infected 9 ml of LB + suitable antibiotic with 30 µl of BBa_J23100-'''BBa_E0240''' and '''BBa_R0051'''-BBa_B0030 glycerol stocks.<br />
<br />
*We picked up all the remaining (3) colonies of BBa_J23100-'''BBa_E0240''' plate to grow 9 ml cultures of transformed bacteria overnight. With these cultures, we wanted to check if there are correctly ligated colonies. We incubated all the 9 cultures at 37°C, 220 rpm overnight.<br />
<br />
*PCR for BBa_J23100-'''BBa_E0240''' and '''BBa_R0051'''-BBa_B0030 to check for contaminations (even this time, we could not check the actual insert length because insert in ligation reaction were too small).<br />
<br />
*We performed electrophoresis on PCR result to check for contaminating plasmids: the plasmids was correct.<br />
</td><br />
</tr><br />
</table><br />
</td><br />
<td valign="top"><br />
[[Image:pv_01_03pcr.png|thumb|300px|right|PCR results for BBa_J23100-'''BBa_E0240''' and '''BBa_R0051'''-BBa_B0030 ligations: plasmid lengths are correct]]<br />
</td><br />
</tr><br />
</table><br />
<br />
*Digestion for:<br />
{|cellpadding="20"<br />
|BBa_E0240 (E-X)<br />
|BBa_B0030 (S-P) <br />
|BBa_E0040 (X-P)<br />
|BBa_E1010 (X-P)<br />
|-<br />
|BBa_C0051 (X-P)<br />
|BBa_C0061 (X-P)<br />
|BBa_C0078 (X-P)<br />
|BBa_J23100 (E-S)<br />
|}<br />
<br />
*Gel run for these parts.<br />
<br />
*Gel extraction for these parts.<br />
<br />
*Antarctic Phosphatase for BBa_E0240(E-X) and BBa_B0030(S-P)<br />
<br />
*Ligation (20 µl final volume):<br />
#BBa_J23100-'''BBa_E0240''' &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; (Pcon(E-S)-'''assGFP(E-X)''')<br />
#<br />
#<br />
#'''BBa_B0030'''-BBa_C0051 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''RBS(S-P)'''-cI(X-P))<br />
#'''BBa_B0030'''-BBa_E1010 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''RBS(S-P)'''-RFP(X-P))<br />
#'''BBa_B0030'''-BBa_C0061 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''RBS(S-P)'''-luxI_LVA(X-P))<br />
#'''BBa_B0030'''-BBa_C0078 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''RBS(S-P)'''-lasI(X-P))<br />
<br />
*We prepared 2 eppendorf tubes for each ligation reaction. We incubated one ligation set at 4°C overnight and the other ligation set at 25°C for 3 hours.<br />
<br />
*We received sequencing results for BBa_I14032: the sequence was 37 bp long, but it was not PlacIQ. We contacted Meagan to try to solve this problem.<br />
<br />
<br><br><br />
'''06/13/08'''<br />
<br><br />
*We prepared 5 glycerol stocks taking 800 µl from 9 ml cultures grown.<br />
<br />
*Miniprep for these cultures<br />
<br />
*We sent all the 5 purified plasmids to Primm for sequencing.<br />
<br />
*We transformed the whole volume of the ligations.<br />
<br />
*We plated the 10 ligations.<br />
<br />
<br><br><br />
'''06/14/08'''<br />
<br><br />
*Only one of 10 ligation plates worked: BBa_B0030-'''BBa_C0078''' showed one colony...Horrible result!<br />
<br />
*We decided to dedicate the following week to debug our protocols and to change something:<br />
**Reduce ligation volume to 10 µl<br />
**Inactivate T4 Ligase after ligation heating at 65°C for 10 min<br />
**Heat at 65°C for 5 min ligation mix before adding T4 Ligase and its buffer<br />
**Use different amounts of vector and insert<br />
**Don't use Antarctic Phosphatase<br />
<br />
<br></div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week3Team:UNIPV-Pavia/Notebook/Week32008-10-26T21:25:23Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 3: 06/03/08 - 06/08/08==<br />
<br />
'''06/03/08'''<br />
<br><br />
*We transformed 60 µl of TOP10 with 2 µl of the 6 parts (DNA + glycerol) we received from IGEM HQ:<br />
<br />
{|cellpadding="20"<br />
|BBa_C0179<br />
|BBa_C0161<br />
|BBa_R0051<br />
|BBa_I14032<br />
|-<br />
|BBa_I15008<br />
|BBa_I15010<br />
|}<br />
<br />
*NOTES: We noticed that IGEM 2007 teams which used luxI or lasR parts choosed BBa_C0061 instead of BBa_C0161 (for luxI) and BBa_C0079 instead of BBa_C0179 (for lasR). So, we decided in addition to amplify BBa_C0061 and BBa_C0079; we shall see later which one to choose for our project between the two luxI and the two lasR.<br />
<br />
*So, we cut paper spots for BBa_C0061 and BBa_C0079 and resuspended them in 10 µl of warmed TE buffer.<br />
<br />
*We transformed these 2 parts using 4 µl of DNA in TE. <br />
<br />
*We plated transformed bacteria and incubated them at 37°C overnight.<br />
<br />
<br><br><br />
