http://2008.igem.org/wiki/index.php?title=Special:Contributions/David.bikard&feed=atom&limit=50&target=David.bikard&year=&month=2008.igem.org - User contributions [en]2024-03-28T15:49:16ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/Team:Paris/ConstructionTeam:Paris/Construction2008-10-30T04:09:30Z<p>David.bikard: /* Oscillating module */</p>
<hr />
<div>{{Paris/Menu}}<br />
'''Have a look at our [[Team:Paris/Notebook|notebook]].'''<br />
<br />
=Model constructions: from the modelling to the characterization=<br />
<br />
Our project BacterioClock is based on an oscillating FIFO synchronized at the population level. To obtain and have preliminary results of this system we divided it in three "little" modules as the modeling team exposed them previously.<br />
==The First is to demonstrate the [[Team:Paris/Analysis#FIFO_behaviour|FIFO]] which constitutes our core system==<br />
<br />
A simple manner is to implement two class II promoter followed by fluorescent reporter genes like GFP in a bacteria strain that is able to produce the flagella. <br />
<br />
By this way, we are able to see in single cell the order of the activation of pFliL then pFlgA then pFlhB and then the inactivation in the same order as a FIFO will do.<br />
<br />
These constructions are equivalent to the experiments realized by Uri Alon in the article "Using quantitative Blueprint to reprogram the Dynamics of the Flagella Gene Network" Kalir S, Alon U. Cell 2004.<br />
<br />
A better way to observe the FIFO is to study the system with an inducible regulator of class II gene in a bacteria strain deleted for this gene. We are extremely grateful for Alon U. that send us the inducible gene ''FlhDC'', and ''FliA'' in pBad18 plasmid.<br />
These plasmids with our own araC/pBad-EnvZ* will allowed us to study the system in mutated strains that we have in our lab (ΔFlhD, ΔFliA, ΔFlgM, ΔEnvZ). By cotransforming in mutated strains the inducible regulators of the class II promoters with one of this promoter associated to a fluorescent protein, we could characterize the influence of the master regulators of the flagella on their promoter. The fluorescence is normalized to the OD<sub>600</sub>.<br />
<br>For example below are some constructions to see and induce the FIFO:<br />
<br><br><br><br />
[[Image:Felipe01.jpg|500px|center|thumb|pFliL alone]]<br><br><br><br />
[[Image:Felipe02.jpg|500px|center|thumb|3 promoters of the FIFO in one Cell]]<br><br><br><br />
[[Image:Felipe03.jpg|500px|center|thumb|FIFO and oscillating constructions in a single cell]]<br><br><br><br />
<br />
<br />
To perform such a cotransformation we take care about the ORI of each low copy plasmid which are often incompatible and we made all our final constructions in the pSB4T5 plasmid that care the only one resistance that was not already used and the pSC101 ORI which is compatible with the ColE1.<br />
<br />
We could see the FIFO in a more convenient manner with the assembly of two class II promoters associated to different fluorophores. Then we could observed two kinds of fluorescents in One Cell along time. <br />
One problem that could occur is the interaction between the different fluorescents proteins due to the cross-over of their emission and excitation spectra and moreover in our final system that count three fluorophores.<br />
<br />
[[Image:Spectre.jpg |600px |thumb| emission and excitation spectra of ECFP, YFP and mRFP]]<br />
<br />
<br>The choice of ECFP, YFP and mRFP is based upon this consideration and upon the order of appearing in the FIFO.<br />
Indeed by putting the mRFP in last position, we improve the visualization of the FIFO like the maturation time of the protein is about of one hour.<br />
<br />
We thank a lot Arnau Montagu, advisor of the iGEM Valencia Team. He advised us, after his team had troubleshooting with fluorescence, to take care about ''spurious FRET''. This phenomenon occurs even with non covered fluorescent spectra when the concentration of two fluorophores is sufficient so they are enough closer to induce some FRET. But it could happen at lower concentration than 40 μM. <br><br />
<br>- "DsRed as a Potential FRET Partner with CFP and GFP" Michael G. Erickson, Daniel L. Moon, and David T. Yue Biophysical Journal 2003. <br />
<br>- "A Comparison of Donor-Acceptor Pairs for Genetically Encoded FRET Sensors: Application to the Epac cAMP<br />
Sensor as an Example" Gerard N. M. van der Krogt, Janneke Ogink, Bas Ponsioen, Kees Jalink PLoS 2008.<br />
<br />
<br><br />
<br />
==[[Team:Paris/Analysis#Oscillations|Oscillating]] module==<br />
Few approaches have been thought in order to create the oscillating module. The pTet/TetR system that allows us to precisely control the inhibition of TetR on the Tet promoter. Another system use the natural inhibition of OmpR and of EnvZ via OmpR. We finally perform this system that we have developed since we know thanks to the modeling team that the oscillations are the only way to see the full development of the FIFO.<br />
<br />
==Synchronization module==<br />
The modeling simulation showed us that to have some oscillations that are difficult to obtain in a simple feedforward loop without the delay introduce by the quorum sensing synchronization, and for us it is more interesting to have the FIFO behavior at the population level. We did not achieved the this module but we started the construction of it and have already some intermediate parts that you will find below. <br />
==The constructions we were able to obtain allow us to see the pictures below of the fluorescence of one of the promoter (pFlgA) of the FIFO Flagella:==<br />
[[Image:ParisGfp.jpg]]Green fluorescence<br />
[[Image:ParisRfp.jpg]]Red Fluorescence<br />
[[Image:ParisCfp.jpg]]Cyan Fluorescence<br />
<br />
== Table of the constructions we realized==<br />
{|<br />
|- style="background: #649cd7;"<br />
!colspan=4 style="background: #649cd7" | FIFO<br />
|- style="background: #649cd7; text-align: center;"<br />
|width=30%| Construction<br />
|width=55%| Description<br />
|width=15%| Validation Date<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136039 pFliL(RBS WT)-ECFP tripart (LVA+)]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFliL is ECFP fused with a LVA tag.<br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>[http://partsregistry.org/Part:BBa_K136037 pFliL(+ RBS WT)-GFP]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFliL is GFP.<br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136012 pFlgA(+ RBS WT) - YFP Tripart (LVA+)]<br />
|align=|Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is EYFP fused with a LVA tag. <br />
|align="center"| [[Team:Paris/August 22|August 22]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136013 pFlgA(+ RBS WT) - YFP Tripart (LVA-)]<br />
|align=|Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is EYFP fused without a LVA tag. <br />
|align="center"| [[Team:Paris/August 22|August 22]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136054 pFlgA(+ RBS WT)-YFP tripart (AAV+)]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is EYFP fused with a AAV tag. <br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>[http://partsregistry.org/Part:BBa_K136053 pFlgA(+ RBS WT)-GFP]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is GFP. <br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136055 pFlhB(+ RBS WT)-mRFP tripart (+LVA)]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlhB is mRFP with a LVA tag. <br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>[http://partsregistry.org/Part:BBa_K136065 pFlhB(+ RBS WT)-GFP]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlhB is GFP. <br />
|align="center"| [[Team:Paris/October 26|October 26]]<br />
|-<br />
|colspan=4 style="background: #649cd7" | <center>'''OSCILLATION'''</center><br />
|-<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136064 pFliL(+ RBS WT)-EnvZ*]<br />
|align=| pFliL is regulated by FlhDC and EnvZ regulates pFlhDC thanks to a negative feedback loop, origin of an oscillation behavior.<br />
|align="center"| [[Team:Paris/October 26|October 26]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136069 pFliL(+ RBS WT)-EnvZ*-GFP generator]<br />
|align=| The same construction as before but with a GFP in order to follow the dynamic of expression of pFliL.<br />
|align="center"| [[Team:Paris/October 29|October 29]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136051 pFlhB(+ RBS WT)-EnvZ*]<br />
|align=| pFlhB is regulated by FlhDC and EnvZ regulates pFlhDC thanks to a negative feedback loop, origin of an oscillation behavior.<br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136070 pFlhB(+ RBS WT)-EnvZ*-GFP generator]<br />
|align=| The same construction as before but with a GFP in order to follow the dynamic of expression of pFlhB.<br />
|align="center"| [[Team:Paris/October 29|October 29]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136066 araC/pBad-EnvZ*]<br />
|align=| A construction made for characterizing the repression power of the mutated EnvZ on our system<br />
|align="center"| [[Team:Paris/October 27|October 27]]<br />
|-<br />
|colspan=4 style="background: #649cd7" | <center>'''SYNCHRONISATION'''</center><br />
|-<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136029 pLas - gfp Tripart]<br />
|align=| Construction for characterization of the pLas promoter<br />
|align="center"| [[Team:Paris/August 1|August 1]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136027 pLas - ECFP]<br />
|align=| The same as before but with another fluorophore<br />
|align="center"| [[Team:Paris/August 1|August 1]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br> [http://partsregistry.org/Part:BBa_K136024 strongest promoter-rbs-lasR-dble ter]<br />
|align=| A construction for a strong constitutive expression of the Las Regulator in order to get maximal expression in presence of AHL<br />
|align="center"| [[Team:Paris/August 20|August 20]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136023 Strong promoter-rbs-lasR-dble ter]<br />
|align=| A derived construction of the previous one for smaller constitutive expression of LasR in the Bacteria.<br />
|align="center"| [[Team:Paris/August 20|August 20]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136044 Strongest RBS (1)- LasR activator (+LVA)]<br />
|align=| Intermediary construction. We uses different rbs for Las R in order to get various response dynamic to the presence of AHL<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136048 rbs-lasR + dbl ter]<br />
|align=| Intermediary construction. We uses different rbs for Las R in order to get various response dynamic to the presence of AHL<br />
|align="center"| [[Team:Paris/August 19|August 19]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136052 lasR activator + LVA - Double Terminator]<br />
|align=| Intermediary construction<br />
|align="center"| [[Team:Paris/August 1|August 1]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136057 RBS lasI - GFP tripart]<br />
|align=| Construction that has to be associated with the FIFO. We will know when LasI is expressed and then when AHL is produced.<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br> [http://partsregistry.org/Part:BBa_K136056 RBS-lasI - ECFP]<br />
|align=| The same as the previous one but with another fluorophore.<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|-<br />
|colspan=4 style="background: #649cd7" | <center>'''NEW GENE AND PROMOTER BIOBRICKS'''</center><br />
|-<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136010 pFliA]<br />
|align=| Natural promoter of E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC.<br />
|align="center"| [[Team:Paris/October 04|October 04]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136009 pFliL (+RBS WT)]<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA.<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136006 pFlgA (+RBS WT)]<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA..<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br> pFlgB (+RBS WT)<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA.<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136008 pFlhB (+RBS WT)]<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA.<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136046 EnvZ*]<br />
|align=| EnvZ is a membrane protein involved into the osmotic response and is interacting with OmpR that will bind the promoter of FlhDC ad will repress it. EnvZ* is a mutated form of the coding sequence, giving a phenotype where pFlhDC is completely repressed. EnvZ has been extracted by PVR for the correct mutant strain.<br />
|align="center"| [[Team:Paris/September 04|September 04]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_coding.png]]<br>FliA<br />
|align=| FliA is a sigma factor involved into the regulation of very various genes, the promoter used in the FIFO were selected to be higly responding to FliA. In order to have the FliA gene possibly used as biobrick, we mutated 2 sites (EcoRI and PstI) inside the FliA sequence by assemby PCR. <br />
|align="center"| [[Team:Paris/September 04|September 04]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" |[[Image:Icon_coding.png]]<br>FlhDC<br />
|align=| FlhDC is the main regulator of the flagella genetic system in E. coli. FlhDC regulates all the promoter of the FIFO.<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|}<br />
<br />
{{Paris/Navig|Team:Paris/Construction}}</div>David.bikardhttp://2008.igem.org/Team:Paris/ConstructionTeam:Paris/Construction2008-10-30T04:09:15Z<p>David.bikard: /* The First is to demonstrate the FIFO which constitutes our core system */</p>
<hr />
<div>{{Paris/Menu}}<br />
'''Have a look at our [[Team:Paris/Notebook|notebook]].'''<br />
<br />
=Model constructions: from the modelling to the characterization=<br />
<br />
Our project BacterioClock is based on an oscillating FIFO synchronized at the population level. To obtain and have preliminary results of this system we divided it in three "little" modules as the modeling team exposed them previously.<br />
==The First is to demonstrate the [[Team:Paris/Analysis#FIFO_behaviour|FIFO]] which constitutes our core system==<br />
<br />
A simple manner is to implement two class II promoter followed by fluorescent reporter genes like GFP in a bacteria strain that is able to produce the flagella. <br />
<br />
By this way, we are able to see in single cell the order of the activation of pFliL then pFlgA then pFlhB and then the inactivation in the same order as a FIFO will do.<br />
<br />
These constructions are equivalent to the experiments realized by Uri Alon in the article "Using quantitative Blueprint to reprogram the Dynamics of the Flagella Gene Network" Kalir S, Alon U. Cell 2004.<br />
<br />
A better way to observe the FIFO is to study the system with an inducible regulator of class II gene in a bacteria strain deleted for this gene. We are extremely grateful for Alon U. that send us the inducible gene ''FlhDC'', and ''FliA'' in pBad18 plasmid.<br />
These plasmids with our own araC/pBad-EnvZ* will allowed us to study the system in mutated strains that we have in our lab (ΔFlhD, ΔFliA, ΔFlgM, ΔEnvZ). By cotransforming in mutated strains the inducible regulators of the class II promoters with one of this promoter associated to a fluorescent protein, we could characterize the influence of the master regulators of the flagella on their promoter. The fluorescence is normalized to the OD<sub>600</sub>.<br />
<br>For example below are some constructions to see and induce the FIFO:<br />
<br><br><br><br />
[[Image:Felipe01.jpg|500px|center|thumb|pFliL alone]]<br><br><br><br />
[[Image:Felipe02.jpg|500px|center|thumb|3 promoters of the FIFO in one Cell]]<br><br><br><br />
[[Image:Felipe03.jpg|500px|center|thumb|FIFO and oscillating constructions in a single cell]]<br><br><br><br />
<br />
<br />
To perform such a cotransformation we take care about the ORI of each low copy plasmid which are often incompatible and we made all our final constructions in the pSB4T5 plasmid that care the only one resistance that was not already used and the pSC101 ORI which is compatible with the ColE1.<br />
<br />
We could see the FIFO in a more convenient manner with the assembly of two class II promoters associated to different fluorophores. Then we could observed two kinds of fluorescents in One Cell along time. <br />
One problem that could occur is the interaction between the different fluorescents proteins due to the cross-over of their emission and excitation spectra and moreover in our final system that count three fluorophores.<br />
<br />
[[Image:Spectre.jpg |600px |thumb| emission and excitation spectra of ECFP, YFP and mRFP]]<br />
<br />
<br>The choice of ECFP, YFP and mRFP is based upon this consideration and upon the order of appearing in the FIFO.<br />
Indeed by putting the mRFP in last position, we improve the visualization of the FIFO like the maturation time of the protein is about of one hour.<br />
<br />
We thank a lot Arnau Montagu, advisor of the iGEM Valencia Team. He advised us, after his team had troubleshooting with fluorescence, to take care about ''spurious FRET''. This phenomenon occurs even with non covered fluorescent spectra when the concentration of two fluorophores is sufficient so they are enough closer to induce some FRET. But it could happen at lower concentration than 40 μM. <br><br />
<br>- "DsRed as a Potential FRET Partner with CFP and GFP" Michael G. Erickson, Daniel L. Moon, and David T. Yue Biophysical Journal 2003. <br />
<br>- "A Comparison of Donor-Acceptor Pairs for Genetically Encoded FRET Sensors: Application to the Epac cAMP<br />
Sensor as an Example" Gerard N. M. van der Krogt, Janneke Ogink, Bas Ponsioen, Kees Jalink PLoS 2008.<br />
<br />
<br><br />
<br />
==[[Team:Paris/Analysis#Oscillations|Oscillating]] module==<br />
Few approaches have been thought in order to create the oscillating module. The pTet/TetR system that allows us to precisely control the inhibition of TetR on the Tet promotor. Another system use the natural inhibition of OmpR and of EnvZ via OmpR. We finally perform this system that we have developed since we know thanks to the modeling team that the oscillations are the only way to see the full development of the FIFO.<br />
<br />
==Synchronization module==<br />
The modeling simulation showed us that to have some oscillations that are difficult to obtain in a simple feedforward loop without the delay introduce by the quorum sensing synchronization, and for us it is more interesting to have the FIFO behavior at the population level. We did not achieved the this module but we started the construction of it and have already some intermediate parts that you will find below. <br />
==The constructions we were able to obtain allow us to see the pictures below of the fluorescence of one of the promoter (pFlgA) of the FIFO Flagella:==<br />
[[Image:ParisGfp.jpg]]Green fluorescence<br />
[[Image:ParisRfp.jpg]]Red Fluorescence<br />
[[Image:ParisCfp.jpg]]Cyan Fluorescence<br />
<br />
== Table of the constructions we realized==<br />
{|<br />
|- style="background: #649cd7;"<br />
!colspan=4 style="background: #649cd7" | FIFO<br />
|- style="background: #649cd7; text-align: center;"<br />
|width=30%| Construction<br />
|width=55%| Description<br />
|width=15%| Validation Date<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136039 pFliL(RBS WT)-ECFP tripart (LVA+)]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFliL is ECFP fused with a LVA tag.<br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>[http://partsregistry.org/Part:BBa_K136037 pFliL(+ RBS WT)-GFP]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFliL is GFP.<br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136012 pFlgA(+ RBS WT) - YFP Tripart (LVA+)]<br />
|align=|Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is EYFP fused with a LVA tag. <br />
|align="center"| [[Team:Paris/August 22|August 22]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136013 pFlgA(+ RBS WT) - YFP Tripart (LVA-)]<br />
|align=|Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is EYFP fused without a LVA tag. <br />
|align="center"| [[Team:Paris/August 22|August 22]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136054 pFlgA(+ RBS WT)-YFP tripart (AAV+)]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is EYFP fused with a AAV tag. <br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>[http://partsregistry.org/Part:BBa_K136053 pFlgA(+ RBS WT)-GFP]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is GFP. <br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136055 pFlhB(+ RBS WT)-mRFP tripart (+LVA)]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlhB is mRFP with a LVA tag. <br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>[http://partsregistry.org/Part:BBa_K136065 pFlhB(+ RBS WT)-GFP]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlhB is GFP. <br />
|align="center"| [[Team:Paris/October 26|October 26]]<br />
|-<br />
|colspan=4 style="background: #649cd7" | <center>'''OSCILLATION'''</center><br />
|-<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136064 pFliL(+ RBS WT)-EnvZ*]<br />
|align=| pFliL is regulated by FlhDC and EnvZ regulates pFlhDC thanks to a negative feedback loop, origin of an oscillation behavior.<br />
|align="center"| [[Team:Paris/October 26|October 26]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136069 pFliL(+ RBS WT)-EnvZ*-GFP generator]<br />
|align=| The same construction as before but with a GFP in order to follow the dynamic of expression of pFliL.<br />
|align="center"| [[Team:Paris/October 29|October 29]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136051 pFlhB(+ RBS WT)-EnvZ*]<br />
|align=| pFlhB is regulated by FlhDC and EnvZ regulates pFlhDC thanks to a negative feedback loop, origin of an oscillation behavior.<br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136070 pFlhB(+ RBS WT)-EnvZ*-GFP generator]<br />
|align=| The same construction as before but with a GFP in order to follow the dynamic of expression of pFlhB.<br />
|align="center"| [[Team:Paris/October 29|October 29]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136066 araC/pBad-EnvZ*]<br />
|align=| A construction made for characterizing the repression power of the mutated EnvZ on our system<br />
|align="center"| [[Team:Paris/October 27|October 27]]<br />
|-<br />
|colspan=4 style="background: #649cd7" | <center>'''SYNCHRONISATION'''</center><br />
|-<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136029 pLas - gfp Tripart]<br />
|align=| Construction for characterization of the pLas promoter<br />