'''06/04/08'''<br />
<br><br />
*After overnight incubation, the following 5 plates showed colonies:<br />
<br />
<table width="100%"><br />
<tr valign="up"><br />
<td><br />
{|cellpadding="20"<br />
|BBa_C0061<br />
|BBa_R0051<br />
|-<br />
|BBa_I14032<br />
|BBa_I15008<br />
|-<br />
|BBa_I15010<br />
|}<br />
</td><br />
<td><br />
{|<br />
|[[Image:pv_dnacol.jpg|thumb|300px|left|BBa_R0051 plate: very high yield from IGEM HQ DNA + glycerol]]<br />
|}<br />
</td><br />
<td><br />
{|<br />
|[[Image:bench_papercol.jpg|thumb|300px|left|BBa_C0061 plate: medium yield from paper spot]]<br />
|}<br />
</td><br />
</tr><br />
</table><br />
<br />
*while the following 3 plates did not:<br />
<br />
{|cellpadding="20"<br />
|BBa_C0079<br />
|BBa_C0179<br />
|-<br />
|BBa_C0161<br />
|}<br />
<br />
*We repeated the transformation for BBa_C0079, BBa_C0179 and C0161. We used 6 µl of DNA in TE for BBa_C0079, while we used 3 µl of DNA + glycerol for BBa_C0179 and BBa_C0161.<br />
<br />
*We plated transformed bacteria and incubated them at 37°C overnight. <br />
<br />
*While we were preparing our 5 ml cultures for the 5 working plates, we noticed that LB + Amp was contaminated! We decided to prepare a big quantity of LB + Amp and also of LB + Kan: we prepared 0.5 l LB + Amp and 0.5 l LB + Kan for liquid cultures; 0.5 l LB + Amp and 0.5 l LB + Kan for plates.<br />
<br />
{|<br />
|<div style="padding:20px;">[[Image:pv_lb_tower.jpg|thumb|300px|left|A lot of just prepared LB plates]]</div><br />
|}<br />
<br />
*We picked up one colony from BBa_C0061, BBa_R0051, BBa_I14032, I15008 and I15010 plates to grow 5 ml cultures of transformed bacteria overnight.<br />
<br />
*We also infected 5 ml of LB + Amp with 15 µl of BBa_B0030 glycerol stock. We incubated the 5 ml culture overnight at 37°C.<br />
<br />
*We received QIAGEN QIAprep Spin Miniprep Kit!!! We will inaugurate it tomorrow on these 5 ml cultures;)<br />
<br />
<br><br><br />
'''06/05/08'''<br />
<br><br />
*We received Euroclone Antarctic Phosphatase: we are going to use it in the afternoon before ligation reaction.<br />
<br />
*We prepared 6 glycerol stocks taking 800 µl from 5 ml cultures containing:<br />
<br />
{|cellpadding="20"<br />
|BBa_R0051<br />
|BBa_I15008<br />
|BBa_I15010<br />
|BBa_I14032<br />
|-<br />
|BBa_C0061<br />
|BBa_B0030<br />
|}<br />
<br />
*We performed miniprep for these 6 parts with our new fantastic kit;) Plasmid quantification confirmed a higher yield than our previous QIAGEN kit.<br />
<br />
*We performed plasmid digestion for these parts (20 of 30 µl).<br />
<br />
*We had to insulate excided fragments for:<br />
{|cellpadding="20"<br />
|BBa_I15008 (X-P)<br />
|BBa_I15010 (X-P)<br />
|BBa_I14032 (X-P)<br />
|BBa_C0061 (X-P)<br />
|-<br />
|BBa_B0030 (X-P)<br />
|}<br />
<br />
*While we had to insulate opened plasmids for:<br />
{|cellpadding="20"<br />
|BBa_R0051 (S-P)<br />
|}<br />
<br />
*Due to the different dimension of the DNA we had to insulate, we ran 3 different gels:<br />
**one for BBa_R0051 (S-P)<br />
**one for BBa_I14032 (X-P) and BBa_B0030 (X-P)<br />
**one for BBa_C0061 (X-P), BBa_I15008 (X-P) and BBa_I15010 (X-P).<br />
<br />
*We could see and cut all the bands we wanted, except for BBa_I14032, for which we couldn't see any band.<br />
<br />
*We performed gel extraction for:<br />
<br />
{|cellpadding="20"<br />
|BBa_R0051 (S-P)<br />
|BBa_B0030 (X-P)<br />
|BBa_C0061 (X-P)<br />
|BBa_I15008 (X-P)<br />
|-<br />
|BBa_I15010 (X-P)<br />
|}<br />
<br />
*All the 3 overnight plates worked (low yield for all).<br />
<br />
*We picked up one colony from BBa_C0161, BBa_C0179 and BBa_C0079 plates to grow 5 ml cultures of transformed bacteria overnight. We did the same thing for BBa_I14032 6/3/08 plate; we didn't use glycerol stock for BBa_I14032 because we thought there was something wrong with the colony we picked on 6/4/08.<br />
<br />
*Antarctic Phosphatase for BBa_E0240 (E-X), BBa_R0051 (S-P), BBa_B0030 (S-P), BBa_B0030 (E-X).<br />
<br />
*We calculated the amount of vector/insert for ligation reaction to have a molar ratio of 2:1 (insert:vector).<br />
<br />
*We performed ligation reaction (30 µl final volume) for '''(vectors are in bold type)''':<br />
#BBa_J23100-'''BBa_E0240''' &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; (Pcon(E-S)-'''assGFP(E-X)''')<br />
#BBa_J23100-'''BBa_B0030''' &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; (Pcon(E-S)-'''RBS(E-X)''')<br />
#'''BBa_R0051'''-BBa_B0030 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''Plam(S-P)'''-RBS(X-P))<br />
#'''BBa_B0030'''-BBa_E0040 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''RBS(S-P)'''-GFP(X-P))<br />
#'''BBa_B0030'''-BBa_C0051 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''RBS(S-P)'''-cI(X-P))<br />
#'''BBa_B0030'''-BBa_E1010 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''RBS(S-P)'''-RFP(X-P))<br />
#'''BBa_B0030'''-BBa_C0061 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''RBS(S-P)'''-luxI_LVA(X-P))<br />
#'''BBa_B0030'''-BBa_C0078 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ('''RBS(S-P)'''-lasI(X-P))<br />
<br />
*We incubated ligation overnight at 16°C.<br />
<br />
<br><br><br />
'''06/06/08'''<br />
<br><br />
<br />
*We prepared 4 glycerol stocks taking 800 µl from 5 ml cultures containing:<br />
<br />
{|cellpadding="20"<br />
|BBa_C0161<br />
|BBa_C0179<br />
|BBa_C0079<br />
|BBa_I14032<br />
|}<br />
<br />
*Miniprep for BBa_C0161, BBa_C0179, BBa_C0079 and BBa_I14032.<br />
<br />
<table width="100%" cellspacing="0" cellpadding="0"><br />
<tr><br />
<td><br />
*We performed plasmid digestion for these 4 parts (20 of 30 µl).<br />
<br />
*We ran two different gels:<br />
**one for BBa_C0161 (X-P), BBa_C0179 (X-P) and BBa_C0079 (X-P)<br />
**one for BBa_I14032 (X-P), that is smaller than the other 3 parts<br />
<br />
*For the second time we couldn't see any band for BBa_I14032.<br />
<br />
*We performed gel extraction for:<br />
<br />
{|cellpadding="20"<br />
|BBa_C0161 (X-P)<br />
|BBa_C0179 (X-P)<br />
|-<br />
|BBa_C0079 (X-P)<br />
|}<br />
<br />
</td><br />
<td align="center"><br />
{|<br />
|[[Image:pv_mattia_gelextr.jpg|thumb|300px|right|Mattia cutting BBa_C0161 (X-P), BBa_C0179 (X-P) and BBa_C0079 (X-P) gel]]<br />
|}<br />
</td><br />
</tr><br />
</table><br />
<br />
*We decided to sequence BBa_I14032 to check if the sequence is correct. Unfortunately we had not enough plasmid and so we picked another colony from 6/3/08 plate, infected 9 ml LB + Kan and incubated it overnight at 37°C. We decided to use 9 ml instead of 5 ml in order to verify if we could increment our yield without saturating the QIAGEN spin column and without performing a midiprep.<br />
<br />
*We transformed the whole ligation volume (30 µl) for all the 8 overnight ligations.<br />
<br />
*We plated transformed bacteria and incubated at 37°C overnight.<br />
<br />
<br><br><br />
'''06/07/08'''<br />
<br><br />
<table width="100%" cellspacing="0" cellpadding="0"><br />
<tr><br />
<td><br />
*We took 800 µl of BBa_I14032 overnight culture to prepare a glycerol stock.<br />
<br />
*Miniprep for BBa_I14032 9 ml culture: absorbance spectrum was very good, so we decided to use 9 ml cultures even the next times.<br />
<br />
*We sent BBa_I14032 sample and VF2 primer to Primm for sequencing.<br />
</td><br />
<td align="center"><br />
{|<br />
|[[Image:pv_nanodrop_9ml_placiq.jpg|thumb|300px|right|NanoDrop output for BBa_I14032 9 ml miniprep]]<br />
|}<br />
</td><br />
</tr><br />
</table><br />
<br />
*Only BBa_J23100-'''BBa_B0030''' (Pcon(E-S)-'''RBS(E-X)''') plate showed colonies. We put it at 4°C.<br />
<br />
*NOTES: obviously we were not happy with this result. So, how could we do to debug this situation?<br />
**Our hypothesis was that we had to focus on plasmid digestion process: we approximately checked the length of every opened plasmid/excided fragment we wanted to insulate on gel and they were right.<br />
**We were sure that restriction enzymes cut both the restriction sites (E-S or X-P) for excided fragments, but we could not be so sure whether opened plasmids had been cut (E-X or S-P) in both restriction sites or only in one site.<br />
**In previous digestion protocols to open plasmids, we performed 2 cutting cycles: one for the first enzyme (1 hour and 30 min) and one for the second enzyme (1 hour and 30 min).<br />
**We supposed there was something wrong with that digestion protocol, so we decided to perform plasmid digestion again for our 4 plasmids to open, following the digestion protocol (read Protocols section for details).<br />
**We also noticed that digestion protocol for excided fragments worked very well for BBa_B0030, whose fragment length is 15 bp, so we hoped that this protocol might actually work to open plasmids.<br />
**In addition, we decided to check if our only working plate was a false positive: next week we will perform PCR/electrophoresis to check for contaminating plasmids (we could not check immediately for insert length by electrophoresis because BBa_J23100 and BBa_B0030 are small parts).<br />
<br />