|align="center"| [[Team:Paris/August 1|August 1]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136027 pLas - ECFP]<br />
|align=| The same as before but with another fluorophore<br />
|align="center"| [[Team:Paris/August 1|August 1]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br> [http://partsregistry.org/Part:BBa_K136024 strongest promoter-rbs-lasR-dble ter]<br />
|align=| A construction for a strong constitutive expression of the Las Regulator in order to get maximal expression in presence of AHL<br />
|align="center"| [[Team:Paris/August 20|August 20]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136023 Strong promoter-rbs-lasR-dble ter]<br />
|align=| A derived construction of the previous one for smaller constitutive expression of LasR in the Bacteria.<br />
|align="center"| [[Team:Paris/August 20|August 20]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136044 Strongest RBS (1)- LasR activator (+LVA)]<br />
|align=| Intermediary construction. We uses different rbs for Las R in order to get various response dynamic to the presence of AHL<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136048 rbs-lasR + dbl ter]<br />
|align=| Intermediary construction. We uses different rbs for Las R in order to get various response dynamic to the presence of AHL<br />
|align="center"| [[Team:Paris/August 19|August 19]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136052 lasR activator + LVA - Double Terminator]<br />
|align=| Intermediary construction<br />
|align="center"| [[Team:Paris/August 1|August 1]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136057 RBS lasI - GFP tripart]<br />
|align=| Construction that has to be associated with the FIFO. We will know when LasI is expressed and then when AHL is produced.<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br> [http://partsregistry.org/Part:BBa_K136056 RBS-lasI - ECFP]<br />
|align=| The same as the previous one but with another fluorophore.<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|-<br />
|colspan=4 style="background: #649cd7" | <center>'''NEW GENE AND PROMOTER BIOBRICKS'''</center><br />
|-<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136010 pFliA]<br />
|align=| Natural promoter of E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC.<br />
|align="center"| [[Team:Paris/October 04|October 04]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136009 pFliL (+RBS WT)]<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA.<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136006 pFlgA (+RBS WT)]<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA..<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br> pFlgB (+RBS WT)<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA.<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136008 pFlhB (+RBS WT)]<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA.<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136046 EnvZ*]<br />
|align=| EnvZ is a membrane protein involved into the osmotic response and is interacting with OmpR that will bind the promoter of FlhDC ad will repress it. EnvZ* is a mutated form of the coding sequence, giving a phenotype where pFlhDC is completely repressed. EnvZ has been extracted by PVR for the correct mutant strain.<br />
|align="center"| [[Team:Paris/September 04|September 04]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_coding.png]]<br>FliA<br />
|align=| FliA is a sigma factor involved into the regulation of very various genes, the promoter used in the FIFO were selected to be higly responding to FliA. In order to have the FliA gene possibly used as biobrick, we mutated 2 sites (EcoRI and PstI) inside the FliA sequence by assemby PCR. <br />
|align="center"| [[Team:Paris/September 04|September 04]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" |[[Image:Icon_coding.png]]<br>FlhDC<br />
|align=| FlhDC is the main regulator of the flagella genetic system in E. coli. FlhDC regulates all the promoter of the FIFO.<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|}<br />
<br />
{{Paris/Navig|Team:Paris/Construction}}</div>David.bikardhttp://2008.igem.org/Team:Paris/ConstructionTeam:Paris/Construction2008-10-30T04:06:55Z<p>David.bikard: </p>
<hr />
<div>{{Paris/Menu}}<br />
'''Have a look at our [[Team:Paris/Notebook|notebook]].'''<br />
<br />
=Model constructions: from the modelling to the characterization=<br />
<br />
Our project BacterioClock is based on an oscillating FIFO synchronized at the population level. To obtain and have preliminary results of this system we divided it in three "little" modules as the modeling team exposed them previously.<br />
==The First is to demonstrate the [[Team:Paris/Analysis#FIFO_behaviour|FIFO]] which constitutes our core system==<br />
<br />
A simple manner is to implement two class II promotor followed by fluorescent reporter genes like GFP in a bacteria strain that is able to produce the flagella. <br />
<br />
By this way, we are able to see in single cell the order of the activation of pFliL then pFlgA then pFlhB and then the inactivation in the same order as a FIFO will do.<br />
<br />
These constructions are equivalent to the experiments realized by Uri Alon in the article "Using quantitative Blueprint to reprogram the Dynamics of the Flagella Gene Network" Kalir S, Alon U. Cell 2004.<br />
<br />
A better way to observe the FIFO is to study the system with an inducible regulator of class II gene in a bacteria strain deleted for this gene. We are extremely grateful for Alon U. that send us the inducible gene ''FlhDC'', and ''FliA'' in pBad18 plasmid.<br />
These plasmids with our own araC/pBad-EnvZ* will allowed us to study the system in mutated strains that we have in our lab (ΔFlhD, ΔFliA, ΔFlgM, ΔEnvZ). By cotransforming in mutated strains the inducibles regulators of the class II promotors with one of this promotor associated to a fluorescent protein, we could characterize the influence of the master regulators of the flagella on their pormoter. The fluorescence is normalized to the OD<sub>600</sub>.<br />
<br>For example below are some constructions to see and induce the FIFO:<br />
<br><br><br><br />
[[Image:Felipe01.jpg|500px|center|thumb|pFliL alone]]<br><br><br><br />
[[Image:Felipe02.jpg|500px|center|thumb|3 promoters of the FIFO in one Cell]]<br><br><br><br />
[[Image:Felipe03.jpg|500px|center|thumb|FIFO and oscillating constructions in a single cell]]<br><br><br><br />
<br />
<br />
To perform such a cotransformation we take care about the ORI of each low copy plasmid which are often incompatible and we made all our final constructions in the pSB4T5 plasmid that care the only one resistance that was not already used and the pSC101 ORI which is compatible with the ColE1.<br />
<br />
We could see the FIFO in a more convenient manner with the assembly of two class II promotors associated to differents fluorophores. Then we could observed two kinds of fluorescents in One Cell along time. <br />
One problem that could occur is the interaction between the differents fluorescents proteins due to the cross-over of their emission and excitation spectra and moreover in our final system that count three fluorophores.<br />
<br />
[[Image:Spectre.jpg |600px |thumb| emission and excitation spectra of ECFP, YFP and mRFP]]<br />
<br />
<br>The choice of ECFP, YFP and mRFP is based upon this consideration and upon the order of appearing in the FIFO.<br />
Indeed by putting the mRFP in last position, we improve the visualization of the FIFO like the maturation time of the protein is about of one hour.<br />
<br />
We thank a lot Arnau Montagu, advisor of the iGEM Valencia Team. He advised us, after his team had troubleshooting with fluorescence, to take care about ''spurious FRET''. This phenomenon occurs even with non covered fluorescent spectra when the concentration of two fluorophores is sufficient so they are enough closer to induce some FRET. But it could happen at lower concentration than 40 μM. <br><br />
<br>- "DsRed as a Potential FRET Partner with CFP and GFP" Michael G. Erickson, Daniel L. Moon, and David T. Yue Biophysical Journal 2003. <br />
<br>- "A Comparison of Donor-Acceptor Pairs for Genetically Encoded FRET Sensors: Application to the Epac cAMP<br />
Sensor as an Example" Gerard N. M. van der Krogt, Janneke Ogink, Bas Ponsioen, Kees Jalink PLoS 2008.<br />
<br />
<br><br />
<br />
==[[Team:Paris/Analysis#Oscillations|Oscillating]] module==<br />
Few approaches have been thought in order to create the oscillating module. The pTet/TetR system that allows us to precisely control the inhibition of TetR on the Tet promotor. Another system use the natural inhibition of OmpR and of EnvZ via OmpR. We finally perform this system that we have developed since we know thanks to the modeling team that the oscillations are the only way to see the full development of the FIFO.<br />
<br />
==Synchronization module==<br />
The modeling simulation showed us that to have some oscillations that are difficult to obtain in a simple feedforward loop without the delay introduce by the quorum sensing synchronization, and for us it is more interesting to have the FIFO behavior at the population level. We did not achieved the this module but we started the construction of it and have already some intermediate parts that you will find below. <br />
The constructions we were able to obtain allow us to see the pictures below of the fluorescence of one of the promoter of the FIFO Flagella:<br />
<br />
== Table of the constructions we realized==<br />
{|<br />
|- style="background: #649cd7;"<br />
!colspan=4 style="background: #649cd7" | FIFO<br />
|- style="background: #649cd7; text-align: center;"<br />
|width=30%| Construction<br />
|width=55%| Description<br />
|width=15%| Validation Date<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136039 pFliL(RBS WT)-ECFP tripart (LVA+)]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFliL is ECFP fused with a LVA tag.<br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>[http://partsregistry.org/Part:BBa_K136037 pFliL(+ RBS WT)-GFP]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFliL is GFP.<br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136012 pFlgA(+ RBS WT) - YFP Tripart (LVA+)]<br />
|align=|Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is EYFP fused with a LVA tag. <br />
|align="center"| [[Team:Paris/August 22|August 22]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136013 pFlgA(+ RBS WT) - YFP Tripart (LVA-)]<br />
|align=|Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is EYFP fused without a LVA tag. <br />
|align="center"| [[Team:Paris/August 22|August 22]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136054 pFlgA(+ RBS WT)-YFP tripart (AAV+)]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is EYFP fused with a AAV tag. <br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>[http://partsregistry.org/Part:BBa_K136053 pFlgA(+ RBS WT)-GFP]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is GFP. <br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136055 pFlhB(+ RBS WT)-mRFP tripart (+LVA)]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlhB is mRFP with a LVA tag. <br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>[http://partsregistry.org/Part:BBa_K136065 pFlhB(+ RBS WT)-GFP]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlhB is GFP. <br />
|align="center"| [[Team:Paris/October 26|October 26]]<br />
|-<br />
|colspan=4 style="background: #649cd7" | <center>'''OSCILLATION'''</center><br />
|-<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136064 pFliL(+ RBS WT)-EnvZ*]<br />
|align=| pFliL is regulated by FlhDC and EnvZ regulates pFlhDC thanks to a negative feedback loop, origin of an oscillation behavior.<br />
|align="center"| [[Team:Paris/October 26|October 26]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136069 pFliL(+ RBS WT)-EnvZ*-GFP generator]<br />
|align=| The same construction as before but with a GFP in order to follow the dynamic of expression of pFliL.<br />
|align="center"| [[Team:Paris/October 29|October 29]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136051 pFlhB(+ RBS WT)-EnvZ*]<br />
|align=| pFlhB is regulated by FlhDC and EnvZ regulates pFlhDC thanks to a negative feedback loop, origin of an oscillation behavior.<br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136070 pFlhB(+ RBS WT)-EnvZ*-GFP generator]<br />
|align=| The same construction as before but with a GFP in order to follow the dynamic of expression of pFlhB.<br />
|align="center"| [[Team:Paris/October 29|October 29]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136066 araC/pBad-EnvZ*]<br />
|align=| A construction made for characterizing the repression power of the mutated EnvZ on our system<br />
|align="center"| [[Team:Paris/October 27|October 27]]<br />
|-<br />
|colspan=4 style="background: #649cd7" | <center>'''SYNCHRONISATION'''</center><br />
|-<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136029 pLas - gfp Tripart]<br />
|align=| Construction for characterization of the pLas promoter<br />
|align="center"| [[Team:Paris/August 1|August 1]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136027 pLas - ECFP]<br />
|align=| The same as before but with another fluorophore<br />
|align="center"| [[Team:Paris/August 1|August 1]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br> [http://partsregistry.org/Part:BBa_K136024 strongest promoter-rbs-lasR-dble ter]<br />
|align=| A construction for a strong constitutive expression of the Las Regulator in order to get maximal expression in presence of AHL<br />
|align="center"| [[Team:Paris/August 20|August 20]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136023 Strong promoter-rbs-lasR-dble ter]<br />
|align=| A derived construction of the previous one for smaller constitutive expression of LasR in the Bacteria.<br />
|align="center"| [[Team:Paris/August 20|August 20]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136044 Strongest RBS (1)- LasR activator (+LVA)]<br />
|align=| Intermediary construction. We uses different rbs for Las R in order to get various response dynamic to the presence of AHL<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136048 rbs-lasR + dbl ter]<br />
|align=| Intermediary construction. We uses different rbs for Las R in order to get various response dynamic to the presence of AHL<br />
|align="center"| [[Team:Paris/August 19|August 19]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136052 lasR activator + LVA - Double Terminator]<br />
|align=| Intermediary construction<br />
|align="center"| [[Team:Paris/August 1|August 1]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136057 RBS lasI - GFP tripart]<br />
|align=| Construction that has to be associated with the FIFO. We will know when LasI is expressed and then when AHL is produced.<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br> [http://partsregistry.org/Part:BBa_K136056 RBS-lasI - ECFP]<br />
|align=| The same as the previous one but with another fluorophore.<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|-<br />
|colspan=4 style="background: #649cd7" | <center>'''NEW GENE AND PROMOTER BIOBRICKS'''</center><br />
|-<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136010 pFliA]<br />
|align=| Natural promoter of E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC.<br />
|align="center"| [[Team:Paris/October 04|October 04]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136009 pFliL (+RBS WT)]<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA.<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136006 pFlgA (+RBS WT)]<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA..<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br> pFlgB (+RBS WT)<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA.<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136008 pFlhB (+RBS WT)]<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA.<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136046 EnvZ*]<br />
|align=| EnvZ is a membrane protein involved into the osmotic response and is interacting with OmpR that will bind the promoter of FlhDC ad will repress it. EnvZ* is a mutated form of the coding sequence, giving a phenotype where pFlhDC is completely repressed. EnvZ has been extracted by PVR for the correct mutant strain.<br />
|align="center"| [[Team:Paris/September 04|September 04]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_coding.png]]<br>FliA<br />
|align=| FliA is a sigma factor involved into the regulation of very various genes, the promoter used in the FIFO were selected to be higly responding to FliA. In order to have the FliA gene possibly used as biobrick, we mutated 2 sites (EcoRI and PstI) inside the FliA sequence by assemby PCR. <br />
|align="center"| [[Team:Paris/September 04|September 04]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" |[[Image:Icon_coding.png]]<br>FlhDC<br />
|align=| FlhDC is the main regulator of the flagella genetic system in E. coli. FlhDC regulates all the promoter of the FIFO.<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|}<br />
<br />
{{Paris/Navig|Team:Paris/Construction}}</div>David.bikardhttp://2008.igem.org/Team:Paris/ConstructionTeam:Paris/Construction2008-10-30T04:06:28Z<p>David.bikard: </p>
<hr />
<div>{{Paris/Menu}}<br />
Have a look at our [[Team:Paris/Notebook|notebook]].<br />
<br />
=Model constructions: from the modelling to the characterization=<br />
<br />
Our project BacterioClock is based on an oscillating FIFO synchronized at the population level. To obtain and have preliminary results of this system we divided it in three "little" modules as the modeling team exposed them previously.<br />
==The First is to demonstrate the [[Team:Paris/Analysis#FIFO_behaviour|FIFO]] which constitutes our core system==<br />
<br />
A simple manner is to implement two class II promotor followed by fluorescent reporter genes like GFP in a bacteria strain that is able to produce the flagella. <br />
<br />
By this way, we are able to see in single cell the order of the activation of pFliL then pFlgA then pFlhB and then the inactivation in the same order as a FIFO will do.<br />
<br />
These constructions are equivalent to the experiments realized by Uri Alon in the article "Using quantitative Blueprint to reprogram the Dynamics of the Flagella Gene Network" Kalir S, Alon U. Cell 2004.<br />
<br />
A better way to observe the FIFO is to study the system with an inducible regulator of class II gene in a bacteria strain deleted for this gene. We are extremely grateful for Alon U. that send us the inducible gene ''FlhDC'', and ''FliA'' in pBad18 plasmid.<br />
These plasmids with our own araC/pBad-EnvZ* will allowed us to study the system in mutated strains that we have in our lab (ΔFlhD, ΔFliA, ΔFlgM, ΔEnvZ). By cotransforming in mutated strains the inducibles regulators of the class II promotors with one of this promotor associated to a fluorescent protein, we could characterize the influence of the master regulators of the flagella on their pormoter. The fluorescence is normalized to the OD<sub>600</sub>.<br />
<br>For example below are some constructions to see and induce the FIFO:<br />
<br><br><br><br />
[[Image:Felipe01.jpg|500px|center|thumb|pFliL alone]]<br><br><br><br />
[[Image:Felipe02.jpg|500px|center|thumb|3 promoters of the FIFO in one Cell]]<br><br><br><br />
[[Image:Felipe03.jpg|500px|center|thumb|FIFO and oscillating constructions in a single cell]]<br><br><br><br />
<br />
<br />
To perform such a cotransformation we take care about the ORI of each low copy plasmid which are often incompatible and we made all our final constructions in the pSB4T5 plasmid that care the only one resistance that was not already used and the pSC101 ORI which is compatible with the ColE1.<br />
<br />
We could see the FIFO in a more convenient manner with the assembly of two class II promotors associated to differents fluorophores. Then we could observed two kinds of fluorescents in One Cell along time. <br />
One problem that could occur is the interaction between the differents fluorescents proteins due to the cross-over of their emission and excitation spectra and moreover in our final system that count three fluorophores.<br />
<br />
[[Image:Spectre.jpg |600px |thumb| emission and excitation spectra of ECFP, YFP and mRFP]]<br />
<br />
<br>The choice of ECFP, YFP and mRFP is based upon this consideration and upon the order of appearing in the FIFO.<br />
Indeed by putting the mRFP in last position, we improve the visualization of the FIFO like the maturation time of the protein is about of one hour.<br />
<br />
We thank a lot Arnau Montagu, advisor of the iGEM Valencia Team. He advised us, after his team had troubleshooting with fluorescence, to take care about ''spurious FRET''. This phenomenon occurs even with non covered fluorescent spectra when the concentration of two fluorophores is sufficient so they are enough closer to induce some FRET. But it could happen at lower concentration than 40 μM. <br><br />
<br>- "DsRed as a Potential FRET Partner with CFP and GFP" Michael G. Erickson, Daniel L. Moon, and David T. Yue Biophysical Journal 2003. <br />
<br>- "A Comparison of Donor-Acceptor Pairs for Genetically Encoded FRET Sensors: Application to the Epac cAMP<br />
Sensor as an Example" Gerard N. M. van der Krogt, Janneke Ogink, Bas Ponsioen, Kees Jalink PLoS 2008.<br />
<br />
<br><br />
<br />
==[[Team:Paris/Analysis#Oscillations|Oscillating]] module==<br />
Few approaches have been thought in order to create the oscillating module. The pTet/TetR system that allows us to precisely control the inhibition of TetR on the Tet promotor. Another system use the natural inhibition of OmpR and of EnvZ via OmpR. We finally perform this system that we have developed since we know thanks to the modeling team that the oscillations are the only way to see the full development of the FIFO.<br />
<br />
==Synchronization module==<br />
The modeling simulation showed us that to have some oscillations that are difficult to obtain in a simple feedforward loop without the delay introduce by the quorum sensing synchronization, and for us it is more interesting to have the FIFO behavior at the population level. We did not achieved the this module but we started the construction of it and have already some intermediate parts that you will find below. <br />
The constructions we were able to obtain allow us to see the pictures below of the fluorescence of one of the promoter of the FIFO Flagella:<br />
<br />
== Table of the constructions we realized==<br />
{|<br />
|- style="background: #649cd7;"<br />