*Wiki updating.<br />
<br />
<br><br><br />
'''06/08/08'''<br />
<br><br />
*We picked up one colony from BBa_J23100-'''BBa_B0030''' plate to grow a 9 ml culture of transformed bacteria overnight.<br />
<br />
*We also infected 9 ml of LB + Amp with 15 µl of BBa_B0030, BBa_B0030 (another time), BBa_R0051, BBa_E0240 glycerol stocks. We incubated 9 ml cultures overnight at 37°C.<br />
<br />
*Wiki updating.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week2Team:UNIPV-Pavia/Notebook/Week22008-10-26T21:25:10Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 2: 05/26/08 - 05/31/08==<br />
<br />
'''05/26/08'''<br />
<br><br />
*Team meeting at Biomedical Informatics Lab to talk about UNIPV-Pavia team presentation at Europe Teachers' Workshop in Paris and to make our first wiki updating.<br />
<br />
*We received restriction enzymes and Agarose Gel DNA Extraction Kit from Roche. Now we are ready to cut;)<br />
<br><br><br />
<br />
'''05/27/08'''<br />
<br><br />
*We performed plasmid digestion for these 9 parts (1 µg):<br />
<br />
{|cellpadding="20"<br />
|BBa_C0078 (X-P)<br />
|BBa_E0040 (X-P)<br />
|BBa_E1010 (X-P)<br />
|BBa_C0051 (X-P)<br />
|-<br />
|BBa_B0030 (X-P)<br />
|BBa_B0030 (E-X)<br />
|BBa_B0030 (S-P)<br />
|BBa_E0240 (E-X)<br />
|-<br />
|BBa_J23100 (E-S)<br />
|}<br />
<br />
*We had to insulate excided fragments for:<br />
{|cellpadding="20"<br />
|BBa_C0078 (X-P)<br />
|BBa_E0040 (X-P)<br />
|BBa_E1010 (X-P)<br />
|BBa_C0051 (X-P)<br />
|-<br />
|BBa_B0030 (X-P)<br />
|BBa_J23100 (E-S)<br />
|}<br />
<br />
*While we had to insulate opened plasmids for:<br />
{|cellpadding="20"<br />
|BBa_B0030 (E-X)<br />
|BBa_B0030 (S-P)<br />
|BBa_E0240 (E-X)<br />
|}<br />
<br />
*We ran two different gels for these two groups, because excided fragments are smaller than opened plasmids.<br />
<br />
*Unfortunately, BBa_B0030 (X-P) and BBa_J23100 (E-S) fragments had smearing appearance because they were smaller than other excided fragments and ran too fast.<br />
<br />
*We performed gel extraction for all the 9 parts, but we decided to repeat cutting/run/extraction process for BBa_B0030 (X-P) and BBa_J23100 (E-S) in the next days, to perform a more efficient extraction for these 2 parts.<br />
<br />
*We took 15 µl from BBa_B0030 and BBa_J23100 glycerol stocks and infected 9 ml LB + Amp for each part. We incubated the 9 ml cultures at 37°C overnight. (We already had at our disposal extracted plasmids for these 2 parts, but we performed plasmid extraction anyway to have more).<br />
<br />
<br><br><br />
'''05/28/08'''<br />
<br><br />
*We performed plasmid digestion for these 4 parts (about 5.5 µg for BBa_C0062; the whole quantity for the others):<br />
{|cellpadding="20"<br />
|BBa_C0062 (X-P)<br />
|BBa_C0012 (X-P)<br />
|BBa_C0040 (X-P)<br />
|BBa_I15009 (X-P)<br />
|}<br />
<br />
*NOTES: to avoid smearing fragments during electrophoresis, we planned experiments in which only excided fragments of the same order of magnitude are ran in the same gel. So, we decided to run these four parts on May 28 (the smallest part is 660 bp and the largest one is 1128 bp). The remaining parts will be digested/gel extracted on May 29 (the smallest part is 39 bp and the largest one is 157 bp).<br />
<br />
*We loaded and ran a gel to separate these 4 excided fragments from the rest of their plasmid.<br />
<br />
*We performed gel extraction.<br />
<br />
*We expected to find the 9 ml culture for BBa_J23100 red, but it was not! So we performed miniprep only for BBa_B0030 9 ml culture, after taking 800 µl from 9 ml BBa_B0030 culture to prepare a new glycerol stock.<br />
<br />
*Unfortunately DNA pellet washing failed...Unlucky day for minipreps!<br />
<br />
*We took 15 µl again from BBa_B0030 and BBa_J23100 glycerol stocks and infected 9 ml LB + Amp for each part. We incubated the 9 ml cultures at 37°C overnight.<br />
<br />
<br><br><br />
'''05/29/08'''<br />
<br><br />
*We received VF2 and VR primers.<br />
<br />
*We received the 6 parts we had requested to IGEM 2008 Headquarters! (5 µl DNA + glycerol for every part) IGEM HQ also sent us a new punch tool:) Thank you very much!<br />
<br />