!colspan=4 style="background: #649cd7" | FIFO<br />
|- style="background: #649cd7; text-align: center;"<br />
|width=30%| Construction<br />
|width=55%| Description<br />
|width=15%| Validation Date<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136039 pFliL(RBS WT)-ECFP tripart (LVA+)]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFliL is ECFP fused with a LVA tag.<br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>[http://partsregistry.org/Part:BBa_K136037 pFliL(+ RBS WT)-GFP]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFliL is GFP.<br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136012 pFlgA(+ RBS WT) - YFP Tripart (LVA+)]<br />
|align=|Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is EYFP fused with a LVA tag. <br />
|align="center"| [[Team:Paris/August 22|August 22]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136013 pFlgA(+ RBS WT) - YFP Tripart (LVA-)]<br />
|align=|Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is EYFP fused without a LVA tag. <br />
|align="center"| [[Team:Paris/August 22|August 22]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136054 pFlgA(+ RBS WT)-YFP tripart (AAV+)]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is EYFP fused with a AAV tag. <br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>[http://partsregistry.org/Part:BBa_K136053 pFlgA(+ RBS WT)-GFP]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is GFP. <br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136055 pFlhB(+ RBS WT)-mRFP tripart (+LVA)]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlhB is mRFP with a LVA tag. <br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>[http://partsregistry.org/Part:BBa_K136065 pFlhB(+ RBS WT)-GFP]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlhB is GFP. <br />
|align="center"| [[Team:Paris/October 26|October 26]]<br />
|-<br />
|colspan=4 style="background: #649cd7" | <center>'''OSCILLATION'''</center><br />
|-<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136064 pFliL(+ RBS WT)-EnvZ*]<br />
|align=| pFliL is regulated by FlhDC and EnvZ regulates pFlhDC thanks to a negative feedback loop, origin of an oscillation behavior.<br />
|align="center"| [[Team:Paris/October 26|October 26]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136069 pFliL(+ RBS WT)-EnvZ*-GFP generator]<br />
|align=| The same construction as before but with a GFP in order to follow the dynamic of expression of pFliL.<br />
|align="center"| [[Team:Paris/October 29|October 29]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136051 pFlhB(+ RBS WT)-EnvZ*]<br />
|align=| pFlhB is regulated by FlhDC and EnvZ regulates pFlhDC thanks to a negative feedback loop, origin of an oscillation behavior.<br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136070 pFlhB(+ RBS WT)-EnvZ*-GFP generator]<br />
|align=| The same construction as before but with a GFP in order to follow the dynamic of expression of pFlhB.<br />
|align="center"| [[Team:Paris/October 29|October 29]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136066 araC/pBad-EnvZ*]<br />
|align=| A construction made for characterizing the repression power of the mutated EnvZ on our system<br />
|align="center"| [[Team:Paris/October 27|October 27]]<br />
|-<br />
|colspan=4 style="background: #649cd7" | <center>'''SYNCHRONISATION'''</center><br />
|-<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136029 pLas - gfp Tripart]<br />
|align=| Construction for characterization of the pLas promoter<br />
|align="center"| [[Team:Paris/August 1|August 1]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136027 pLas - ECFP]<br />
|align=| The same as before but with another fluorophore<br />
|align="center"| [[Team:Paris/August 1|August 1]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br> [http://partsregistry.org/Part:BBa_K136024 strongest promoter-rbs-lasR-dble ter]<br />
|align=| A construction for a strong constitutive expression of the Las Regulator in order to get maximal expression in presence of AHL<br />
|align="center"| [[Team:Paris/August 20|August 20]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136023 Strong promoter-rbs-lasR-dble ter]<br />
|align=| A derived construction of the previous one for smaller constitutive expression of LasR in the Bacteria.<br />
|align="center"| [[Team:Paris/August 20|August 20]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136044 Strongest RBS (1)- LasR activator (+LVA)]<br />
|align=| Intermediary construction. We uses different rbs for Las R in order to get various response dynamic to the presence of AHL<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136048 rbs-lasR + dbl ter]<br />
|align=| Intermediary construction. We uses different rbs for Las R in order to get various response dynamic to the presence of AHL<br />
|align="center"| [[Team:Paris/August 19|August 19]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136052 lasR activator + LVA - Double Terminator]<br />
|align=| Intermediary construction<br />
|align="center"| [[Team:Paris/August 1|August 1]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136057 RBS lasI - GFP tripart]<br />
|align=| Construction that has to be associated with the FIFO. We will know when LasI is expressed and then when AHL is produced.<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br> [http://partsregistry.org/Part:BBa_K136056 RBS-lasI - ECFP]<br />
|align=| The same as the previous one but with another fluorophore.<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|-<br />
|colspan=4 style="background: #649cd7" | <center>'''NEW GENE AND PROMOTER BIOBRICKS'''</center><br />
|-<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136010 pFliA]<br />
|align=| Natural promoter of E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC.<br />
|align="center"| [[Team:Paris/October 04|October 04]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136009 pFliL (+RBS WT)]<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA.<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136006 pFlgA (+RBS WT)]<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA..<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br> pFlgB (+RBS WT)<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA.<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136008 pFlhB (+RBS WT)]<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA.<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136046 EnvZ*]<br />
|align=| EnvZ is a membrane protein involved into the osmotic response and is interacting with OmpR that will bind the promoter of FlhDC ad will repress it. EnvZ* is a mutated form of the coding sequence, giving a phenotype where pFlhDC is completely repressed. EnvZ has been extracted by PVR for the correct mutant strain.<br />
|align="center"| [[Team:Paris/September 04|September 04]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_coding.png]]<br>FliA<br />
|align=| FliA is a sigma factor involved into the regulation of very various genes, the promoter used in the FIFO were selected to be higly responding to FliA. In order to have the FliA gene possibly used as biobrick, we mutated 2 sites (EcoRI and PstI) inside the FliA sequence by assemby PCR. <br />
|align="center"| [[Team:Paris/September 04|September 04]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" |[[Image:Icon_coding.png]]<br>FlhDC<br />
|align=| FlhDC is the main regulator of the flagella genetic system in E. coli. FlhDC regulates all the promoter of the FIFO.<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|}<br />
<br />
{{Paris/Navig|Team:Paris/Construction}}</div>David.bikardhttp://2008.igem.org/Team:Paris/CharacterizationTeam:Paris/Characterization2008-10-30T04:06:17Z<p>David.bikard: </p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
{{Paris/Header|Characterization Approach : Presentation}}<br />
{{Paris/Section_contents_characterization}}<br />
<br />
<br />
In the [[Team:Paris/Analysis|Analysis]] section, we developed a model based on available experimental data and extended previously published models.<br />
This model has proven useful for the initial design stage, in the sense that it has suggested ways to improve the oscillatory behavior. <br />
<br />
However, it relies on parameters collected from various sources and obtained under different conditions. So, this model based on bibliography has probably limited predictive capabilities.<br />
The challenge we address here is to obtain a '''predictive model of our system'''.<br />
Such a model would be a unique tool to tune and optimize the behavior of our system.<br />
<br />
This model is constructed using a '''bottom-up approach'''. Each part is experimentally characterized using specific constructs. <br />
Key parameters are then obtained by fitting data to simple models of each part. These models should capture key features of gene regulation and must only involve experimentally measurable parameters.<br />
We consequently used '''Hill-type-like ODE models'''. However, to establish these models, we started from molecular reactions to obtain our non-linear model so as to explicit underlying assumptions and possible limitations.<br />
The overall model is then obtained by using a compositionality assumption: the model of the full system is obtained using models of each parts.<br />
<br />
It is important to note that we do not follow the Standard Promoter Unit approach in which each part is characterized in normallized conditions to improve reusability of the characteisations across different labs.<br />
In contrast, our characterisations are made in conditions that are as close as possible to the experimental conditions in which our system is supposed to work (same strain, same growth conditions, same plasmid...).<br />
We stress that the goal in our case is to '''improve predictability of the resulting model'''<br />
<br />
This work essentially amounts to quantifying promoter activities as a function of its transcription factors.<br />
Because many promoters need to be characterized, we '''designed a workflow that allows to carry out constructs, experiments and parameter estimation in a rational approach'''.<br />
<br />
<center><br />
*[[Team:Paris/Modeling/Workflow_Example| The whole approach is illustrated with a simple example> ]]<br />
</center>.<br />
<br />
{{Paris/Navig|Team:Paris/Modeling/Workflow_Example}}</div>David.bikardhttp://2008.igem.org/Team:Paris/CharacterizationTeam:Paris/Characterization2008-10-30T04:05:33Z<p>David.bikard: </p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
{{Paris/Header|Characterization Approach : Presentation}}<br />
{{Paris/Section_contents_characterization}}<br />
<br />
Have a look at our [[Team:Paris/Notebook|notebook]].<br />
<br />
In the [[Team:Paris/Analysis|Analysis]] section, we developed a model based on available experimental data and extended previously published models.<br />
This model has proven useful for the initial design stage, in the sense that it has suggested ways to improve the oscillatory behavior. <br />
<br />
However, it relies on parameters collected from various sources and obtained under different conditions. So, this model based on bibliography has probably limited predictive capabilities.<br />
The challenge we address here is to obtain a '''predictive model of our system'''.<br />
Such a model would be a unique tool to tune and optimize the behavior of our system.<br />
<br />
This model is constructed using a '''bottom-up approach'''. Each part is experimentally characterized using specific constructs. <br />
Key parameters are then obtained by fitting data to simple models of each part. These models should capture key features of gene regulation and must only involve experimentally measurable parameters.<br />
We consequently used '''Hill-type-like ODE models'''. However, to establish these models, we started from molecular reactions to obtain our non-linear model so as to explicit underlying assumptions and possible limitations.<br />
The overall model is then obtained by using a compositionality assumption: the model of the full system is obtained using models of each parts.<br />
<br />
It is important to note that we do not follow the Standard Promoter Unit approach in which each part is characterized in normallized conditions to improve reusability of the characteisations across different labs.<br />
In contrast, our characterisations are made in conditions that are as close as possible to the experimental conditions in which our system is supposed to work (same strain, same growth conditions, same plasmid...).<br />
We stress that the goal in our case is to '''improve predictability of the resulting model'''<br />
<br />
This work essentially amounts to quantifying promoter activities as a function of its transcription factors.<br />
Because many promoters need to be characterized, we '''designed a workflow that allows to carry out constructs, experiments and parameter estimation in a rational approach'''.<br />
<br />
<center><br />
*[[Team:Paris/Modeling/Workflow_Example| The whole approach is illustrated with a simple example> ]]<br />
</center>.<br />
<br />
{{Paris/Navig|Team:Paris/Modeling/Workflow_Example}}</div>David.bikardhttp://2008.igem.org/Team:Paris/First_pageTeam:Paris/First page2008-10-29T23:57:00Z<p>David.bikard: /* The BacterioClock */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br><br />
<center><html><div style="color:#275D96; font-size:2em;">Grosse GROSSE Pomme de Terre !</div></html></center><br />
<br><br />
<br />
= The BacterioClock = <br />
<br />
Were you ever dreaming about a new way of thinking everyday life objects ? Be sure .this will come true, under the form of our BacterioClock ! A simple tube which contains the modified bacteria we have created will give you the time, directly from living organisms.<br />
<center><br />
<html><br />
<embed width="426" height="320" src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=true&amp;repeat&amp;file=http://perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" allowfullscreen="true" /><br />
<br />
<br />
</html><br />
</center><br />
To achieve this incredible project, we have decided to rely on a genetic structure that allows a specific sequence of lighting (Firt In - First Out behaviour) to occur. The models designed have shown that simply adding a negative feedback from a lighting gene to the first activator will not yield oscillations ! Therefore, we have proposed to induce a necessary delay, through a synchronization process among the population, as represented below :<br />
[[Image:Image_qui_dechire_touuuut|center]]<br />
<br />
More precisely, the oscillations obtained will allow a precise control of the period of our clock. Moreover, thanks to the Biobricks proposed during the iGEM competition, one can imagine to have a self-tuning according to daylight for instance.<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. If you manage to wait for the updates, you might even get the version which automatically react to daylight saving time ;)</div>David.bikardhttp://2008.igem.org/Team:Paris/First_pageTeam:Paris/First page2008-10-29T23:54:52Z<p>David.bikard: /* The BacterioClock */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br><br />
<center><html><div style="color:#275D96; font-size:2em;">Grosse GROSSE Pomme de Terre !</div></html></center><br />
<br><br />
<br />
= The BacterioClock = <br />
<br />
Were you ever dreaming about a new way of thinking everyday life objects ? Be sure .this will come true, under the form of our BacterioClock ! A simple tube which contains the modified bacteria we have created will give you the time, directly from living organisms.<br />
<center><br />
<html><br />
<embed width="426" height="320" src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=false&amp;file=http://perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" allowfullscreen="true" /><br />
<br />
<br />
</html><br />
</center><br />
To achieve this incredible project, we have decided to rely on a genetic structure that allows a specific sequence of lighting (Firt In - First Out behaviour) to occur. The models designed have shown that simply adding a negative feedback from a lighting gene to the first activator will not yield oscillations ! Therefore, we have proposed to induce a necessary delay, through a synchronization process among the population, as represented below :<br />
[[Image:Image_qui_dechire_touuuut|center]]<br />
<br />
More precisely, the oscillations obtained will allow a precise control of the period of our clock. Moreover, thanks to the Biobricks proposed during the iGEM competition, one can imagine to have a self-tuning according to daylight for instance.<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. If you manage to wait for the updates, you might even get the version which automatically react to daylight saving time ;)</div>David.bikardhttp://2008.igem.org/Team:Paris/First_pageTeam:Paris/First page2008-10-29T23:54:01Z<p>David.bikard: /* The BacterioClock */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br><br />
<center><html><div style="color:#275D96; font-size:2em;">Grosse GROSSE Pomme de Terre !</div></html></center><br />
<br><br />
<br />
= The BacterioClock = <br />
<br />
Were you dreaming about a new way of thinking everyday life objects ? Be sure that this will come true, under the form of our BacterioClock ! A simple tube which contains the modified bacteria we have created will give you the time, directly coming from living organisms.<br />
<center><br />
<html><br />
<embed width="426" height="320" src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=false&amp;file=http://perso.strepsiade.org/~gvieira/out2.flv" mode="loop" quality="high" type="application/x-shockwave-flash" allowfullscreen="true" /><br />
<br />
<br />
</html><br />
</center><br />
To achieve this incredible project, we have decided to rely on a genetic structure that allows a specific sequence of lighting (Firt In - First Out behaviour) to occur. The models designed have shown that simply adding a negative feedback from a lighting gene to the first activator will not yield oscillations ! Therefore, we have proposed to induce a necessary delay, through a synchronization process among the population, as represented below :<br />
[[Image:Image_qui_dechire_touuuut|center]]<br />
<br />
More precisely, the oscillations obtained will allow a precise control of the period of our clock. Moreover, thanks to the Biobricks proposed during the iGEM competition, one can imagine to have a self-tuning according to daylight for instance.<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. If you manage to wait for the updates, you might even get the version which automatically react to daylight saving time ;)</div>David.bikardhttp://2008.igem.org/Team:Paris/First_pageTeam:Paris/First page2008-10-29T23:52:50Z<p>David.bikard: /* The BacterioClock */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br><br />
<center><html><div style="color:#275D96; font-size:2em;">Grosse GROSSE Pomme de Terre !</div></html></center><br />
<br><br />
<br />
= The BacterioClock = <br />
<br />
Were you dreaming about a new way of thinking everyday life objects ? Be sure that this will come true, under the form of our BacterioClock ! A simple tube which contains the modified bacteria we have created will give you the time, directly coming from living organisms.<br />
<center><br />
<html><br />
<embed width="426" height="320" src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=false&amp;file=http://perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" allowfullscreen="true" /><br />
<br />
<br />
</html><br />
</center><br />
To achieve this incredible project, we have decided to rely on a genetic structure that allows a specific sequence of lighting (Firt In - First Out behaviour) to occur. The models designed have shown that simply adding a negative feedback from a lighting gene to the first activator will not yield oscillations ! Therefore, we have proposed to induce a necessary delay, through a synchronization process among the population, as represented below :<br />
[[Image:Image_qui_dechire_touuuut|center]]<br />
<br />
More precisely, the oscillations obtained will allow a precise control of the period of our clock. Moreover, thanks to the Biobricks proposed during the iGEM competition, one can imagine to have a self-tuning according to daylight for instance.<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. If you manage to wait for the updates, you might even get the version which automatically react to daylight saving time ;)</div>David.bikardhttp://2008.igem.org/Team:Paris/First_pageTeam:Paris/First page2008-10-29T23:49:54Z<p>David.bikard: /* The BacterioClock */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br><br />
<center><html><div style="color:#275D96; font-size:2em;">Grosse GROSSE Pomme de Terre !</div></html></center><br />
<br><br />
<br />
= The BacterioClock = <br />
<br />
Were you dreaming about a new way of thinking everyday life objects ? Be sure that this will come true, under the form of our BacterioClock ! A simple tube which contains the modified bacteria we have created will give you the time, directly coming from living organisms.<br />
<center><br />
<html><br />
<embed width="426" height="320" <br />
src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=false&amp;file=http:/perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" <br />
allowfullscreen="true" /><br />
<br />
</html><br />
</center><br />
To achieve this incredible project, we have decided to rely on a genetic structure that allows a specific sequence of lighting (Firt In - First Out behaviour) to occur. The models designed have shown that simply adding a negative feedback from a lighting gene to the first activator will not yield oscillations ! Therefore, we have proposed to induce a necessary delay, through a synchronization process among the population, as represented below :<br />
[[Image:Image_qui_dechire_touuuut|center]]<br />
<br />
More precisely, the oscillations obtained will allow a precise control of the period of our clock. Moreover, thanks to the Biobricks proposed during the iGEM competition, one can imagine to have a self-tuning according to daylight for instance.<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. If you manage to wait for the updates, you might even get the version which automatically react to daylight saving time ;)</div>David.bikardhttp://2008.igem.org/Team:Paris/First_pageTeam:Paris/First page2008-10-29T23:49:24Z<p>David.bikard: /* The BacterioClock */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br><br />