*9 ml cultures were grown correctly and BBa_J23100 culture was red. So we could perform miniprep for these 2 parts.<br />
<br />
{|<br />
|<div style="padding:20px;">[[Image:pv_red_pellet.jpg|thumb|300px|left|Red pellet for BBa_J23100 culture after a 15 min 4°C 6000 x g centrifuging]]</div><br />
|}<br />
<br />
*Unfortunately, plasmids extracted from BBa_B0030 culture were not pure: quantification at spectrophotometer showed an extra peak at 280 nm, which corresponds to protein contaminations.<br />
<br />
*We will repeat miniprep for BBa_B0030 next week, hoping to be luckier than this week...and hoping that QIAGEN miniprep kit will arrive soon!<br />
<br />
*We performed plasmid digestion for these 5 parts:<br />
{|cellpadding="20"<br />
|BBa_R0079 (X-P)<br />
|BBa_R0040 (X-P)<br />
|BBa_R0082 (X-P)<br />
|BBa_R0062 (X-P)<br />
|BBa_J23100 (E-S)<br />
|}<br />
<br />
*We loaded and ran a gel to separate these 5 excided fragments from the rest of their plasmid.<br />
<br />
*We performed gel extraction.<br />
<br />
*Wiki updating<br />
<br />
<br><br><br />
'''05/30/08'''<br />
<br><br />
*Wiki updating<br />
<br />
*We received TOP10 stocks in the afternoon: next week we will transform the 6 parts we received on May 29.<br />
<br />
<br><br><br />
'''05/31/08'''<br />
<br><br />
*Europe Teachers' Workshop in Paris<br />
<br></div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week1Team:UNIPV-Pavia/Notebook/Week12008-10-26T21:24:59Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 1: 05/19/08 - 05/23/08==<br />
<br />
'''05/19/08'''<br />
<br><br />
*Let’s start our IGEM 2008 experience! At first, we broke the punch tool…:)<br />
<br />
*We used a scalpel to cut and resuspend the following 22 paper spots:<br />
<br />
<table width=100%><br />
<tr><td><br />
{|cellpadding="20"<br />
|BBa_I14032<br />
|BBa_R0079<br />
|BBa_R0062<br />
|BBa_R0040<br />
|-<br />
|BBa_R0082<br />
|BBa_R0051<br />
|BBa_J23100<br />
|BBa_C0161<br />
|-<br />
|BBa_C0062<br />
|BBa_C0078<br />
|BBa_C0179<br />
|BBa_C0051<br />
|-<br />
|BBa_C0012<br />
|BBa_C0040<br />
|BBa_I15010<br />
|BBa_I15008<br />
|-<br />
|BBa_I15009<br />
|BBa_E0040<br />
|BBa_E1010<br />
|BBa_B0030<br />
|-<br />
|BBa_B1006<br />
|BBa_E0240<br />
|}<br />
</td><br />
<td align="center"><br />
{|<br />
|[[Image:bench_registry.jpg|thumb|300px|left|Registry of Standard Parts 2008 in our lab]]<br />
|}<br />
<br />
</td></tr><br />
</table><br />
*We used tweezers to put the cut paper into tubes containing 10 μl of warmed TE buffer.<br />
<br />
*We transformed 60 µl of TOP10 E. coli with 4 µl of DNA in TE for all 22 parts, plated transformed bacteria and incubated overnight at 37°C.<br />
<br />
{|<br />
|<div style="padding:20px;">[[Image:pv_22plates.jpg|thumb|300px|left|Our 22 parts at 37°C]]</div><br />
|}<br />
<br />
*We used LB medium previously prepared, with the suitable antibiotic added.<br />
<br><br><br />
'''05/20/08'''<br />
<br><br />
*After overnight incubation, the following 14 plates showed colonies:<br />
<br />
<table width="100%"><br />
<tr><td><br />
{|cellpadding="20"<br />
|BBa_R0079<br />
|BBa_R0062<br />
|BBa_R0040<br />
|BBa_R0082<br />
|-<br />
|BBa_J23100<br />
|BBa_C0062<br />
|BBa_C0051<br />
|BBa_C0012<br />
|-<br />
|BBa_C0040<br />
|BBa_E0040<br />
|BBa_E1010<br />
|BBa_B0030<br />
|-<br />
|BBa_B1006<br />
|BBa_E0240<br />
|}<br />
</td><br />
<td align="center"><br />
{|<br />
|[[Image:plategfp.jpg|thumb|300px|left|BBa_E0240]]<br />
|}<br />
</td></tr><br />
</table><br />
<br />
*While the following plates did not:<br />
<br />
{|cellpadding="20"<br />
|BBa_I14032<br />
|BBa_R0051<br />
|BBa_C0161<br />
|BBa_C0078<br />
|-<br />
|BBa_C0179<br />
|BBa_I15008<br />
|BBa_I15009<br />
|BBa_I15010<br />
|}<br />
<br />
<br><br />
*Plate containing BBa_J23100 showed red colonies, as we expected: this part is inserted into plasmid BBa_J61002 which places the RFP downstream of the inserted part, which is a constitutive promoter.<br />
<br />
*We picked up one colony from every working plate to grow 5 ml cultures of transformed bacteria overnight.<br />
<br />
*We re-transformed 60 µl of TOP10 with the remaining 6 µl of DNA in TE for BBa_I14032, BBa_R0051, BBa_I15008, BBa_I15009, BBa_I15010, BBa_C0161, BBa_C0078, BBa_C0179.<br />
<br />
*We plated transformed bacteria and incubated them at 37°C overnight.<br />
<br />
*We ordered primers VF2 and VR. We also ordered new TOP10 stocks to Invitrogen, because our stock was about to run out.<br />