<center><html><div style="color:#275D96; font-size:2em;">Grosse GROSSE Pomme de Terre !</div></html></center><br />
<br><br />
<br />
= The BacterioClock = <br />
<br />
Were you dreaming about a new way of thinking everyday life objects ? Be sure that this will come true, under the form of our BacterioClock ! A simple tube which contains the modified bacteria we have created will give you the time, directly coming from living organisms.<br />
<center><br />
<html><br />
<embed width="426" height="320" <br />
image=http://perso.strepsiade.org/~gvieira/iGEM/groupe/photo_003b.JPG&amp;<br />
src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=false&amp;file=http:/perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" <br />
allowfullscreen="true" /><br />
<br />
</html><br />
</center><br />
To achieve this incredible project, we have decided to rely on a genetic structure that allows a specific sequence of lighting (Firt In - First Out behaviour) to occur. The models designed have shown that simply adding a negative feedback from a lighting gene to the first activator will not yield oscillations ! Therefore, we have proposed to induce a necessary delay, through a synchronization process among the population, as represented below :<br />
[[Image:Image_qui_dechire_touuuut|center]]<br />
<br />
More precisely, the oscillations obtained will allow a precise control of the period of our clock. Moreover, thanks to the Biobricks proposed during the iGEM competition, one can imagine to have a self-tuning according to daylight for instance.<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. If you manage to wait for the updates, you might even get the version which automatically react to daylight saving time ;)</div>David.bikardhttp://2008.igem.org/Team:Paris/First_pageTeam:Paris/First page2008-10-29T23:49:03Z<p>David.bikard: /* The BacterioClock */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br><br />
<center><html><div style="color:#275D96; font-size:2em;">Grosse GROSSE Pomme de Terre !</div></html></center><br />
<br><br />
<br />
= The BacterioClock = <br />
<br />
Were you dreaming about a new way of thinking everyday life objects ? Be sure that this will come true, under the form of our BacterioClock ! A simple tube which contains the modified bacteria we have created will give you the time, directly coming from living organisms.<br />
<center><br />
<html><br />
<embed width="426" height="320" <br />
src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=false&amp;file=http:/perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" <br />
allowfullscreen="true" /><br />
image=http://perso.strepsiade.org/~gvieira/iGEM/groupe/photo_003b.JPG&amp;<br />
</html><br />
</center><br />
To achieve this incredible project, we have decided to rely on a genetic structure that allows a specific sequence of lighting (Firt In - First Out behaviour) to occur. The models designed have shown that simply adding a negative feedback from a lighting gene to the first activator will not yield oscillations ! Therefore, we have proposed to induce a necessary delay, through a synchronization process among the population, as represented below :<br />
[[Image:Image_qui_dechire_touuuut|center]]<br />
<br />
More precisely, the oscillations obtained will allow a precise control of the period of our clock. Moreover, thanks to the Biobricks proposed during the iGEM competition, one can imagine to have a self-tuning according to daylight for instance.<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. If you manage to wait for the updates, you might even get the version which automatically react to daylight saving time ;)</div>David.bikardhttp://2008.igem.org/Team:Paris/First_pageTeam:Paris/First page2008-10-29T23:48:26Z<p>David.bikard: /* The BacterioClock */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br><br />
<center><html><div style="color:#275D96; font-size:2em;">Grosse GROSSE Pomme de Terre !</div></html></center><br />
<br><br />
<br />
= The BacterioClock = <br />
<br />
Were you dreaming about a new way of thinking everyday life objects ? Be sure that this will come true, under the form of our BacterioClock ! A simple tube which contains the modified bacteria we have created will give you the time, directly coming from living organisms.<br />
<center><br />
<html><br />
<embed width="426" height="320" <br />
<br />
src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=false&amp;file=http:/perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" <br />
<br />
allowfullscreen="true" /><br />
</html><br />
</center><br />
To achieve this incredible project, we have decided to rely on a genetic structure that allows a specific sequence of lighting (Firt In - First Out behaviour) to occur. The models designed have shown that simply adding a negative feedback from a lighting gene to the first activator will not yield oscillations ! Therefore, we have proposed to induce a necessary delay, through a synchronization process among the population, as represented below :<br />
[[Image:Image_qui_dechire_touuuut|center]]<br />
<br />
More precisely, the oscillations obtained will allow a precise control of the period of our clock. Moreover, thanks to the Biobricks proposed during the iGEM competition, one can imagine to have a self-tuning according to daylight for instance.<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. If you manage to wait for the updates, you might even get the version which automatically react to daylight saving time ;)</div>David.bikardhttp://2008.igem.org/Team:Paris/First_pageTeam:Paris/First page2008-10-29T23:47:45Z<p>David.bikard: /* The BacterioClock */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br><br />
<center><html><div style="color:#275D96; font-size:2em;">Grosse GROSSE Pomme de Terre !</div></html></center><br />
<br><br />
<br />
= The BacterioClock = <br />
<br />
Were you dreaming about a new way of thinking everyday life objects ? Be sure that this will come true, under the form of our BacterioClock ! A simple tube which contains the modified bacteria we have created will give you the time, directly coming from living organisms.<br />
<center><br />
[http://www.igem.org Video_de_ouf_!]<br />
<html><br />
<embed width="426" height="320" <br />
<br />
src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=false&amp;file=http:/perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" <br />
<br />
allowfullscreen="true" /><br />
</html><br />
</center><br />
To achieve this incredible project, we have decided to rely on a genetic structure that allows a specific sequence of lighting (Firt In - First Out behaviour) to occur. The models designed have shown that simply adding a negative feedback from a lighting gene to the first activator will not yield oscillations ! Therefore, we have proposed to induce a necessary delay, through a synchronization process among the population, as represented below :<br />
[[Image:Image_qui_dechire_touuuut|center]]<br />
<br />
More precisely, the oscillations obtained will allow a precise control of the period of our clock. Moreover, thanks to the Biobricks proposed during the iGEM competition, one can imagine to have a self-tuning according to daylight for instance.<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. If you manage to wait for the updates, you might even get the version which automatically react to daylight saving time ;)</div>David.bikardhttp://2008.igem.org/Team:Paris/First_pageTeam:Paris/First page2008-10-29T23:47:29Z<p>David.bikard: /* The BacterioClock */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br><br />
<center><html><div style="color:#275D96; font-size:2em;">Grosse GROSSE Pomme de Terre !</div></html></center><br />
<br><br />
<br />
= The BacterioClock = <br />
<br />
Were you dreaming about a new way of thinking everyday life objects ? Be sure that this will come true, under the form of our BacterioClock ! A simple tube which contains the modified bacteria we have created will give you the time, directly coming from living organisms.<br />
<center><br />
[http://www.igem.org Video_de_ouf_!]<br />
<embed width="426" height="320" <br />
<br />
src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=false&amp;file=http:/perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" <br />
<br />
allowfullscreen="true" /><br />
<br />
</center><br />
To achieve this incredible project, we have decided to rely on a genetic structure that allows a specific sequence of lighting (Firt In - First Out behaviour) to occur. The models designed have shown that simply adding a negative feedback from a lighting gene to the first activator will not yield oscillations ! Therefore, we have proposed to induce a necessary delay, through a synchronization process among the population, as represented below :<br />
[[Image:Image_qui_dechire_touuuut|center]]<br />
<br />
More precisely, the oscillations obtained will allow a precise control of the period of our clock. Moreover, thanks to the Biobricks proposed during the iGEM competition, one can imagine to have a self-tuning according to daylight for instance.<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. If you manage to wait for the updates, you might even get the version which automatically react to daylight saving time ;)</div>David.bikardhttp://2008.igem.org/Team:Paris/First_pageTeam:Paris/First page2008-10-29T23:46:48Z<p>David.bikard: /* The BacterioClock */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br><br />
<center><html><div style="color:#275D96; font-size:2em;">Grosse GROSSE Pomme de Terre !</div></html></center><br />
<br><br />
<br />
= The BacterioClock = <br />
<br />
Were you dreaming about a new way of thinking everyday life objects ? Be sure that this will come true, under the form of our BacterioClock ! A simple tube which contains the modified bacteria we have created will give you the time, directly coming from living organisms.<br />
<center><br />
[http://www.igem.org Video_de_ouf_!]<br />
<embed width="426" height="320" <br />
<br />
src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=false&amp;file=http<br />
<br />
://perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" <br />
<br />
allowfullscreen="true" /><br />
<br />
</center><br />
To achieve this incredible project, we have decided to rely on a genetic structure that allows a specific sequence of lighting (Firt In - First Out behaviour) to occur. The models designed have shown that simply adding a negative feedback from a lighting gene to the first activator will not yield oscillations ! Therefore, we have proposed to induce a necessary delay, through a synchronization process among the population, as represented below :<br />
[[Image:Image_qui_dechire_touuuut|center]]<br />
<br />
More precisely, the oscillations obtained will allow a precise control of the period of our clock. Moreover, thanks to the Biobricks proposed during the iGEM competition, one can imagine to have a self-tuning according to daylight for instance.<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. If you manage to wait for the updates, you might even get the version which automatically react to daylight saving time ;)</div>David.bikardhttp://2008.igem.org/Team:Paris/Modeling/Workflow_ExampleTeam:Paris/Modeling/Workflow Example2008-10-29T23:41:17Z<p>David.bikard: </p>
<hr />
<div>{{Paris/Menu}}<br />
{{Paris/Header|Characterization Approach : Generic Workflow on an Example}}<br />
<br />
Here, we will explain the generic protocol that leads to the modelisation "by characterization", of one of the parts of our system. The choosen example is '''the activity of the promoter ''pFliL'' ''' (that leads to the production of the '''Fluorescent Protein FP1''') '''in function of the transcription factors ''FlhDC'' and ''FliA'' '''.<br />
We know that ''the hexamere FlhD<sub>4</sub>C<sub>2</sub>'' and the protein ''FliA'' act both on the promoter ''pFliL'' as inducers... how do we evaluate this phenomenon? <br><br />
As it is illustrated below; starting from ''molecular reactions'' '''(1)''', we develop a ''model of our system'' '''(2)''', and then we identify the ''parameters'' '''(3)''' that needs to be estimated. Essentially, it amounts to estimate ''Promoters Activities as functions of their Transcription Factors''. To do so, we propose ''constructs'' '''(4)''', ''protocols'' '''(5)''' and ''programs'' '''(6)'''.<br />
<br><br />
<center><span style="color: red; font-weight: bold; font-size: 16">CLICK on the images below for further informations on each step!</span></center> <br />
<br><br />
<center><br />
{|border="0" style="text-align: center" cellspacing="-1"<br />
|'''Modelisation'''<br />
|'''Experiments'''<br />
|-<br />
|<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Molecular_Reactions"><img src="https://static.igem.org/mediawiki/2008/e/e3/Molecular_Reactions.jpg" width=480></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/FromMolReactToNLOde"><img src="https://static.igem.org/mediawiki/2008/b/b1/HypothesisModel.jpg" width=480></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/FromMolReactToNLOde#Mathematical_Interpretation_and_Simulation_of_the_Molecular_Reactions"><img src="https://static.igem.org/mediawiki/2008/8/85/TemporalVariation.jpg" width=480></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/FromMolReactToNLOde#Mathematical_Interpretation_and_Simulation_of_the_Molecular_Reactions"><img src="https://static.igem.org/mediawiki/2008/4/40/RQSS.jpg" width=240></a></html><html><a href = "https://2008.igem.org/Team:Paris/Modeling/Implementation#Parameters_Finder_Programs"><img src="https://static.igem.org/mediawiki/2008/b/b4/Programs.jpg" width=240></a></html> <br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Implementation#All_Algorithm"><img src="https://static.igem.org/mediawiki/2008/3/31/Goal.jpg" width=480></a></html><br />
|<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Protocol_Of_Characterization"><img src="https://static.igem.org/mediawiki/2008/f/fd/Principles.jpg" width=410></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Protocol_Of_Characterization"><img src="https://static.igem.org/mediawiki/2008/5/52/Plasmid.jpg" width=410></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Protocol_Of_Characterization"><img src="https://static.igem.org/mediawiki/2008/7/78/HypothesisCharact.jpg" width=410></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Implementation#Parameters_Finder_Programs"><img src="https://static.igem.org/mediawiki/2008/7/78/Graph.jpg" width=410></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Implementation#All_Algorithms"><img src="https://static.igem.org/mediawiki/2008/8/83/FullSystem.jpg" width=410></a></html><br />
|}<br />
</center><br />
<br />
Once we got every parameters for every functions involved in our Full System, we have got a "Modular Virtual Lab" of our System that aims to be ''predictive''.<br />
<br />
<br><br />
<br />
<div style="text-align: center"><br />
{{Paris/Toggle|Details on the Steps|Team:Paris/Modeling/Rubriques}} <br />
</div><br />
<br />
<br><br />
<br />
<center><br />
[[Team:Paris/Modeling/Characterization_Approach| <Back - to the "Presentation" ]]|[[Team:Paris/Modeling/Characterization_Approach_Conclu| Next - to the "Conclusion"> ]]<br />
</center></div>David.bikardhttp://2008.igem.org/Team:Paris/Modeling/Workflow_ExampleTeam:Paris/Modeling/Workflow Example2008-10-29T23:40:54Z<p>David.bikard: </p>
<hr />
<div>{{Paris/Menu}}<br />
{{Paris/Header|Characterization Approach : Generic Workflow on an Example}}<br />
<br />
Here, we will explain the generic protocol that leads to the modelisation "by characterization", of one of the parts of our system. The choosen example is '''the activity of the promoter ''pFliL'' ''' (that leads to the production of the '''Fluorescent Protein FP1''') '''in function of the transcription factors ''FlhDC'' and ''FliA'' '''.<br />
We know that ''the hexamere FlhD<sub>4</sub>C<sub>2</sub>'' and the protein ''FliA'' act both on the promoter ''pFliL'' as inducers... how do we evaluate this phenomenon? <br><br />
As it is illustrated below; starting from ''molecular reactions'' '''(1)''', we develop a ''model of our system'' '''(2)''', and then we identify the ''parameters'' '''(3)''' that needs to be estimated. Essentially, it amounts to estimate ''Promoters Activities as functions of their Transcription Factors''. To do so, we propose ''constructs'' '''(4)''', ''protocols'' '''(5)''' and ''programs'' '''(6)'''.<br />
<br><br />
<center><span style="color: red; font-weight: bold; font-size: 16">''CLICK on the images below for further informations on each step!''</span></center> <br />
<br><br />
<center><br />
{|border="0" style="text-align: center" cellspacing="-1"<br />
|'''Modelisation'''<br />
|'''Experiments'''<br />
|-<br />
|<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Molecular_Reactions"><img src="https://static.igem.org/mediawiki/2008/e/e3/Molecular_Reactions.jpg" width=480></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/FromMolReactToNLOde"><img src="https://static.igem.org/mediawiki/2008/b/b1/HypothesisModel.jpg" width=480></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/FromMolReactToNLOde#Mathematical_Interpretation_and_Simulation_of_the_Molecular_Reactions"><img src="https://static.igem.org/mediawiki/2008/8/85/TemporalVariation.jpg" width=480></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/FromMolReactToNLOde#Mathematical_Interpretation_and_Simulation_of_the_Molecular_Reactions"><img src="https://static.igem.org/mediawiki/2008/4/40/RQSS.jpg" width=240></a></html><html><a href = "https://2008.igem.org/Team:Paris/Modeling/Implementation#Parameters_Finder_Programs"><img src="https://static.igem.org/mediawiki/2008/b/b4/Programs.jpg" width=240></a></html> <br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Implementation#All_Algorithm"><img src="https://static.igem.org/mediawiki/2008/3/31/Goal.jpg" width=480></a></html><br />
|<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Protocol_Of_Characterization"><img src="https://static.igem.org/mediawiki/2008/f/fd/Principles.jpg" width=410></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Protocol_Of_Characterization"><img src="https://static.igem.org/mediawiki/2008/5/52/Plasmid.jpg" width=410></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Protocol_Of_Characterization"><img src="https://static.igem.org/mediawiki/2008/7/78/HypothesisCharact.jpg" width=410></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Implementation#Parameters_Finder_Programs"><img src="https://static.igem.org/mediawiki/2008/7/78/Graph.jpg" width=410></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Implementation#All_Algorithms"><img src="https://static.igem.org/mediawiki/2008/8/83/FullSystem.jpg" width=410></a></html><br />
|}<br />
</center><br />
<br />
Once we got every parameters for every functions involved in our Full System, we have got a "Modular Virtual Lab" of our System that aims to be ''predictive''.<br />
<br />
<br><br />
<br />
<div style="text-align: center"><br />
{{Paris/Toggle|Details on the Steps|Team:Paris/Modeling/Rubriques}} <br />
</div><br />
<br />
<br><br />
<br />
<center><br />
[[Team:Paris/Modeling/Characterization_Approach| <Back - to the "Presentation" ]]|[[Team:Paris/Modeling/Characterization_Approach_Conclu| Next - to the "Conclusion"> ]]<br />
</center></div>David.bikardhttp://2008.igem.org/Team:Paris/Modeling/Workflow_ExampleTeam:Paris/Modeling/Workflow Example2008-10-29T23:40:32Z<p>David.bikard: </p>
<hr />
<div>{{Paris/Menu}}<br />
{{Paris/Header|Characterization Approach : Generic Workflow on an Example}}<br />
<br />
Here, we will explain the generic protocol that leads to the modelisation "by characterization", of one of the parts of our system. The choosen example is '''the activity of the promoter ''pFliL'' ''' (that leads to the production of the '''Fluorescent Protein FP1''') '''in function of the transcription factors ''FlhDC'' and ''FliA'' '''.<br />
We know that ''the hexamere FlhD<sub>4</sub>C<sub>2</sub>'' and the protein ''FliA'' act both on the promoter ''pFliL'' as inducers... how do we evaluate this phenomenon? <br><br />
As it is illustrated below; starting from ''molecular reactions'' '''(1)''', we develop a ''model of our system'' '''(2)''', and then we identify the ''parameters'' '''(3)''' that needs to be estimated. Essentially, it amounts to estimate ''Promoters Activities as functions of their Transcription Factors''. To do so, we propose ''constructs'' '''(4)''', ''protocols'' '''(5)''' and ''programs'' '''(6)'''.<br />
<br><br />
<center><span style="color: red; font-weight: bold; font-size: 16">''CLICK on the images or buttons below for further information on each step!''</span></center> <br />
<br><br />
<center><br />
{|border="0" style="text-align: center" cellspacing="-1"<br />
|'''Modelisation'''<br />
|'''Experiments'''<br />
|-<br />
|<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Molecular_Reactions"><img src="https://static.igem.org/mediawiki/2008/e/e3/Molecular_Reactions.jpg" width=480></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/FromMolReactToNLOde"><img src="https://static.igem.org/mediawiki/2008/b/b1/HypothesisModel.jpg" width=480></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/FromMolReactToNLOde#Mathematical_Interpretation_and_Simulation_of_the_Molecular_Reactions"><img src="https://static.igem.org/mediawiki/2008/8/85/TemporalVariation.jpg" width=480></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/FromMolReactToNLOde#Mathematical_Interpretation_and_Simulation_of_the_Molecular_Reactions"><img src="https://static.igem.org/mediawiki/2008/4/40/RQSS.jpg" width=240></a></html><html><a href = "https://2008.igem.org/Team:Paris/Modeling/Implementation#Parameters_Finder_Programs"><img src="https://static.igem.org/mediawiki/2008/b/b4/Programs.jpg" width=240></a></html> <br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Implementation#All_Algorithm"><img src="https://static.igem.org/mediawiki/2008/3/31/Goal.jpg" width=480></a></html><br />
|<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Protocol_Of_Characterization"><img src="https://static.igem.org/mediawiki/2008/f/fd/Principles.jpg" width=410></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Protocol_Of_Characterization"><img src="https://static.igem.org/mediawiki/2008/5/52/Plasmid.jpg" width=410></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Protocol_Of_Characterization"><img src="https://static.igem.org/mediawiki/2008/7/78/HypothesisCharact.jpg" width=410></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Implementation#Parameters_Finder_Programs"><img src="https://static.igem.org/mediawiki/2008/7/78/Graph.jpg" width=410></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Implementation#All_Algorithms"><img src="https://static.igem.org/mediawiki/2008/8/83/FullSystem.jpg" width=410></a></html><br />
|}<br />
</center><br />
<br />
Once we got every parameters for every functions involved in our Full System, we have got a "Modular Virtual Lab" of our System that aims to be ''predictive''.<br />
<br />
<br><br />
<br />
<div style="text-align: center"><br />
{{Paris/Toggle|Details on the Steps|Team:Paris/Modeling/Rubriques}} <br />
</div><br />
<br />
<br><br />
<br />
<center><br />
[[Team:Paris/Modeling/Characterization_Approach| <Back - to the "Presentation" ]]|[[Team:Paris/Modeling/Characterization_Approach_Conclu| Next - to the "Conclusion"> ]]<br />
</center></div>David.bikardhttp://2008.igem.org/Team:Paris/Modeling/Workflow_ExampleTeam:Paris/Modeling/Workflow Example2008-10-29T23:39:54Z<p>David.bikard: </p>
<hr />
<div>{{Paris/Menu}}<br />
{{Paris/Header|Characterization Approach : Generic Workflow on an Example}}<br />
<br />
Here, we will explain the generic protocol that leads to the modelisation "by characterization", of one of the parts of our system. The choosen example is '''the activity of the promoter ''pFliL'' ''' (that leads to the production of the '''Fluorescent Protein FP1''') '''in function of the transcription factors ''FlhDC'' and ''FliA'' '''.<br />
We know that ''the hexamere FlhD<sub>4</sub>C<sub>2</sub>'' and the protein ''FliA'' act both on the promoter ''pFliL'' as inducers... how do we evaluate this phenomenon? <br><br />
As it is illustrated below; starting from ''molecular reactions'' '''(1)''', we develop a ''model of our system'' '''(2)''', and then we identify the ''parameters'' '''(3)''' that needs to be estimated. Essentially, it amounts to estimate ''Promoters Activities as functions of their Transcription Factors''. To do so, we propose ''constructs'' '''(4)''', ''protocols'' '''(5)''' and ''programs'' '''(6)'''.<br />
<br><br />
<center><span style="color: red; font-weight: bold; font-size: 16">''CLICK on the images or buttons below for further information''</span></center> <br />
<br><br />
<center><br />
{|border="0" style="text-align: center" cellspacing="-1"<br />
|'''Modelisation'''<br />
|'''Experiments'''<br />
|-<br />
|<html><a href = "https://2008.igem.org/Team:Paris/Modeling/Molecular_Reactions"><img src="https://static.igem.org/mediawiki/2008/e/e3/Molecular_Reactions.jpg" width=480></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/FromMolReactToNLOde"><img src="https://static.igem.org/mediawiki/2008/b/b1/HypothesisModel.jpg" width=480></a></html><br><br />
<html><a href = "https://2008.igem.org/Team:Paris/Modeling/FromMolReactToNLOde#Mathematical_Interpretation_and_Simulation_of_the_Molecular_Reactions"><img src="https://static.igem.org/mediawiki/2008/8/85/TemporalVariation.jpg" width=480></a></html><br><br />
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|}<br />
</center><br />
<br />
Once we got every parameters for every functions involved in our Full System, we have got a "Modular Virtual Lab" of our System that aims to be ''predictive''.<br />
<br />
<br><br />
<br />
<div style="text-align: center"><br />
{{Paris/Toggle|Details on the Steps|Team:Paris/Modeling/Rubriques}} <br />
</div><br />
<br />
<br><br />
<br />
<center><br />
[[Team:Paris/Modeling/Characterization_Approach| <Back - to the "Presentation" ]]|[[Team:Paris/Modeling/Characterization_Approach_Conclu| Next - to the "Conclusion"> ]]<br />
</center></div>David.bikardhttp://2008.igem.org/Team:Paris/Analysis/Construction2Team:Paris/Analysis/Construction22008-10-29T23:19:47Z<p>David.bikard: /* Introduction */</p>
<hr />
<div>{{Paris/Menu}}<br />
{{Paris/Header|Model Construction}}<br />
<br />
== Introduction ==<br />
<br />
In this section, we investigate possible improvements of the [[Team:Paris/Network_analysis_and_design/Core_system/Model_construction|core system]]. Our objective is twofold : the system has to provide sustained oscillations and these oscillations should be synchronized amongst a population of cells. To this aim we explore designs inspired by quorum sensing and model in a chemostat cell growth and species diffusion outside cells. We consider two models relying on different designs principles.<br />
<br />
{{Paris/Toggle|To join our quest for alternatives click here!|Team:Paris/DescriptionMoreModelConstruction}}<br />
<br />
== Modeling Alternatives ==<br />
<br />
The proposed systems are:<br />
{|align="center" cellpadding="0" cellspacing="20"<br />
|-style="background: #D4E2EF; text-align: center;"<br />
|'''HSL mediated coupled oscillators'''<br />
|'''HSL mediated simple oscillator'''<br />
|-style="background: #ffffff; text-align: center;"<br />
|[[Image:Bimo.png|250px]]<br />
|[[Image:Unimo.png|250px]]<br />
|}<br />
<br />
*In the first system, we use a modular design. Since the core system is a 'poor' oscillator we hope that by coupling it with a 'good' oscillator we could obtain sustained oscillations in the whole system. Thus, we consider that the core system is one of the modules of the system and that the other module is a two gene oscillator system presented in [[Team:Paris/Bibliography|[2]]] that accounts for quorum sensing. We call this alternative the 'HSL mediated coupled oscillators'.<br />
<br />
*In the second system, namely the 'HSL mediated simple oscillator', we rewire the architecture of the core system to introduce delay via HSL export in the environment in a single circuit.<br />
<br />
Both the coupled and simple oscillators describe events that happen not only at the cellular level (as in the core system) but also at the population level due to interactions needed between a cell and its environment.<br />
<br />
In the following sections, we first describe the [[Team:Paris/Network_analysis_and_design/System_improvements/Description#Common_Description|common]] part among the two proposed models and then focus our attention to the [[Team:Paris/Network_analysis_and_design/System_improvements/Description#Alternatives_Description| alternatives description]] where the characteristics that are specific to each of the modeling alternatives are presented.<br />
<!-- MORE DETAILS ABOUT THE CHEMOST<br />
* A chemostat is generally used to keep bacteria volume constant in the medium. The constant conditions provided by the chemostat help us to control bacteria growth rate. <br />
<br />
* We assume a logistic model to determine bacteria growth in the chemostat, in agreement with standar procedures (reference). This hypothesis implies that bacteria growth rate has to be proportional to the existing bacteria population size and to the amount of available resources in the medium.<br />
--><br />
<!-- MORE DETAILS ABOUT QS<br />
* As discussed [[Team:Paris/Network_analysis_and_design/System_improvements|previously]], we choose to use quorum sensing as a way to improve the oscillating behaviour of our [[Team:Paris/Network_analysis_and_design/Core_system|core system]] and, at the same time, as a way to archeive population synchronization. When a cultive of bacteria is synchronized, it means that every single cell express in average the same genes in unison. As a result, a maximum level of fluorescense is obtained.<br />
--><br />
<br />
== Common Description ==<br />
{|align="center"<br />
|-style="background: #ffffff; text-align: left;"<br />
|Logistic growth in chemostat:<br />
|-style="background: #ffffff; text-align: center;"<br />
|[[Image:DescriptionCommonDynamicsPart1.png|center|600px]]<br />
|-style="background: #ffffff; text-align: left;"<br />
|Quorum sensing by HSL diffusion:<br />
|-style="background: #ffffff; text-align: center;"<br />
|[[Image:DescriptionCommonDynamicsPart2.png|center|600px]]<br />
|-style="background: #ffffff; text-align: left;"<br />
|As in [[Team:Paris/Network_analysis_and_design/Core_system|core system]] flhDC inhibited by EnvZ and fliA activated by FlhDC and FliA:<br />
|-style="background: #ffffff; text-align: center;"<br />
|[[Image:DescriptionCommonDynamicsPart3.png|center|600px]]<br />
|}<br />
{{Paris/Toggle|read more on common dynamics...|Team:Paris/DescriptionDetailsS4-S2Part1}}<br />
<br />
== Alternatives Description ==<br />
{|align="center" cellpadding="2" cellspacing="10"<br />
|-style="background: #D4E2EF; text-align: center;"<br />
|'''HSL mediated coupled oscillators'''<br />
|'''HSL mediated simple oscillator'''<br />
|-style="background: #ffffff; text-align: left;"<br />
|Core system coupled with an oscillator:<br />
|Modified core system that accounts for quorum sensing:<br />
|-style="background: #ffffff; text-align: center;"<br />
|[[Image:SumaryBiMo.png|450px]]<br />
|[[Image:SumaryUniMo.png|350px]]<br />
|}<br />
{{Paris/Toggle|read more on alternatives description...|Team:Paris/DescriptionDetailsS4-S2Part2}}<br />
<br />
== Kinetic parameter values==<br />
<br />
Remarkably, almost all parameter values are available from experimental measurements in [[Team:Paris/Bibliography|[1]]] and from the work of Garcia-Ojalvo in [[Team:Paris/Bibliography|[2]]]. Additionally minimizing the number of parameters is possible by rescaling as done for the core system. Relevant values for the sole two missing parameters are found by exploiting a modularity assumption as described in the [[Team:Paris/Network_analysis_and_design/System_improvements/Analysis|next section]]. The following table summarize our findings:<br />
<br />
<br />
<center><br />
{|<br />
|- style="background: #649CD7;"<br />
! colspan="8" style="background: #649CD7;" | Parameters<br />
|- style="background: #ffffff;"<br />
! colspan="8" | &nbsp;<br />
|- style="background: #649CD7;" |<br />
|style="background: #ffffff; text-align:center;" width=20% |'''Chemostat'''<br />
|Parameter<br />
|Meaning<br />
|Original Value<br />
|Normalized Value<br />
|Unit<br />
|Source <br />
|- style="background: #dddddd;"| <br />
| ! rowspan="5" style="background: #ffffff;" | <br />
|style="background: #D4E2EF;" | α<sub>cell</sub><br />
|style="background: #dddddd;"| Growth rate<br />
| style="background: #dddddd;"|0.0198<br />
| style="background: #dddddd;"|1<br />
|style="background: #dddddd;"|min<sup>-1</sup><br />
|style="background: #dddddd;"| [[Team:Paris/Remarks|wet-lab]] <br />
|- style="background: #dddddd;"| <br />
| style="background: #D4E2EF;"| c<sub>max</sub><br />
| style="background: #dddddd;"| Carrying capacity for cell growth<br />
| style="background: #dddddd;"| 0.1<br />
| style="background: #dddddd;"| 0.1<br />
| style="background: #dddddd;"| µm<sup>3</sup><br />
| style="background: #dddddd;"| [[Team:Paris/Bibliography|[3]]]<br />
|- style="background: #dddddd;"| <br />
| style="background: #D4E2EF;"| D<sub>renewal</sub><br />
| style="background: #dddddd;"| Dilution rate<br />
| style="background: #dddddd;"| 0.00198<br />
| style="background: #dddddd;"| 0.1<br />
| style="background: #dddddd;"| min<sup>-1</sup><br />
| style="background: #dddddd;"| [[Team:Paris/Remarks|wet-lab]] ([[Team:Paris/Bibliography|[3]]])<br />
|- style="background: #dddddd;"| <br />
| style="background: #D4E2EF;"| d<br />
| style="background: #dddddd;"| Death rate<br />
| style="background: #dddddd;"| 0.0099<br />
| style="background: #dddddd;"| 0.5<br />
| style="background: #dddddd;"| min<sup>-1</sup><br />
| style="background: #dddddd;"|[[Team:Paris/Remarks|wet-lab]]<br />
|- style="background: #ffffff;"<br />
! colspan="8" | &nbsp;<br />
|- style="background: #649CD7;" |<br />
|style="background: #ffffff; text-align:center;" |'''Quorum Sensing'''<br />
|Parameter<br />
|Meaning<br />
|Original Value<br />
|Normalized Value<br />
|Unit<br />
|Source <br />
|- style="background: #dddddd;"| <br />
| ! rowspan="6" style="background: #ffffff;" | <br />
| style="background: #D4E2EF;" |γ<sub>HSL</sub> <br />
| style="background: #dddddd;"|Degradation rate<br />
| style="background: #dddddd;"|0.0053<br />
| style="background: #dddddd;"|0.2690<br />
| style="background: #dddddd;"|min<sup>-1</sup><br />
| style="background: #dddddd;"|[[Team:Paris/Remarks|see remarks]] <br />
|- style="background: #dddddd;"| <br />
| style="background: #D4E2EF;"|γ<sub>HSL<sub>ext</sub></sub> <br> <br><br />
| style="background: #dddddd;"|Degradation rate<br />
| style="background: #dddddd;"|0.0106<br />
| style="background: #dddddd;"|0.5380<br />
|style="background: #dddddd;"| min<sup>-1</sup><br />
| style="background: #dddddd;"|[[Team:Paris/Bibliography|[2]]]<br />
|- style="background: #dddddd;"| <br />
| style="background: #D4E2EF;" |β<sub>HSL</sub> <br />
| style="background: #dddddd;"|Production rate<br />
| style="background: #dddddd;"|0.3168<br />
| style="background: #dddddd;"|16<br />
| style="background: #dddddd;"|min<sup>-1</sup><br />
| style="background: #dddddd;"|[[Team:Paris/Bibliography|∅]]<br />
|- style="background: #dddddd;"| <br />
| style="background: #D4E2EF;" |η<br />
| style="background: #dddddd;"|Diffusion rate<br />
| style="background: #dddddd;"|10<br />
| style="background: #dddddd;"|505<br />
| style="background: #dddddd;"|min<sup>-1</sup><br />
| style="background: #dddddd;"|[[Team:Paris/Bibliography|[2]]]<br />
|- style="background: #dddddd;"| <br />
| style="background: #D4E2EF;" |n<sub>HSL</sub><br />
| style="background: #dddddd;"|Hill coefficient<br />
| style="background: #dddddd;"|&nbsp;<br />
| style="background: #dddddd;"|4<br />
| style="background: #dddddd;"|&nbsp;<br />
| style="background: #dddddd;"|[[Team:Paris/Bibliography|[3]]]<br />
|- style="background: #dddddd;"| <br />
| style="background: #D4E2EF;" |θ<sub>HSL</sub><br />
| style="background: #dddddd;"|Hill characteristic concentration for the second operator<br />
| style="background: #dddddd;"|&nbsp;<br />
| style="background: #dddddd;"|0.5<br />
| style="background: #dddddd;"|[[Team:Paris/Remarks|c.u.]]<br />
| style="background: #dddddd;"|[[Team:Paris/Bibliography|[3]]]<br />
|}</center></div>David.bikardhttp://2008.igem.org/Team:Paris/PerspectivesTeam:Paris/Perspectives2008-10-28T10:25:33Z<p>David.bikard: </p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
== Biosynthetic Bottum-up approach ==<br />
<br />
==A.DNA nanocar factory ==<br />
<br />
<br />
<br />
The field of artificial molecular machines and motors is growing at an astonishing rate and is attracting a great deal of interest in nanoscience.<br />
Research in the last decade has shown that species made of components like DNA or proteins are attractive candidates.<br />
Our aims is to build by a bottom-up approched DNA nanocars that have the properties to produce energy in vitro. <br />
<br />
<span style="color:blue">You have to explain what you mean by "produce energy in vitro". What energy does it use? to convert into what other energy? or to use in what way? Also, why in vitro? Do you plan to use our system in cell-free extracts or in living cells?</span><br />
<br />
Our system is more efficient than a classic system because the FIFO order is very important: the first part will bind by complementarity with part 2 this new part will make the binding site for part 3. <br />
<br />
Moreover,in the FIFO the first in is the first out meaning that the 2-3 genes is still activate cause the one went off ,in our construction we success to bind only the higher part ,to built the lower part we need to express 2-3 gene that will finalize the car structure. <br />
<span style="color:blue"> This sentence is poorly constructed and full of mistakes </span><br />
<br />
The oscillation of our system can be assimilized to a factory when a certain amount of car his made, we need a little time off to permit them to move ,that will make the place for a new one ,his similar to make cookies when they cook you need to move them to put new ones in the oven.<br />
<span style="color:blue"> This really is farfetched. What will make the cars move? from where to where? Is diffusion not enough?</span><br />
<br />
<br />
<br />
[[Image:car2.jpg|center]]<br />
<br />
Fig.1:biosynthesis by sequential expression of 3 DNA origami<br />
<br />
If we add in vitro a complementary miRNA to the red regions (fig.2), the competition of the miRNA will open the DNA strands by broking the hydrogen backbound this processes deliveres minimun ten time more energy than ATP biodegradation (winfree et.al), the instability of the miRNA may make this processe reversible.<br />
<br />
<span style="color:blue">What is this red region? How will it allow to use the delivered energy? Plus it's not enough to cite a paper to prove that energy is delivered. If the miRNA is complementary to a region that is already paired, the energy you need to unpair this region is the same that you get when you pair it with the miRNA. Result: 0 energy gain.</span><br />
<br />
<br />
In conclusion, these kinds of DNA structures can be suitable, reversible and metastable DNA fuels and a new kind of cargo delivery machine.<br />
<span style="color:blue"> How will they move? I doubt that turning their wheels will be of any help...</span><br />
<br />
== B.Artificial virus factory ==<br />
<br />
<br />
We can also produce a lot of differentes kinds of self-assembly structures like virus for example,HIV:<br />
<br />
<br />
<br />
[[Image:HIV.jpg|center]]<br />
<br />
Fig2: genetic organization of HIV virus <br />
<br />
HIV can be produce by sequential expression of 3 genes gag,env and pol (Fig.2)those genes will be cleaved by protease P10 and than the subunits of the virus will self-assembled into mature virus.<br />
In our case, we will expressed gag p10 and finaly Env (without Pol to avoid pathogenicity) the FIFO in this case will be essential.<br />
<br />
If you ask us why?<br />
<br />
For 3 main reasons :<br />
<br />
* First, when they genes 1 and 2 will be expressed, the protease P10 will cleave gag that will self-assemble.<br />
<br />
* Then, when 123 will be expressed Env will get cleaved and self assemble with the products of gag and P10.<br />
<br />
* Finaly, when 23 are activated alone, we will increase the quantity of subunits delivered from the clivage of Env.<br />
<br />
In conclusion, our sytem will be very useful for the developpement of new drugs against HIV or in new kinds of vectors for genetic therapy. <br />
<span style="color: blue"> Please avoid this kind of over enthousiastic phrases... you cannot just state this boldly without constructing an argument. </span><br />
<br />
== Metabolic engineering of polyhydroxyalkanoate biosynthesis pathways ==<br />
Human overpopulation combined with the current lifestyle urges the rational, efficient, and sustainable use of natural resources to produce environmentally friendly plastic materials such as polyhydroxyalkanoic acids (PHAs), whose production/degradation cycle reduces undesirable wastes and emissions.<br />
Our study is a new metabolic strategy to generate PHA-hyperproducer strains, that have the properties to be a sequential metabolic pathway, we believe the sequential expression may increases the production of purified PHA.<br />
<br />
Our strategy consists on replacing the RFP,CFP and YFP genes by the PhaA ,PhaB and PhaC genes in our final system (containing oscillation,FIFO,synchronisation modules).<br />
<br />
This strategy is more efficient than a constitutive activation for 3 main reasons:<br />
*First, in this application the NADPH, which is a cellular metastable fuel, is used by the PhaB to synthesize bioplastic. This molecule is very important for many metabolic pathways in bacteria. A NADPH recuperation step is then needed to ensure other metabolic activities to go on. We can then make the hypothesis that if the PhaB is always activated the bacteria will get exhausted and die quickly. In our system, the bacteria will have a NADPH recuperation step, this is why we hope bacteria will live more than in an usual chemoreactor.<br />
<br />
*Secondly, we hope by the order of the FIFO to make a synchronized and sequential turnover of the enzymatic expression in order to increase the rate of the PHA biosynthesis.<br />
<span style="color:blue">I'm not sure turnover has this meaning</span><br />
<br />
*Third, as the quality of the bioplastic increases with time, we could predict the quality of the final product since we would know the duration of a production cycle thanks to the periodicity of our system. <br />
<span style="color: blue"> Again, you cannot just state this boldly without constructing an argument. </span><br />
<br />
<span style="color: blue"> You never explain why your system is better than simply expressing all the genes continuously at a lower level </span><br />
<br />
<span style="color: blue"> More generally, on this whole page: We don't expect you to give very detailed projects. You just need to give simple ideas for which the interest of the FIFO seems obvious (or at least you need to try making it seem obvious). We are not so much interested in the precise mechanisms or genes involved, but rather in the principles that make your projects interesting.</span><br />
<br />
[[Image:PHA.jpg|center]]<br />
<br />
== Bibliography ==<br />
* Folding DNA to create nanoscale shapes and patterns<br />
Paul W. K. Rothemund <br />
* Design of DNA origami<br />
Paul W. K. Rothemund <br />
* An autonomous polymerization motor powered by DNA hybridization.<br />
Suvir Venkataraman, Robert M. Dirks, Paul W. K. Rothemund, Erik Winfree, Niles A. Pierce.<br />
* Catalyzed Relaxation of a Metastable DNA Fuel.<br />
Georg Seelig, Bernard Yurke, Erik Winfree.</div>David.bikardhttp://2008.igem.org/Team:Paris/PerspectivesTeam:Paris/Perspectives2008-10-28T09:04:17Z<p>David.bikard: </p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
== Biosynthetic Bottum-up approach ==<br />
<br />
==A.DNA nanocar factory ==<br />
<br />
<br />
<br />
The field of artificial molecular machines and motors is growing at an astonishing rate and is attracting a great deal of interest in nanoscience.<br />
Research in the last decade has shown that species made of components like DNA or proteins are attractive candidates.<br />
Our aims is to build by a bottom-up approched DNA nanocars that have the properties to produce energy in vitro. <br />
<br />
<span style="color:blue">You have to explain what you mean by "produce energy in vitro". What energy does it use? to convert into what other energy? or to use in what way? Also, why in vitro? Do you plan to use our system in cell-free extracts or in living cells?</span><br />
<br />
Our system is more efficient than a classic system because the FIFO order is very important: the first part will bind by complementarity with part 2 this new part will make the binding site for part 3. <br />