<br />
<br><br><br />
'''05/21/08'''<br />
<br><br />
*Only BBa_I15009 and BBa_C0078 plates showed colonies and for the remaining 6 plates there were no colonies again.<br />
<br />
*We picked up one colony from BBa_I15009 and BBa_C0078 plates to grow 5 ml cultures of transformed bacteria overnight.<br />
<br />
*We re-cut paper spots for BBa_R0051, BBa_I14032, BBa_I15008, BBa_I15010, BBa_C0161, BBa_C0179 and resuspended them again in 10 µl of warmed TE buffer.<br />
<br />
*We repeated the transformation for these 6 parts using 4 µl of DNA in TE.<br />
<br />
*We plated transformed bacteria and incubated them at 37°C overnight.<br />
<br />
*We prepared 14 glycerol stocks taking 800 µl from 5 ml cultures containing:<br />
<br />
<table width="100%"><br />
<tr><td><br />
{|cellpadding="20"<br />
|BBa_R0079<br />
|BBa_R0062<br />
|BBa_R0040<br />
|BBa_R0082<br />
|-<br />
|BBa_J23100<br />
|BBa_C0062<br />
|BBa_C0051<br />
|BBa_C0012<br />
|-<br />
|BBa_C0040<br />
|BBa_E0040<br />
|BBa_E1010<br />
|BBa_B0030<br />
|-<br />
|BBa_B1006<br />
|BBa_E0240<br />
|}<br />
</td><br />
<td align="center"><br />
{|<br />
|[[Image:pv_red_culture.jpg|thumb|300px|left|Two glycerol stocks: BBa_J23100 red culture and BBa_B0030 normal color culture]]<br />
|}<br />
</td></tr><br />
</table><br />
<br />
*We performed plasmid purification for these 14 parts.<br />
<br />
<br><br><br />
'''05/22/08'''<br />
<br><br />
*For the third time, there were no colonies in the 6 plates.<br />
<br />
*We contacted IGEM Headquarters to explain our problem for these 6 parts. We thank Meagan Lizarazo for her kind attention! IGEM Headquarters will send us the 6 parts.<br />
<br />
*We tried to transform these 6 parts for the last time, using the remaining 6 µl of DNA in TE. We used our last 6 TOP10 stocks.<br />
<br />
*We prepared 2 glycerol stocks taking 800 µl from 5 ml cultures containing BBa_I15009 and BBa_C0078.<br />
<br />
*We performed plasmid purification for these 2 parts.<br />
<br />
*We had QIAGEN mini kit at our disposal, instead of QIAGEN miniprep kit for plasmid purification. We noticed that QIAGEN mini kit performed a low yield extraction (40-80ng/µl instead of 200-500ng/µl normally yielded by miniprep kit): quantified plasmid concentrations measured with NanoDrop spectrophotometer were very low.<br />
<br />
*For this reason we decided to re-perform plasmid extraction with a higher culture volume: 9 ml instead of 5 ml. Anyway, we are waiting to receive QIAGEN miniprep kit in order to perform more efficient plasmid purifications.<br />
<br />
*We took 15 µl from all our 16 glycerol stocks and infected 9 ml LB + suitable antibiotic for each part we had. We incubated the 9 ml cultures at 37°C overnight.<br />
<br />
<br><br><br />
'''05/23/08'''<br />
<br><br />
*For the fourth time, there were no colonies in the 6 plates. We decided to wait for the 6 paper spots from IGEM Headquarters, instead of performing another cut/transformation.<br />
<br />
*We prepared 14 glycerol stocks taking 800 µl from our 10 ml cultures.<br />
<br />
*We performed plasmid purification for all cultures.<br />
<br />
*Quantification of plasmid concentration showed that 9 ml cultures yielded higher amounts of plasmid than using 5 ml cultures (100-150ng/µl instead of 40-80ng/µl).<br />
<br />
*Center for Tissue Engineering (CIT) meeting: we explained our first results to CIT members.<br />
<br></div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/NotebookTeam:UNIPV-Pavia/Notebook2008-10-26T21:24:46Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]<br />
|}<br />
<br />
<br><br></div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week21Team:UNIPV-Pavia/Notebook/Week212008-10-26T21:23:06Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 19: 10/6/08 - 10/10/08==<br />
<br />
Mathematical modeling using Matlab and Simulink. Our aim was to build a scaffold for future quantitative standard characterization.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week20Team:UNIPV-Pavia/Notebook/Week202008-10-26T21:20:43Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 20: 09/29/08 - 10/3/08==<br />
<br />
'''09/29/08'''<br />
<br><br />
*Ligations:<br />
**Lig.34-Lig.15 (=TR)(our RFP protein generator downstream, to check Lig.34 functionality)<br />
**T5-Lig.15 (=TT)(our RFP protein generator after our GFP protein generator, to check terminator efficiency)<br />
*We incubated ligations at 16°C overnight.<br />
<br />
'''09/30/08'''<br />
<br><br />
*We transformed the two overnight ligations and plated transformed bacteria. We incubated the two plates at 37°C overnight.<br />
<br />
'''10/1/08'''<br />
<br><br />
*We received sequencing results for Lig.31 and Lig.36. Sequences were OK!!! Notice that both of the two parts are long. So, sequencing could only confirm partially the nucleotides composing these two parts.<br />
<br />
*Plates were ok!<br />
<br />
*We performed colony PCR on the two plates. Medium and large gel could not be ran because instrumentation was buisy. We could ran two small gels (8 wells), so the maximum number of colonies was 13 (considering two markers and one blank). Whe chose to screen 6 colonies for TT and 7 colonies for TR.<br />
<br />
*Gel results:<br />
**TT - 2nd colony<br />
**TR - 1st colony (gel picture not available...sorry!)<br />
<br />
{|<br />
|[[Image:pv_colonypcr_TT.jpg|thumb|370px|left|Marker 1Kb, blank, R0051-B0030-C0062-B1006-R0062-B0030-E0040-B1006-B0030-E1010-B1006 (6 colonies)]]<br />
|}<br />
<br />
*We incubated the chosen colonies at 37°C, 220 rpm overnight.<br />
<br />
'''10/2/08'''<br />
<br><br />
*Glycerol stocks for TT-2 and TR-1.<br />
<br />
*TR fluorescence test. Experiment details and results are reported in The Project section, Experiments.<br />
<br />
*Data processing.<br />
<br />
'''10/3/08'''<br />
<br><br />
*We prepared 0.5 l of LB + Amp for liquid cultures because our LB was looked turbid.<br />
<br />
*Wiki updating.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week17Team:UNIPV-Pavia/Notebook/Week172008-10-26T21:19:18Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 17: 09/8/08 - 09/12/08==<br />
<br />
'''09/8/08'''<br />
<br><br />
*We prepared 0.5 l of LB + Amp for liquid cultures.<br />
<br />
*We infected 9 ml of LB + Amp with 30 µl of Lig.30(3), Lig.31, Lig.22, Lig.13 and two falcon tubes for Lig.a.<br />
<br />
*We received 3OC6HSL from Sigma! we resuspended it in ddH2O, we prepared some stocks (2 mM) and stored them at -20°C.<br />
<br />
'''09/9/08'''<br />
<br><br />
*Glycerol stocks/miniprep for Lig.30(3), Lig.31, Lig.22 and Lig.13.<br />
<br />
*Plasmid digestion for:<br />
**Lig.30 (S-P)<br />
**Lig.31 (S-P)<br />
**Lig.30 (X-P)<br />
**Lig.22 (X-P)<br />
**13 (X-P)<br />
<br />
*Run/gel extraction.<br />
<br />
*Ligations:<br />
**Lig.31-Lig.30 (="Lig.34")<br />
**Lig.31-Lig.22 (="Lig.35")<br />
**Lig.30-Lig.13 (="Lig.T5" for green fluorescence test)<br />
<br />
*We induced one of the two Lig.a overnight culture with 3OC6HSL 1 µM. We incubated the two cultures for 1 hour and then watched TRITC channel at microscope. (We didn't synchronize the two cultures, but performed a qualitative test for luxR mutated protein integrity evaluation).<br />
<br />
*Fluorescence test results: HSL induced Lig.a show RFP expression, while Lig.a without HSL showed a weak red fluorescence. Result pictures are not available at the moment.<br />
<br />
'''09/10/08'''<br />
<br><br />
*We transformed/plated ligations. We decided to perform two transformations for each ligation:<br />
**one normal transformation (1 µl of ligation);<br />
**one diluted ligation (1:10)<br />
*We decided to try diluted transformations to have less colonies in the plate and to avoid streaking single colonies plates when colonies are not insulated.<br />
<br />
'''09/11/08'''<br />
<br><br />
*Plates grew correctly and diluted transformation showed less colonies.<br />
<br />
*Colony PCR for Lig.34, Lig.35 and Lig.T5 (4 colonies for normal transformation plates and 4 colonies for diluted transformation plates).<br />
<br />
*Gel results (gel picture not available, we're sorry...!):<br />
**Lig.34 (1st,3rd,4th colonies for normal transformation plate; 1st,2nd colonies for diluted transformation plate)<br />
**Lig.35 (3rd,4th colonies for normal transformation plate; 1st,4th colonies for diluted transformation plate) (this time there were not unexpected contaminants!)<br />
**Lig.T5 (1st colony)<br />
<br />
*Comments: we chose MANY colonies because Lig.34 and Lig.35 are our final assemblies and we want to be sure that the parts were correct. We decided to extract plasmids from all these colonies and to sequence all of them.<br />
<br />
*We incubated the chosen colonies at 37°C, 220 rpm overnight.<br />
<br />
'''09/12/08'''<br />
<br><br />
*Glycerol stocks/miniprep for our ten overnight cultures.<br />
<br />