<br />
Moreover,in the FIFO the first in is the first out meaning that the 2-3 genes is still activate cause the one went off ,in our construction we success to bind only the higher part ,to built the lower part we need to express 2-3 gene that will finalize the car structure. <br />
<span style="color:blue"> This sentence is poorly constructed and full of mistakes </span><br />
<br />
The oscillation of our system can be assimilized to a factory when a certain amount of car his made, we need a little time off to permit them to move ,that will make the place for a new one ,his similar to make cookies when they cook you need to move them to put new ones in the oven.<br />
<span style="color:blue"> This really is farfetched. What will make the cars moove? from where to where? Is diffusion not enough?</span><br />
<br />
<br />
<br />
[[Image:car2.jpg|center]]<br />
<br />
Fig.1:biosynthesis by sequential expression of 3 DNA origami<br />
<br />
If we add in vitro a complementary miRNA to the red regions (fig.2), the competition of the miRNA will open the DNA strands by broking the hydrogen backbound this processes deliveres minimun ten time more energy than ATP biodegradation (winfree et.al), the instability of the miRNA may make this processe reversible.<br />
<br />
<span style="color:blue">What is this red region? How will it allow to use the delivered energy? Plus it's not enough to cite a paper to prove that energy is delivered. If the miRNA is complementary to a region that is already paired, the energy you need to unpair this region is the same that you get when you pair it with the miRNA. Result: 0 energy gain.</span><br />
<br />
<br />
In conclusion, these kinds of DNA structures can be suitable, reversible and metastable DNA fuels and a new kind of cargo delivery machine.<br />
<span style="color:blue"> How will they moves? I doubt that turning their wheels will be of any help...</span><br />
<br />
== B.Artificial virus factory ==<br />
<br />
<br />
We can also produce a lot of differentes kinds of self-assembly structures like virus for example,HIV:<br />
<br />
<br />
<br />
[[Image:HIV.jpg|center]]<br />
<br />
Fig2: genetic organization of HIV virus <br />
<br />
HIV can be produce by sequential expression of 3 genes gag,env and pol (Fig.2)those genes will be cleaved by protease P10 and than the subunits of the virus will self-assembled into mature virus.<br />
In our case, we will expressed gag p10 and finaly Env (without Pol to avoid pathogenicity) the FIFO in this case will be essential.<br />
<br />
If you ask us why?<br />
<br />
For 3 main reasons :<br />
<br />
* First, when they genes 1 and 2 will be expressed, the protease P10 will cleave gag that will self-assemble.<br />
<br />
* Then, when 123 will be expressed Env will get cleaved and self assemble with the products of gag and P10.<br />
<br />
* Finaly, when 23 are activated alone, we will increase the quantity of subunits delivered from the clivage of Env.<br />
<br />
<span style="color:blue">what are 1, 2, 3 ? There is no way to make sense out of this...</span><br />
<br />
In conclusion, our sytem will be very useful for the developpement of new drugs against HIV or in new kinds of vectors for genetic therapy. <br />
<span style="color: blue"> Please avoid this kind of over enthousiastic phrases... you cannot just state this boldly without constructing an argument. </span><br />
<br />
== Metabolic engineering of polyhydroxyalkanoate biosynthesis pathways ==<br />
Human overpopulation combined with the current lifestyle urges the rational, efficient, and sustainable use of natural resources to produce environmentally friendly plastic materials such as polyhydroxyalkanoic acids (PHAs), whose production/degradation cycle reduces undesirable wastes and emissions.<br />
Our study is a new metabolic strategy to generate PHA-hyperproducer strains, that have the properties to be a sequential metabolic pathway, we believe the sequential expression may increases the production of purified PHA.<br />
<br />
Our strategy consists on replacing the RFP,CFP and YFP genes by the PhaA ,PhaB and PhaC genes in our final system (containing oscillation,FIFO,synchronisation modules).<br />
<br />
This strategy is more efficient than a constitutive activation for 3 main reasons:<br />
*First, in this application the NADPH, which is a cellular metastable fuel, is used by the PhaB to synthesize bioplastic. This molecule is very important for many metabolic pathways in bacteria. A NADPH recuperation step is then needed to ensure other metabolic activities to go on. We can then make the hypothesis that if the PhaB is always activated the bacteria will get exhausted and die quickly. In our system, the bacteria will have a NADPH recuperation step, this is why we hope bacteria will live more than in an usual chemoreactor.<br />
<br />
*Secondly, we hope by the order of the FIFO to make a synchronized and sequential turnover of the enzymatic expression in order to increase the rate of the PHA biosynthesis.<br />
<span style="color:blue">I'm not sure turnover has this meaning</span><br />
<br />
*Third, as the quality of the bioplastic increases with time, we could predict the quality of the final product since we would know the duration of a production cycle thanks to the periodicity of our system. <br />
<span style="color: blue"> Again, you cannot just state this boldly without constructing an argument. </span><br />
<br />
<span style="color: blue"> You never explain why your system is better than simply expressing all the genes continuously at a lower level </span><br />
<br />
<span style="color: blue"> More generally, on this whole page: We don't expect you to give very detailed projects. You just need to give simple ideas for which the interest of the FIFO seems obvious (or at least you need to try making it seem obvious). We are not so much interested in the precise mechanisms or genes involved, but rather in the principles that make your projects interesting.</span><br />
<br />
[[Image:PHA.jpg|center]]<br />
<br />
== Bibliography ==<br />
* Folding DNA to create nanoscale shapes and patterns<br />
Paul W. K. Rothemund <br />
* Design of DNA origami<br />
Paul W. K. Rothemund <br />
* An autonomous polymerization motor powered by DNA hybridization.<br />
Suvir Venkataraman, Robert M. Dirks, Paul W. K. Rothemund, Erik Winfree, Niles A. Pierce.<br />
* Catalyzed Relaxation of a Metastable DNA Fuel.<br />
Georg Seelig, Bernard Yurke, Erik Winfree.</div>David.bikardhttp://2008.igem.org/Team:Paris/PerspectivesTeam:Paris/Perspectives2008-10-28T08:50:02Z<p>David.bikard: /* B.Artificial virus factory */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
== Biosynthetic Bottum-up approach ==<br />
<br />
==A.DNA nanocar factory ==<br />
<br />
<br />
<br />
The field of artificial molecular machines and motors is growing at an astonishing rate and is attracting a great deal of interest in nanoscience.<br />
Research in the last decade has shown that species made of components like DNA or proteins are attractive candidates.<br />
Our aims is to build by a bottom-up approched DNA nanocars that have the properties to produce energy in vitro. <br />
<br />
<span style="color:blue">You have to explain what you mean by "produce energy in vitro". What energy does it use? to convert into what other energy? or to use in what way? Also, why in vitro? Do you plan to use our system in cell-free extracts or in living cells?</span><br />
<br />
Our system is more efficient than a classic system because the FIFO order is very important: the first part will bind by complementarity with part 2 this new part will make the binding site for part 3. <br />
<br />
Moreover,in the FIFO the first in is the first out meaning that the 2-3 genes is still activate cause the one went off ,in our construction we success to bind only the higher part ,to built the lower part we need to express 2-3 gene that will finalize the car structure. <br />
<span style="color:blue"> This sentence is poorly constructed and full of mistakes </span><br />
<br />
The oscillation of our system can be assimilized to a factory when a certain amount of car his made, we need a little time off to permit them to move ,that will make the place for a new one ,his similar to make cookies when they cook you need to move them to put new ones in the oven.<br />
<span style="color:blue"> This really is farfetched. What will make the cars moove? from where to where? Is diffusion not enough?</span><br />
<br />
<br />
<br />
[[Image:car2.jpg|center]]<br />
<br />
Fig.1:biosynthesis by sequential expression of 3 DNA origami<br />
<br />
If we add in vitro a complementary miRNA to the red regions (fig.2), the competition of the miRNA will open the DNA strands by broking the hydrogen backbound this processes deliveres minimun ten time more energy than ATP biodegradation (winfree et.al), the instability of the miRNA may make this processe reversible.<br />
<br />
<span style="color:blue">What is this red region? How will it allow to use the delivered energy? Plus it's not enough to cite a paper to prove that energy is delivered. If the miRNA is complementary to a region that is already paired, the energy you need to unpair this region is the same that you get when you pair it with the miRNA. Result: 0 energy gain.</span><br />
<br />
<br />
In conclusion, these kinds of DNA structures can be suitable, reversible and metastable DNA fuels and a new kind of cargo delivery machine.<br />
<span style="color:blue"> How will they moves? I doubt that turning their wheels will be of any help...</span><br />
<br />
== B.Artificial virus factory ==<br />
<br />
<br />
We can also produce a lot of differentes kinds of self-assembly structures like virus for example,HIV:<br />
<br />
<br />
<br />
[[Image:HIV.jpg|center]]<br />
<br />
Fig2: genetic organization of HIV virus <br />
<br />
HIV can be produce by sequential expression of 3 genes gag,env and pol (Fig.2)those genes will be cleaved by protease P10 and than the subunits of the virus will self-assembled into mature virus.<br />
In our case, we will expressed gag p10 and finaly Env (without Pol to avoid pathogenicity) the FIFO in this case will be essential.<br />
<br />
If you ask us why?<br />
<br />
For 3 main reasons :<br />
<br />
* First, when they genes 1 and 2 will be expressed, the protease P10 will cleave gag that will self-assemble.<br />
<br />
* Then, when 123 will be expressed Env will get cleaved and self assemble with the products of gag and P10.<br />
<br />
* Finaly, when 23 are activated alone, we will increase the quantity of subunits delivered from the clivage of Env.<br />
<br />
In conclusion, our sytem will be very useful for the developpement of new drugs against HIV or in new kinds of vectors for genetic therapy. <br />
<span style="text-color: blue;"> Please avoid this kind of over enthousiastic phrases... you cannot just state this boldly without constructing an argument. </span><br />
<br />
== Metabolic engineering of polyhydroxyalkanoate biosynthesis pathways ==<br />
Human overpopulation combined with the current lifestyle urges the rational, efficient, and sustainable use of natural resources to produce environmentally friendly plastic materials such as polyhydroxyalkanoic acids (PHAs), whose production/degradation cycle reduces undesirable wastes and emissions.<br />
Our study is a new metabolic strategy to generate PHA-hyperproducer strains, that have the properties to be a sequential metabolic pathway, we believe the sequential expression may increases the production of purified PHA.<br />
<br />
Our strategy consists on replacing the RFP,CFP and YFP genes by the PhaA ,PhaB and PhaC genes in our final system (containing oscillation,FIFO,synchronisation modules).<br />
<br />
This strategy is more efficient than a constitutive activation for 3 main reasons:<br />
*First, in this application the NADPH, which is a cellular metastable fuel, is used by the PhaB to synthesize bioplastic. This molecule is very important for many metabolic pathways in bacteria. A NADPH recuperation step is then needed to ensure other metabolic activities to go on. We can then make the hypothesis that if the PhaB is always activated the bacteria will get exhausted and die quickly (4-5 days). In our system, the bacteria will have a NADPH recuperation step, this is why we hope bacteria will live more than in an usual chemoreactor.<br />
<br />
*Secondly, we hope by the order of the FIFO to make a synchronized and sequential turn-over of the enzymatic expression in order to increase the rate of the PHA biosynthesis.<br />
<br />
*Third, as the quality of the bioplastic increases with time, we could predict the quality of the final product since we would know the duration of a production cycle thanks to the periodicity of our system. <br />
<br />
[[Image:PHA.jpg|center]]<br />
<br />
== Bibliography ==<br />
* Folding DNA to create nanoscale shapes and patterns<br />
Paul W. K. Rothemund <br />
* Design of DNA origami<br />
Paul W. K. Rothemund <br />
* An autonomous polymerization motor powered by DNA hybridization.<br />
Suvir Venkataraman, Robert M. Dirks, Paul W. K. Rothemund, Erik Winfree, Niles A. Pierce.<br />
* Catalyzed Relaxation of a Metastable DNA Fuel.<br />
Georg Seelig, Bernard Yurke, Erik Winfree.</div>David.bikardhttp://2008.igem.org/Team:Paris/PerspectivesTeam:Paris/Perspectives2008-10-28T08:49:22Z<p>David.bikard: /* B.Artificial virus factory */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
== Biosynthetic Bottum-up approach ==<br />
<br />
==A.DNA nanocar factory ==<br />
<br />
<br />
<br />
The field of artificial molecular machines and motors is growing at an astonishing rate and is attracting a great deal of interest in nanoscience.<br />
Research in the last decade has shown that species made of components like DNA or proteins are attractive candidates.<br />
Our aims is to build by a bottom-up approched DNA nanocars that have the properties to produce energy in vitro. <br />
<br />
<span style="color:blue">You have to explain what you mean by "produce energy in vitro". What energy does it use? to convert into what other energy? or to use in what way? Also, why in vitro? Do you plan to use our system in cell-free extracts or in living cells?</span><br />
<br />
Our system is more efficient than a classic system because the FIFO order is very important: the first part will bind by complementarity with part 2 this new part will make the binding site for part 3. <br />
<br />
Moreover,in the FIFO the first in is the first out meaning that the 2-3 genes is still activate cause the one went off ,in our construction we success to bind only the higher part ,to built the lower part we need to express 2-3 gene that will finalize the car structure. <br />
<span style="color:blue"> This sentence is poorly constructed and full of mistakes </span><br />
<br />
The oscillation of our system can be assimilized to a factory when a certain amount of car his made, we need a little time off to permit them to move ,that will make the place for a new one ,his similar to make cookies when they cook you need to move them to put new ones in the oven.<br />
<span style="color:blue"> This really is farfetched. What will make the cars moove? from where to where? Is diffusion not enough?</span><br />
<br />
<br />
<br />
[[Image:car2.jpg|center]]<br />
<br />
Fig.1:biosynthesis by sequential expression of 3 DNA origami<br />
<br />
If we add in vitro a complementary miRNA to the red regions (fig.2), the competition of the miRNA will open the DNA strands by broking the hydrogen backbound this processes deliveres minimun ten time more energy than ATP biodegradation (winfree et.al), the instability of the miRNA may make this processe reversible.<br />
<br />
<span style="color:blue">What is this red region? How will it allow to use the delivered energy? Plus it's not enough to cite a paper to prove that energy is delivered. If the miRNA is complementary to a region that is already paired, the energy you need to unpair this region is the same that you get when you pair it with the miRNA. Result: 0 energy gain.</span><br />
<br />
<br />
In conclusion, these kinds of DNA structures can be suitable, reversible and metastable DNA fuels and a new kind of cargo delivery machine.<br />
<span style="color:blue"> How will they moves? I doubt that turning their wheels will be of any help...</span><br />
<br />
== B.Artificial virus factory ==<br />
<br />
<br />
We can also produce a lot of differentes kinds of self-assembly structures like virus for example,HIV:<br />
<br />
<br />
<br />
[[Image:HIV.jpg|center]]<br />
<br />
Fig2: genetic organization of HIV virus <br />
<br />
HIV can be produce by sequential expression of 3 genes gag,env and pol (Fig.2)those genes will be cleaved by protease P10 and than the subunits of the virus will self-assembled into mature virus.<br />
In our case, we will expressed gag p10 and finaly Env (without Pol to avoid pathogenicity) the FIFO in this case will be essential.<br />
<br />
If you ask us why?<br />
<br />
For 3 main reasons :<br />
<br />
* First, when they genes 1 and 2 will be expressed, the protease P10 will cleave gag that will self-assemble.<br />
<br />
* Then, when 123 will be expressed Env will get cleaved and self assemble with the products of gag and P10.<br />
<br />
* Finaly, when 23 are activated alone, we will increase the quantity of subunits delivered from the clivage of Env.<br />
<br />
In conclusion, our sytem will be very useful for the developpement of new drugs against HIV or in new kinds of vectors for genetic therapy. <span style="text-color: blue"> Please avoid this kind of over enthousiastic phrases... you cannot just state this boldly without constructing an argument. </span><br />
<br />
== Metabolic engineering of polyhydroxyalkanoate biosynthesis pathways ==<br />
Human overpopulation combined with the current lifestyle urges the rational, efficient, and sustainable use of natural resources to produce environmentally friendly plastic materials such as polyhydroxyalkanoic acids (PHAs), whose production/degradation cycle reduces undesirable wastes and emissions.<br />
Our study is a new metabolic strategy to generate PHA-hyperproducer strains, that have the properties to be a sequential metabolic pathway, we believe the sequential expression may increases the production of purified PHA.<br />
<br />
Our strategy consists on replacing the RFP,CFP and YFP genes by the PhaA ,PhaB and PhaC genes in our final system (containing oscillation,FIFO,synchronisation modules).<br />
<br />
This strategy is more efficient than a constitutive activation for 3 main reasons:<br />
*First, in this application the NADPH, which is a cellular metastable fuel, is used by the PhaB to synthesize bioplastic. This molecule is very important for many metabolic pathways in bacteria. A NADPH recuperation step is then needed to ensure other metabolic activities to go on. We can then make the hypothesis that if the PhaB is always activated the bacteria will get exhausted and die quickly (4-5 days). In our system, the bacteria will have a NADPH recuperation step, this is why we hope bacteria will live more than in an usual chemoreactor.<br />
<br />
*Secondly, we hope by the order of the FIFO to make a synchronized and sequential turn-over of the enzymatic expression in order to increase the rate of the PHA biosynthesis.<br />
<br />
*Third, as the quality of the bioplastic increases with time, we could predict the quality of the final product since we would know the duration of a production cycle thanks to the periodicity of our system. <br />
<br />
[[Image:PHA.jpg|center]]<br />
<br />
== Bibliography ==<br />
* Folding DNA to create nanoscale shapes and patterns<br />
Paul W. K. Rothemund <br />
* Design of DNA origami<br />
Paul W. K. Rothemund <br />
* An autonomous polymerization motor powered by DNA hybridization.<br />
Suvir Venkataraman, Robert M. Dirks, Paul W. K. Rothemund, Erik Winfree, Niles A. Pierce.<br />
* Catalyzed Relaxation of a Metastable DNA Fuel.<br />
Georg Seelig, Bernard Yurke, Erik Winfree.</div>David.bikardhttp://2008.igem.org/Team:Paris/PerspectivesTeam:Paris/Perspectives2008-10-28T08:42:31Z<p>David.bikard: /* A.DNA nanocar factory */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
== Biosynthetic Bottum-up approach ==<br />
<br />
==A.DNA nanocar factory ==<br />
<br />
<br />
<br />
The field of artificial molecular machines and motors is growing at an astonishing rate and is attracting a great deal of interest in nanoscience.<br />
Research in the last decade has shown that species made of components like DNA or proteins are attractive candidates.<br />
Our aims is to build by a bottom-up approched DNA nanocars that have the properties to produce energy in vitro. <br />
<br />
<span style="color:blue">You have to explain what you mean by "produce energy in vitro". What energy does it use? to convert into what other energy? or to use in what way? Also, why in vitro? Do you plan to use our system in cell-free extracts or in living cells?</span><br />
<br />
Our system is more efficient than a classic system because the FIFO order is very important: the first part will bind by complementarity with part 2 this new part will make the binding site for part 3. <br />
<br />
Moreover,in the FIFO the first in is the first out meaning that the 2-3 genes is still activate cause the one went off ,in our construction we success to bind only the higher part ,to built the lower part we need to express 2-3 gene that will finalize the car structure. <br />
<span style="color:blue"> This sentence is poorly constructed and full of mistakes </span><br />
<br />
The oscillation of our system can be assimilized to a factory when a certain amount of car his made, we need a little time off to permit them to move ,that will make the place for a new one ,his similar to make cookies when they cook you need to move them to put new ones in the oven.<br />
<span style="color:blue"> This really is farfetched. What will make the cars moove? from where to where? Is diffusion not enough?</span><br />
<br />
<br />
<br />
[[Image:car2.jpg|center]]<br />
<br />
Fig.1:biosynthesis by sequential expression of 3 DNA origami<br />
<br />
If we add in vitro a complementary miRNA to the red regions (fig.2), the competition of the miRNA will open the DNA strands by broking the hydrogen backbound this processes deliveres minimun ten time more energy than ATP biodegradation (winfree et.al), the instability of the miRNA may make this processe reversible.<br />
<br />
<span style="color:blue">What is this red region? How will it allow to use the delivered energy? Plus it's not enough to cite a paper to prove that energy is delivered. If the miRNA is complementary to a region that is already paired, the energy you need to unpair this region is the same that you get when you pair it with the miRNA. Result: 0 energy gain.</span><br />
<br />
<br />
In conclusion, these kinds of DNA structures can be suitable, reversible and metastable DNA fuels and a new kind of cargo delivery machine.<br />
<span style="color:blue"> How will they moves? I doubt that turning their wheels will be of any help...</span><br />
<br />
== B.Artificial virus factory ==<br />
<br />
<br />
We can also produce a lot of differentes kinds of self-assembly structures like virus for example,HIV:<br />
<br />
<br />
<br />
[[Image:HIV.jpg|center]]<br />
<br />
Fig2: genetic organization of HIV virus <br />
<br />
HIV can be produce by sequential expression of 3 genes gag,env and pol (Fig.2)those genes will be cleaved by protease P10 and than the subunits of the virus will self-assembled into mature virus.<br />
In our case, we will expressed gag p10 and finaly Env (without Pol to avoid pathogenicity) the FIFO in this case will be essential.<br />
<br />
If you ask us why?<br />
<br />
For 3 main reasons :<br />
<br />
* First, when they genes 1 and 2 will be expressed the protease P10 will cleave gag that will self-assembled.<br />
<br />
* Than when 123 will be expresse Env will get cleaved and self assembled with they products of gag and P10.<br />
<br />
* Finaly, when 23 get activate we will increase the quantity of subunits delivered from the clivage of Env.<br />
<br />
In conclusion, our sytem will be very useful for the developpement of new drugs against HIV or in new kinds of vectors for genetic therapy.<br />
<br />
== Metabolic engineering of polyhydroxyalkanoate biosynthesis pathways ==<br />
Human overpopulation combined with the current lifestyle urges the rational, efficient, and sustainable use of natural resources to produce environmentally friendly plastic materials such as polyhydroxyalkanoic acids (PHAs), whose production/degradation cycle reduces undesirable wastes and emissions.<br />
Our study is a new metabolic strategy to generate PHA-hyperproducer strains, that have the properties to be a sequential metabolic pathway, we believe the sequential expression may increases the production of purified PHA.<br />
<br />
Our strategy consists on replacing the RFP,CFP and YFP genes by the PhaA ,PhaB and PhaC genes in our final system (containing oscillation,FIFO,synchronisation modules).<br />
<br />
This strategy is more efficient than a constitutive activation for 3 main reasons:<br />
*First, in this application the NADPH, which is a cellular metastable fuel, is used by the PhaB to synthesize bioplastic. This molecule is very important for many metabolic pathways in bacteria. A NADPH recuperation step is then needed to ensure other metabolic activities to go on. We can then make the hypothesis that if the PhaB is always activated the bacteria will get exhausted and die quickly (4-5 days). In our system, the bacteria will have a NADPH recuperation step, this is why we hope bacteria will live more than in an usual chemoreactor.<br />
<br />
*Secondly, we hope by the order of the FIFO to make a synchronized and sequential turn-over of the enzymatic expression in order to increase the rate of the PHA biosynthesis.<br />
<br />
*Third, as the quality of the bioplastic increases with time, we could predict the quality of the final product since we would know the duration of a production cycle thanks to the periodicity of our system. <br />
<br />
[[Image:PHA.jpg|center]]<br />
<br />
== Bibliography ==<br />
* Folding DNA to create nanoscale shapes and patterns<br />
Paul W. K. Rothemund <br />
* Design of DNA origami<br />
Paul W. K. Rothemund <br />
* An autonomous polymerization motor powered by DNA hybridization.<br />
Suvir Venkataraman, Robert M. Dirks, Paul W. K. Rothemund, Erik Winfree, Niles A. Pierce.<br />
* Catalyzed Relaxation of a Metastable DNA Fuel.<br />
Georg Seelig, Bernard Yurke, Erik Winfree.</div>David.bikardhttp://2008.igem.org/Team:Paris/PerspectivesTeam:Paris/Perspectives2008-10-28T08:41:57Z<p>David.bikard: /* A.DNA nanocar factory */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
== Biosynthetic Bottum-up approach ==<br />
<br />
==A.DNA nanocar factory ==<br />
<br />
<br />
<br />
The field of artificial molecular machines and motors is growing at an astonishing rate and is attracting a great deal of interest in nanoscience.<br />
Research in the last decade has shown that species made of components like DNA or proteins are attractive candidates.<br />
Our aims is to build by a bottom-up approched DNA nanocars that have the properties to produce energy in vitro. <br />
<br />
<span style="color:blue">You have to explain what you mean by "produce energy in vitro". What energy does it use? to convert into what other energy? or to use in what way? Also, why in vitro? Do you plan to use our system in cell-free extracts or in living cells?</span><br />
<br />
Our system is more efficient than a classic system because the FIFO order is very important: the first part will bind by complementarity with part 2 this new part will make the binding site for part 3. <br />
<br />
Moreover,in the FIFO the first in is the first out meaning that the 2-3 genes is still activate cause the one went off ,in our construction we success to bind only the higher part ,to built the lower part we need to express 2-3 gene that will finalize the car structure. <br />
<span style="color:blue"> This sentence is poorly constructed and full of mistakes </span><br />
<br />
The oscillation of our system can be assimilized to a factory when a certain amount of car his made, we need a little time off to permit them to move ,that will make the place for a new one ,his similar to make cookies when they cook you need to move them to put new ones in the oven.<br />
<span style="color:blue"> This really is farfetched. What will make the cars moove? Is diffusion not enough? from were to were?</span><br />
<br />
<br />
<br />
[[Image:car2.jpg|center]]<br />
<br />
Fig.1:biosynthesis by sequential expression of 3 DNA origami<br />
<br />
If we add in vitro a complementary miRNA to the red regions (fig.2), the competition of the miRNA will open the DNA strands by broking the hydrogen backbound this processes deliveres minimun ten time more energy than ATP biodegradation (winfree et.al), the instability of the miRNA may make this processe reversible.<br />
<br />
<span style="color:blue">What is this red region? How will it allow to use the delivered energy? Plus it's not enough to cite a paper to prove that energy is delivered. If the miRNA is complementary to a region that is already paired, the energy you need to unpair this region is the same that you get when you pair it with the miRNA. Result: 0 energy gain.</span><br />
<br />
<br />
In conclusion, these kinds of DNA structures can be suitable, reversible and metastable DNA fuels and a new kind of cargo delivery machine.<br />
<span style="color:blue"> How will they moves? I doubt that turning their wheels will be of any help...</span><br />
<br />
== B.Artificial virus factory ==<br />
<br />
<br />
We can also produce a lot of differentes kinds of self-assembly structures like virus for example,HIV:<br />
<br />
<br />
<br />
[[Image:HIV.jpg|center]]<br />
<br />
Fig2: genetic organization of HIV virus <br />
<br />
HIV can be produce by sequential expression of 3 genes gag,env and pol (Fig.2)those genes will be cleaved by protease P10 and than the subunits of the virus will self-assembled into mature virus.<br />
In our case, we will expressed gag p10 and finaly Env (without Pol to avoid pathogenicity) the FIFO in this case will be essential.<br />
<br />
If you ask us why?<br />
<br />
For 3 main reasons :<br />
<br />
* First, when they genes 1 and 2 will be expressed the protease P10 will cleave gag that will self-assembled.<br />
<br />
* Than when 123 will be expresse Env will get cleaved and self assembled with they products of gag and P10.<br />
<br />
* Finaly, when 23 get activate we will increase the quantity of subunits delivered from the clivage of Env.<br />
<br />
In conclusion, our sytem will be very useful for the developpement of new drugs against HIV or in new kinds of vectors for genetic therapy.<br />
<br />
== Metabolic engineering of polyhydroxyalkanoate biosynthesis pathways ==<br />
Human overpopulation combined with the current lifestyle urges the rational, efficient, and sustainable use of natural resources to produce environmentally friendly plastic materials such as polyhydroxyalkanoic acids (PHAs), whose production/degradation cycle reduces undesirable wastes and emissions.<br />
Our study is a new metabolic strategy to generate PHA-hyperproducer strains, that have the properties to be a sequential metabolic pathway, we believe the sequential expression may increases the production of purified PHA.<br />
<br />
Our strategy consists on replacing the RFP,CFP and YFP genes by the PhaA ,PhaB and PhaC genes in our final system (containing oscillation,FIFO,synchronisation modules).<br />
<br />
This strategy is more efficient than a constitutive activation for 3 main reasons:<br />
*First, in this application the NADPH, which is a cellular metastable fuel, is used by the PhaB to synthesize bioplastic. This molecule is very important for many metabolic pathways in bacteria. A NADPH recuperation step is then needed to ensure other metabolic activities to go on. We can then make the hypothesis that if the PhaB is always activated the bacteria will get exhausted and die quickly (4-5 days). In our system, the bacteria will have a NADPH recuperation step, this is why we hope bacteria will live more than in an usual chemoreactor.<br />
<br />
*Secondly, we hope by the order of the FIFO to make a synchronized and sequential turn-over of the enzymatic expression in order to increase the rate of the PHA biosynthesis.<br />
<br />
*Third, as the quality of the bioplastic increases with time, we could predict the quality of the final product since we would know the duration of a production cycle thanks to the periodicity of our system. <br />
<br />
[[Image:PHA.jpg|center]]<br />
<br />
== Bibliography ==<br />
* Folding DNA to create nanoscale shapes and patterns<br />
Paul W. K. Rothemund <br />
* Design of DNA origami<br />
Paul W. K. Rothemund <br />
* An autonomous polymerization motor powered by DNA hybridization.<br />
Suvir Venkataraman, Robert M. Dirks, Paul W. K. Rothemund, Erik Winfree, Niles A. Pierce.<br />
* Catalyzed Relaxation of a Metastable DNA Fuel.<br />
Georg Seelig, Bernard Yurke, Erik Winfree.</div>David.bikardhttp://2008.igem.org/Team:Paris/PerspectivesTeam:Paris/Perspectives2008-10-28T08:40:45Z<p>David.bikard: /* A.DNA nanocar factory */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
== Biosynthetic Bottum-up approach ==<br />
<br />
==A.DNA nanocar factory ==<br />
<br />
<br />
<br />
The field of artificial molecular machines and motors is growing at an astonishing rate and is attracting a great deal of interest in nanoscience.<br />
Research in the last decade has shown that species made of components like DNA or proteins are attractive candidates.<br />
Our aims is to build by a bottom-up approched DNA nanocars that have the properties to produce energy in vitro. <br />
<br />
<span style="color:blue">You have to explain what you mean by "produce energy in vitro". What energy does it use? to convert into what other energy? or to use in what way? Also, why in vitro? Do you plan to use our system in cell-free extracts or in living cells?</span><br />
<br />
Our system is more efficient than a classic system because the FIFO order is very important: the first part will bind by complementarity with part 2 this new part will make the binding site for part 3. <br />
<br />
Moreover,in the FIFO the first in is the first out meaning that the 2-3 genes is still activate cause the one went off ,in our construction we success to bind only the higher part ,to built the lower part we need to express 2-3 gene that will finalize the car structure. <br />
<span style="color:blue"> This sentence is poorly constructed and full of mistakes </span><br />
<br />
The oscillation of our system can be assimilized to a factory when a certain amount of car his made, we need a little time off to permit them to move ,that will make the place for a new one ,his similar to make cookies when they cook you need to move them to put new ones in the oven.<br />
<span style="color:blue"> This really is farfetched. What will make the cars moove? from were to were?</span><br />
<br />
<br />
<br />
[[Image:car2.jpg|center]]<br />
<br />
Fig.1:biosynthesis by sequential expression of 3 DNA origami<br />
<br />
If we add in vitro a complementary miRNA to the red regions (fig.2), the competition of the miRNA will open the DNA strands by broking the hydrogen backbound this processes deliveres minimun ten time more energy than ATP biodegradation (winfree et.al), the instability of the miRNA may make this processe reversible.<br />
<br />
<span style="color:blue">What is this red region? How will it allow to use the delivered energy? Plus it's not enough to cite a paper to prove that energy is delivered. If the miRNA is complementary to a region that is already paired, the energy you need to unpair this region is the same that you get when you pair it with the miRNA. Result: 0 energy gain.</span><br />
<br />
<br />
In conclusion, these kinds of DNA structures can be suitable, reversible and metastable DNA fuels and a new kind of cargo delivery machine.<br />
<span style="color:blue"> How will they moves? I doubt that turning their wheels will be of any help...</span><br />
<br />
== B.Artificial virus factory ==<br />
<br />
<br />
We can also produce a lot of differentes kinds of self-assembly structures like virus for example,HIV:<br />
<br />
<br />
<br />
[[Image:HIV.jpg|center]]<br />
<br />
Fig2: genetic organization of HIV virus <br />
<br />
HIV can be produce by sequential expression of 3 genes gag,env and pol (Fig.2)those genes will be cleaved by protease P10 and than the subunits of the virus will self-assembled into mature virus.<br />
In our case, we will expressed gag p10 and finaly Env (without Pol to avoid pathogenicity) the FIFO in this case will be essential.<br />
<br />
If you ask us why?<br />
<br />
For 3 main reasons :<br />
<br />
* First, when they genes 1 and 2 will be expressed the protease P10 will cleave gag that will self-assembled.<br />
<br />
* Than when 123 will be expresse Env will get cleaved and self assembled with they products of gag and P10.<br />
<br />
* Finaly, when 23 get activate we will increase the quantity of subunits delivered from the clivage of Env.<br />
<br />
In conclusion, our sytem will be very useful for the developpement of new drugs against HIV or in new kinds of vectors for genetic therapy.<br />
<br />
== Metabolic engineering of polyhydroxyalkanoate biosynthesis pathways ==<br />
Human overpopulation combined with the current lifestyle urges the rational, efficient, and sustainable use of natural resources to produce environmentally friendly plastic materials such as polyhydroxyalkanoic acids (PHAs), whose production/degradation cycle reduces undesirable wastes and emissions.<br />
Our study is a new metabolic strategy to generate PHA-hyperproducer strains, that have the properties to be a sequential metabolic pathway, we believe the sequential expression may increases the production of purified PHA.<br />
<br />
Our strategy consists on replacing the RFP,CFP and YFP genes by the PhaA ,PhaB and PhaC genes in our final system (containing oscillation,FIFO,synchronisation modules).<br />
<br />
This strategy is more efficient than a constitutive activation for 3 main reasons:<br />
*First, in this application the NADPH, which is a cellular metastable fuel, is used by the PhaB to synthesize bioplastic. This molecule is very important for many metabolic pathways in bacteria. A NADPH recuperation step is then needed to ensure other metabolic activities to go on. We can then make the hypothesis that if the PhaB is always activated the bacteria will get exhausted and die quickly (4-5 days). In our system, the bacteria will have a NADPH recuperation step, this is why we hope bacteria will live more than in an usual chemoreactor.<br />
<br />
*Secondly, we hope by the order of the FIFO to make a synchronized and sequential turn-over of the enzymatic expression in order to increase the rate of the PHA biosynthesis.<br />
<br />
*Third, as the quality of the bioplastic increases with time, we could predict the quality of the final product since we would know the duration of a production cycle thanks to the periodicity of our system. <br />
<br />
[[Image:PHA.jpg|center]]<br />
<br />
== Bibliography ==<br />
* Folding DNA to create nanoscale shapes and patterns<br />
Paul W. K. Rothemund <br />
* Design of DNA origami<br />
Paul W. K. Rothemund <br />
* An autonomous polymerization motor powered by DNA hybridization.<br />
Suvir Venkataraman, Robert M. Dirks, Paul W. K. Rothemund, Erik Winfree, Niles A. Pierce.<br />
* Catalyzed Relaxation of a Metastable DNA Fuel.<br />
Georg Seelig, Bernard Yurke, Erik Winfree.</div>David.bikardhttp://2008.igem.org/Team:Paris/PerspectivesTeam:Paris/Perspectives2008-10-28T08:40:18Z<p>David.bikard: /* A.DNA nanocar factory */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
== Biosynthetic Bottum-up approach ==<br />
<br />
==A.DNA nanocar factory ==<br />
<br />
<br />
<br />
The field of artificial molecular machines and motors is growing at an astonishing rate and is attracting a great deal of interest in nanoscience.<br />
Research in the last decade has shown that species made of components like DNA or proteins are attractive candidates.<br />
Our aims is to build by a bottom-up approched DNA nanocars that have the properties to produce energy in vitro. <br />
<br />
<span style="color:blue">You have to explain what you mean by "produce energy in vitro". What energy does it use? to convert into what other energy? or to use in what way? <br />
<br />
Also why in vitro? Do you plan to use our system in cell-free extracts or in living cells?</span><br />
<br />
Our system is more efficient than a classic system because the FIFO order is very important: the first part will bind by complementarity with part 2 this new part will make the binding site for part 3. <br />
<br />
Moreover,in the FIFO the first in is the first out meaning that the 2-3 genes is still activate cause the one went off ,in our construction we success to bind only the higher part ,to built the lower part we need to express 2-3 gene that will finalize the car structure. <br />
<span style="color:blue"> This sentence is poorly constructed and full of mistakes </span><br />
<br />
The oscillation of our system can be assimilized to a factory when a certain amount of car his made, we need a little time off to permit them to move ,that will make the place for a new one ,his similar to make cookies when they cook you need to move them to put new ones in the oven.<br />
<span style="color:blue"> This really is farfetched. What will make the cars moove? from were to were?</span><br />
<br />
<br />
<br />
[[Image:car2.jpg|center]]<br />
<br />
Fig.1:biosynthesis by sequential expression of 3 DNA origami<br />
<br />
If we add in vitro a complementary miRNA to the red regions (fig.2), the competition of the miRNA will open the DNA strands by broking the hydrogen backbound this processes deliveres minimun ten time more energy than ATP biodegradation (winfree et.al), the instability of the miRNA may make this processe reversible.<br />
<br />
<span style="color:blue">What is this red region? How will it allow to use the delivered energy? Plus it's not enough to cite a paper to prove that energy is delivered. If the miRNA is complementary to a region that is already paired, the energy you need to unpair this region is the same that you get when you pair it with the miRNA. Result: 0 energy gain.</span><br />
<br />
<br />
In conclusion, these kinds of DNA structures can be suitable, reversible and metastable DNA fuels and a new kind of cargo delivery machine.<br />
<span style="color:blue"> How will they moves? I doubt that turning their wheels will be of any help...</span><br />
<br />
== B.Artificial virus factory ==<br />
<br />
<br />
We can also produce a lot of differentes kinds of self-assembly structures like virus for example,HIV:<br />
<br />
<br />
<br />
[[Image:HIV.jpg|center]]<br />
<br />
Fig2: genetic organization of HIV virus <br />
<br />
HIV can be produce by sequential expression of 3 genes gag,env and pol (Fig.2)those genes will be cleaved by protease P10 and than the subunits of the virus will self-assembled into mature virus.<br />
In our case, we will expressed gag p10 and finaly Env (without Pol to avoid pathogenicity) the FIFO in this case will be essential.<br />
<br />
If you ask us why?<br />
<br />
For 3 main reasons :<br />
<br />
* First, when they genes 1 and 2 will be expressed the protease P10 will cleave gag that will self-assembled.<br />
<br />
* Than when 123 will be expresse Env will get cleaved and self assembled with they products of gag and P10.<br />
<br />
* Finaly, when 23 get activate we will increase the quantity of subunits delivered from the clivage of Env.<br />
<br />
In conclusion, our sytem will be very useful for the developpement of new drugs against HIV or in new kinds of vectors for genetic therapy.<br />
<br />
== Metabolic engineering of polyhydroxyalkanoate biosynthesis pathways ==<br />
Human overpopulation combined with the current lifestyle urges the rational, efficient, and sustainable use of natural resources to produce environmentally friendly plastic materials such as polyhydroxyalkanoic acids (PHAs), whose production/degradation cycle reduces undesirable wastes and emissions.<br />
Our study is a new metabolic strategy to generate PHA-hyperproducer strains, that have the properties to be a sequential metabolic pathway, we believe the sequential expression may increases the production of purified PHA.<br />
<br />
Our strategy consists on replacing the RFP,CFP and YFP genes by the PhaA ,PhaB and PhaC genes in our final system (containing oscillation,FIFO,synchronisation modules).<br />
<br />