*We sent:<br />
**Lig.34-1<br />
**Lig.34-3<br />
**Lig.34-4<br />
**Lig.34dil-1<br />
**Lig.34dil-2<br />
**Lig.35-3<br />
**Lig.35-4<br />
**Lig.35dil-1<br />
**Lig.35dil-4<br />
*to Primm for sequencing.</div>Magnihttp://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week20Team:UNIPV-Pavia/Notebook/Week202008-10-26T21:15:35Z<p>Magni: </p>
<hr />
<div><!--{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center" --><br />
<br />
{| style="color:#000000;background-color:#ffffff;" cellpadding="3" cellspacing="1" border="3" bordercolor="#3128" width="80%" align="center"<br />
!align="center"|[[Image:home.jpg|30px]] [[Team:UNIPV-Pavia|Home]]<br />
!align="center"|[[Image:unipv_logo.jpg|30px]] [[Team:UNIPV-Pavia/Team|The Team]]<br />
!align="center"|[[Image:and.jpg|30px]] [[Team:UNIPV-Pavia/Project|The Project]]<br />
!align="center"|[[Image:safety.jpg|30px]] [[Team:UNIPV-Pavia/Safety|Biological Safety]]<br />
!align="center"|[[Image:dna.png|30px]] [[Team:UNIPV-Pavia/Parts|Parts Submitted to the Registry]]<br />
|-<br />
!align="center"|[[Image:laptop.jpg|30px]] [[Team:UNIPV-Pavia/Dry_Lab|Dry Lab]]<br />
!align="center"|[[Image:pipette.jpg|30px]] [[Team:UNIPV-Pavia/Wet_Lab|Wet Lab]]<br />
!align="center"|[[Image:math.gif|30px]] [[Team:UNIPV-Pavia/Modeling|Modeling]]<br />
!align="center"|[[Image:note.jpg|30px]] [[Team:UNIPV-Pavia/Protocols|Protocols]]<br />
!align="center"|[[Image:notebook.gif|30px]] [[Team:UNIPV-Pavia/Notebook|Activity Notebook]]<br />
|}<br />
<br />
<br><br />
<br />
<br />
==Notebook==<br />
<br />
<br><br><br />
<br />
{|align="center" cellpadding="30"<br />
!|[[Team:UNIPV-Pavia/Notebook/Week1|Week 1]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week2|Week 2]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week3|Week 3]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week4|Week 4]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week5|Week 5]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week6|Week 6]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week7|Week 7]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week8|Week 8]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week9|Week 9]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week10|Week 10]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week11|Week 11]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week12|Week 12]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]<br />
|-<br />
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]<br />
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]<br />
|}<br />
<br />
<br><br><br />
<br />
==Week 20: 09/29/08 - 10/3/08==<br />
<br />
'''09/29/08'''<br />
<br><br />
*Ligations:<br />
**Lig.34-Lig.15 (=TR)(our RFP protein generator downstream, to check Lig.34 functionality)<br />
**T5-Lig.15 (=TT)(our RFP protein generator after our GFP protein generator, to check terminator efficiency)<br />
*We incubated ligations at 16°C overnight.<br />
<br />
'''09/30/08'''<br />
<br><br />
*We transformed the two overnight ligations and plated transformed bacteria. We incubated the two plates at 37°C overnight.<br />
<br />
'''10/1/08'''<br />
<br><br />
*We received sequencing results for Lig.31 and Lig.36. Sequences were OK!!! Notice that both of the two parts are long. So, sequencing could only confirm partially the nucleotides composing these two parts.<br />
<br />
*Plates were ok!<br />
<br />
*We performed colony PCR on the two plates. Medium and large gel could not be ran because instrumentation was buisy. We could ran two small gels (8 wells), so the maximum number of colonies was 13 (considering two markers and one blank). Whe chose to screen 6 colonies for TT and 7 colonies for TR.<br />
<br />
*Gel results:<br />
**TT - 2nd colony<br />
**TR - 1st colony (gel picture not available...sorry!)<br />
<br />
{|<br />
|[[Image:pv_colonypcr_TT.jpg|thumb|370px|left|Marker 1Kb, blank, R0051-B0030-C0062-B1006-R0062-B0030-E0040-B1006-B0030-E1010-B1006 (6 colonies)]]<br />
|}<br />
<br />
*We incubated the chosen colonies at 37°C, 220 rpm overnight.<br />
<br />
'''10/2/08'''<br />
<br><br />
*Glycerol stocks for TT-2 and TR-1.<br />
<br />
*TR fluorescence test. Experiment details and results are reported in The Project section, Experiments.<br />
<br />
*Data processing.<br />
<br />
'''10/2/08'''<br />
<br><br />
*We prepared 0.5 l of LB + Amp for liquid cultures.<br />
<br />
*Wiki updating.</div>Magni