This strategy is more efficient than a constitutive activation for 3 main reasons:<br />
*First, in this application the NADPH, which is a cellular metastable fuel, is used by the PhaB to synthesize bioplastic. This molecule is very important for many metabolic pathways in bacteria. A NADPH recuperation step is then needed to ensure other metabolic activities to go on. We can then make the hypothesis that if the PhaB is always activated the bacteria will get exhausted and die quickly (4-5 days). In our system, the bacteria will have a NADPH recuperation step, this is why we hope bacteria will live more than in an usual chemoreactor.<br />
<br />
*Secondly, we hope by the order of the FIFO to make a synchronized and sequential turn-over of the enzymatic expression in order to increase the rate of the PHA biosynthesis.<br />
<br />
*Third, as the quality of the bioplastic increases with time, we could predict the quality of the final product since we would know the duration of a production cycle thanks to the periodicity of our system. <br />
<br />
[[Image:PHA.jpg|center]]<br />
<br />
== Bibliography ==<br />
* Folding DNA to create nanoscale shapes and patterns<br />
Paul W. K. Rothemund <br />
* Design of DNA origami<br />
Paul W. K. Rothemund <br />
* An autonomous polymerization motor powered by DNA hybridization.<br />
Suvir Venkataraman, Robert M. Dirks, Paul W. K. Rothemund, Erik Winfree, Niles A. Pierce.<br />
* Catalyzed Relaxation of a Metastable DNA Fuel.<br />
Georg Seelig, Bernard Yurke, Erik Winfree.</div>David.bikardhttp://2008.igem.org/Team:Paris/PerspectivesTeam:Paris/Perspectives2008-10-28T08:40:06Z<p>David.bikard: /* A.DNA nanocar factory */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
== Biosynthetic Bottum-up approach ==<br />
<br />
==A.DNA nanocar factory ==<br />
<br />
<br />
<br />
The field of artificial molecular machines and motors is growing at an astonishing rate and is attracting a great deal of interest in nanoscience.<br />
Research in the last decade has shown that species made of components like DNA or proteins are attractive candidates.<br />
Our aims is to build by a bottom-up approched DNA nanocars that have the properties to produce energy in vitro. <br />
<br />
<span style="color:blue">You have to explain what you mean by "produce energy in vitro". What energy does it use? to convert into what other energy? or to use in what way? <br />
<br />
Also why in vitro? Do you plan to use our system in cell-free extracts or in living cells?</span><br />
<br />
Our system is more efficient than a classic system because the FIFO order is very important: the first part will bind by complementarity with part 2 this new part will make the binding site for part 3. <br />
<br />
Moreover,in the FIFO the first in is the first out meaning that the 2-3 genes is still activate cause the one went off ,in our construction we success to bind only the higher part ,to built the lower part we need to express 2-3 gene that will finalize the car structure. <br />
<span style="color:blue"> This sentence is poorly constructed and full of mistakes </span><br />
<br />
The oscillation of our system can be assimilized to a factory when a certain amount of car his made, we need a little time off to permit them to move ,that will make the place for a new one ,his similar to make cookies when they cook you need to move them to put new ones in the oven.<br />
<span style="color:blue"> This really is farfetched. What will make the cars moove? from were to were?</span><br />
<br />
<br />
<br />
[[Image:car2.jpg|center]]<br />
<br />
Fig.1:biosynthesis by sequential expression of 3 DNA origami<br />
<br />
If we add in vitro a complementary miRNA to the red regions (fig.2), the competition of the miRNA will open the DNA strands by broking the hydrogen backbound this processes deliveres minimun ten time more energy than ATP biodegradation (winfree et.al), the instability of the miRNA may make this processe reversible.<br />
<br />
<span style="color:blue">What is this red region? How will it allow to use the delivered energy? Plus it's not enough to cite a paper to prove that energy is delivered. If the miRNA is complementary to a region that is already paired, the energy you need to unpair this region is the same that you get when you pair it with the miRNA. Result: 0 energy gain.</span><br />
<br />
<br />
In conclusion, these kinds of DNA structures can be suitable, reversible and metastable DNA fuels and a new kind of cargo delivery machine.<br />
<span style="color:blue"> How will they moves? I doubt that turning their wheels will be of any help...</span><br />
<br />
== B.Artificial virus factory ==<br />
<br />
<br />
We can also produce a lot of differentes kinds of self-assembly structures like virus for example,HIV:<br />
<br />
<br />
<br />
[[Image:HIV.jpg|center]]<br />
<br />
Fig2: genetic organization of HIV virus <br />
<br />
HIV can be produce by sequential expression of 3 genes gag,env and pol (Fig.2)those genes will be cleaved by protease P10 and than the subunits of the virus will self-assembled into mature virus.<br />
In our case, we will expressed gag p10 and finaly Env (without Pol to avoid pathogenicity) the FIFO in this case will be essential.<br />
<br />
If you ask us why?<br />
<br />
For 3 main reasons :<br />
<br />
* First, when they genes 1 and 2 will be expressed the protease P10 will cleave gag that will self-assembled.<br />
<br />
* Than when 123 will be expresse Env will get cleaved and self assembled with they products of gag and P10.<br />
<br />
* Finaly, when 23 get activate we will increase the quantity of subunits delivered from the clivage of Env.<br />
<br />
In conclusion, our sytem will be very useful for the developpement of new drugs against HIV or in new kinds of vectors for genetic therapy.<br />
<br />
== Metabolic engineering of polyhydroxyalkanoate biosynthesis pathways ==<br />
Human overpopulation combined with the current lifestyle urges the rational, efficient, and sustainable use of natural resources to produce environmentally friendly plastic materials such as polyhydroxyalkanoic acids (PHAs), whose production/degradation cycle reduces undesirable wastes and emissions.<br />
Our study is a new metabolic strategy to generate PHA-hyperproducer strains, that have the properties to be a sequential metabolic pathway, we believe the sequential expression may increases the production of purified PHA.<br />
<br />
Our strategy consists on replacing the RFP,CFP and YFP genes by the PhaA ,PhaB and PhaC genes in our final system (containing oscillation,FIFO,synchronisation modules).<br />
<br />
This strategy is more efficient than a constitutive activation for 3 main reasons:<br />
*First, in this application the NADPH, which is a cellular metastable fuel, is used by the PhaB to synthesize bioplastic. This molecule is very important for many metabolic pathways in bacteria. A NADPH recuperation step is then needed to ensure other metabolic activities to go on. We can then make the hypothesis that if the PhaB is always activated the bacteria will get exhausted and die quickly (4-5 days). In our system, the bacteria will have a NADPH recuperation step, this is why we hope bacteria will live more than in an usual chemoreactor.<br />
<br />
*Secondly, we hope by the order of the FIFO to make a synchronized and sequential turn-over of the enzymatic expression in order to increase the rate of the PHA biosynthesis.<br />
<br />
*Third, as the quality of the bioplastic increases with time, we could predict the quality of the final product since we would know the duration of a production cycle thanks to the periodicity of our system. <br />
<br />
[[Image:PHA.jpg|center]]<br />
<br />
== Bibliography ==<br />
* Folding DNA to create nanoscale shapes and patterns<br />
Paul W. K. Rothemund <br />
* Design of DNA origami<br />
Paul W. K. Rothemund <br />
* An autonomous polymerization motor powered by DNA hybridization.<br />
Suvir Venkataraman, Robert M. Dirks, Paul W. K. Rothemund, Erik Winfree, Niles A. Pierce.<br />
* Catalyzed Relaxation of a Metastable DNA Fuel.<br />
Georg Seelig, Bernard Yurke, Erik Winfree.</div>David.bikardhttp://2008.igem.org/Team:Paris/PerspectivesTeam:Paris/Perspectives2008-10-28T08:39:47Z<p>David.bikard: /* A.DNA nanocar factory */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
== Biosynthetic Bottum-up approach ==<br />
<br />
==A.DNA nanocar factory ==<br />
<br />
<br />
<br />
The field of artificial molecular machines and motors is growing at an astonishing rate and is attracting a great deal of interest in nanoscience.<br />
Research in the last decade has shown that species made of components like DNA or proteins are attractive candidates.<br />
Our aims is to build by a bottom-up approched DNA nanocars that have the properties to produce energy in vitro. <br />
<br />
<span style="color:blue">You have to explain what you mean by "produce energy in vitro". What energy does it use? to convert into what other energy? or to use in what way? </br> Also why in vitro? Do you plan to use our system in cell-free extracts or in living cells?</span><br />
<br />
Our system is more efficient than a classic system because the FIFO order is very important: the first part will bind by complementarity with part 2 this new part will make the binding site for part 3. <br />
<br />
Moreover,in the FIFO the first in is the first out meaning that the 2-3 genes is still activate cause the one went off ,in our construction we success to bind only the higher part ,to built the lower part we need to express 2-3 gene that will finalize the car structure. <br />
<span style="color:blue"> This sentence is poorly constructed and full of mistakes </span><br />
<br />
The oscillation of our system can be assimilized to a factory when a certain amount of car his made, we need a little time off to permit them to move ,that will make the place for a new one ,his similar to make cookies when they cook you need to move them to put new ones in the oven.<br />
<span style="color:blue"> This really is farfetched. What will make the cars moove? from were to were?</span><br />
<br />
<br />
<br />
[[Image:car2.jpg|center]]<br />
<br />
Fig.1:biosynthesis by sequential expression of 3 DNA origami<br />
<br />
If we add in vitro a complementary miRNA to the red regions (fig.2), the competition of the miRNA will open the DNA strands by broking the hydrogen backbound this processes deliveres minimun ten time more energy than ATP biodegradation (winfree et.al), the instability of the miRNA may make this processe reversible.<br />
<br />
<span style="color:blue">What is this red region? How will it allow to use the delivered energy? Plus it's not enough to cite a paper to prove that energy is delivered. If the miRNA is complementary to a region that is already paired, the energy you need to unpair this region is the same that you get when you pair it with the miRNA. Result: 0 energy gain.</span><br />
<br />
<br />
In conclusion, these kinds of DNA structures can be suitable, reversible and metastable DNA fuels and a new kind of cargo delivery machine.<br />
<span style="color:blue"> How will they moves? I doubt that turning their wheels will be of any help...</span><br />
<br />
== B.Artificial virus factory ==<br />
<br />
<br />
We can also produce a lot of differentes kinds of self-assembly structures like virus for example,HIV:<br />
<br />
<br />
<br />
[[Image:HIV.jpg|center]]<br />
<br />
Fig2: genetic organization of HIV virus <br />
<br />
HIV can be produce by sequential expression of 3 genes gag,env and pol (Fig.2)those genes will be cleaved by protease P10 and than the subunits of the virus will self-assembled into mature virus.<br />
In our case, we will expressed gag p10 and finaly Env (without Pol to avoid pathogenicity) the FIFO in this case will be essential.<br />
<br />
If you ask us why?<br />
<br />
For 3 main reasons :<br />
<br />
* First, when they genes 1 and 2 will be expressed the protease P10 will cleave gag that will self-assembled.<br />
<br />
* Than when 123 will be expresse Env will get cleaved and self assembled with they products of gag and P10.<br />
<br />
* Finaly, when 23 get activate we will increase the quantity of subunits delivered from the clivage of Env.<br />
<br />
In conclusion, our sytem will be very useful for the developpement of new drugs against HIV or in new kinds of vectors for genetic therapy.<br />
<br />
== Metabolic engineering of polyhydroxyalkanoate biosynthesis pathways ==<br />
Human overpopulation combined with the current lifestyle urges the rational, efficient, and sustainable use of natural resources to produce environmentally friendly plastic materials such as polyhydroxyalkanoic acids (PHAs), whose production/degradation cycle reduces undesirable wastes and emissions.<br />
Our study is a new metabolic strategy to generate PHA-hyperproducer strains, that have the properties to be a sequential metabolic pathway, we believe the sequential expression may increases the production of purified PHA.<br />
<br />
Our strategy consists on replacing the RFP,CFP and YFP genes by the PhaA ,PhaB and PhaC genes in our final system (containing oscillation,FIFO,synchronisation modules).<br />
<br />
This strategy is more efficient than a constitutive activation for 3 main reasons:<br />
*First, in this application the NADPH, which is a cellular metastable fuel, is used by the PhaB to synthesize bioplastic. This molecule is very important for many metabolic pathways in bacteria. A NADPH recuperation step is then needed to ensure other metabolic activities to go on. We can then make the hypothesis that if the PhaB is always activated the bacteria will get exhausted and die quickly (4-5 days). In our system, the bacteria will have a NADPH recuperation step, this is why we hope bacteria will live more than in an usual chemoreactor.<br />
<br />
*Secondly, we hope by the order of the FIFO to make a synchronized and sequential turn-over of the enzymatic expression in order to increase the rate of the PHA biosynthesis.<br />
<br />
*Third, as the quality of the bioplastic increases with time, we could predict the quality of the final product since we would know the duration of a production cycle thanks to the periodicity of our system. <br />
<br />
[[Image:PHA.jpg|center]]<br />
<br />
== Bibliography ==<br />
* Folding DNA to create nanoscale shapes and patterns<br />
Paul W. K. Rothemund <br />
* Design of DNA origami<br />
Paul W. K. Rothemund <br />
* An autonomous polymerization motor powered by DNA hybridization.<br />
Suvir Venkataraman, Robert M. Dirks, Paul W. K. Rothemund, Erik Winfree, Niles A. Pierce.<br />
* Catalyzed Relaxation of a Metastable DNA Fuel.<br />
Georg Seelig, Bernard Yurke, Erik Winfree.</div>David.bikardhttp://2008.igem.org/Team:Paris/PerspectivesTeam:Paris/Perspectives2008-10-28T08:38:57Z<p>David.bikard: /* A.DNA nanocar factory */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
== Biosynthetic Bottum-up approach ==<br />
<br />
==A.DNA nanocar factory ==<br />
<br />
<br />
<br />
The field of artificial molecular machines and motors is growing at an astonishing rate and is attracting a great deal of interest in nanoscience.<br />
Research in the last decade has shown that species made of components like DNA or proteins are attractive candidates.<br />
Our aims is to build by a bottom-up approched DNA nanocars that have the properties to produce energy in vitro. <br />
<br />
<span style="color:blue">You have to explain what you mean by "produce energy in vitro". What energy does it use to convert into what other energy, or to use it in what way? </br> Also why in vitro? Do you plan to use our system in cell-free extracts or in living cells?</span><br />
<br />
Our system is more efficient than a classic system because the FIFO order is very important: the first part will bind by complementarity with part 2 this new part will make the binding site for part 3. <br />
<br />
Moreover,in the FIFO the first in is the first out meaning that the 2-3 genes is still activate cause the one went off ,in our construction we success to bind only the higher part ,to built the lower part we need to express 2-3 gene that will finalize the car structure. <br />
<span style="color:blue"> This sentence is poorly constructed and full of mistakes </span><br />
<br />
The oscillation of our system can be assimilized to a factory when a certain amount of car his made, we need a little time off to permit them to move ,that will make the place for a new one ,his similar to make cookies when they cook you need to move them to put new ones in the oven.<br />
<span style="color:blue"> This really is farfetched. What will make the cars moove? from were to were?</span><br />
<br />
<br />
<br />
[[Image:car2.jpg|center]]<br />
<br />
Fig.1:biosynthesis by sequential expression of 3 DNA origami<br />
<br />
If we add in vitro a complementary miRNA to the red regions (fig.2), the competition of the miRNA will open the DNA strands by broking the hydrogen backbound this processes deliveres minimun ten time more energy than ATP biodegradation (winfree et.al), the instability of the miRNA may make this processe reversible.<br />
<br />
<span style="color:blue">What is this red region? How will it allow to use the delivered energy? Plus it's not enough to cite a paper to prove that energy is delivered. If the miRNA is complementary to a region that is already paired, the energy you need to unpair this region is the same that you get when you pair it with the miRNA. Result: 0 energy gain.</span><br />
<br />
<br />
In conclusion, these kinds of DNA structures can be suitable, reversible and metastable DNA fuels and a new kind of cargo delivery machine.<br />
<span style="color:blue"> How will they moves? I doubt that turning their wheels will be of any help...</span><br />
<br />
== B.Artificial virus factory ==<br />
<br />
<br />
We can also produce a lot of differentes kinds of self-assembly structures like virus for example,HIV:<br />
<br />
<br />
<br />
[[Image:HIV.jpg|center]]<br />
<br />
Fig2: genetic organization of HIV virus <br />
<br />
HIV can be produce by sequential expression of 3 genes gag,env and pol (Fig.2)those genes will be cleaved by protease P10 and than the subunits of the virus will self-assembled into mature virus.<br />
In our case, we will expressed gag p10 and finaly Env (without Pol to avoid pathogenicity) the FIFO in this case will be essential.<br />
<br />
If you ask us why?<br />
<br />
For 3 main reasons :<br />
<br />
* First, when they genes 1 and 2 will be expressed the protease P10 will cleave gag that will self-assembled.<br />
<br />
* Than when 123 will be expresse Env will get cleaved and self assembled with they products of gag and P10.<br />
<br />
* Finaly, when 23 get activate we will increase the quantity of subunits delivered from the clivage of Env.<br />
<br />
In conclusion, our sytem will be very useful for the developpement of new drugs against HIV or in new kinds of vectors for genetic therapy.<br />
<br />
== Metabolic engineering of polyhydroxyalkanoate biosynthesis pathways ==<br />
Human overpopulation combined with the current lifestyle urges the rational, efficient, and sustainable use of natural resources to produce environmentally friendly plastic materials such as polyhydroxyalkanoic acids (PHAs), whose production/degradation cycle reduces undesirable wastes and emissions.<br />
Our study is a new metabolic strategy to generate PHA-hyperproducer strains, that have the properties to be a sequential metabolic pathway, we believe the sequential expression may increases the production of purified PHA.<br />
<br />
Our strategy consists on replacing the RFP,CFP and YFP genes by the PhaA ,PhaB and PhaC genes in our final system (containing oscillation,FIFO,synchronisation modules).<br />
<br />
This strategy is more efficient than a constitutive activation for 3 main reasons:<br />
*First, in this application the NADPH, which is a cellular metastable fuel, is used by the PhaB to synthesize bioplastic. This molecule is very important for many metabolic pathways in bacteria. A NADPH recuperation step is then needed to ensure other metabolic activities to go on. We can then make the hypothesis that if the PhaB is always activated the bacteria will get exhausted and die quickly (4-5 days). In our system, the bacteria will have a NADPH recuperation step, this is why we hope bacteria will live more than in an usual chemoreactor.<br />
<br />
*Secondly, we hope by the order of the FIFO to make a synchronized and sequential turn-over of the enzymatic expression in order to increase the rate of the PHA biosynthesis.<br />
<br />
*Third, as the quality of the bioplastic increases with time, we could predict the quality of the final product since we would know the duration of a production cycle thanks to the periodicity of our system. <br />
<br />
[[Image:PHA.jpg|center]]<br />
<br />
== Bibliography ==<br />
* Folding DNA to create nanoscale shapes and patterns<br />
Paul W. K. Rothemund <br />
* Design of DNA origami<br />
Paul W. K. Rothemund <br />
* An autonomous polymerization motor powered by DNA hybridization.<br />
Suvir Venkataraman, Robert M. Dirks, Paul W. K. Rothemund, Erik Winfree, Niles A. Pierce.<br />
* Catalyzed Relaxation of a Metastable DNA Fuel.<br />
Georg Seelig, Bernard Yurke, Erik Winfree.</div>David.bikardhttp://2008.igem.org/Paris/FFLParis/FFL2008-10-25T14:54:17Z<p>David.bikard: New page: A Feed-Forward Loop is a genetic network composed of three nodes. This strong network motif is composed of a transcription factor X that regulates a second transcription factor, Y, and bot...</p>
<hr />
<div>A Feed-Forward Loop is a genetic network composed of three nodes. This strong network motif is composed of a transcription factor X that regulates a second transcription factor, Y, and both X and Y regulate Z (Figure 1).<br />
<br />
Depending on the type of regulations between the different nodes, we can define eight types of FFL that can be classified into two groups : ''coherent'' and ''incoherent'' FFLs. In ''coherent'' FFLs, the indirect path has the same overall sign as the direct path. The most abundant FFL is the type-1 coherent FFL (C1-FFL).<br />
<br />
In addition to the signs of the edges, to understand the dynamics of the FFL, we must also know how the inputs from the two regulators X and Y are integrated at the promoter of the gene Z. Uri ALON considers that there are two biologically reasonable logic functions : "AND" logic, in which ''both'' X and Y activities are need to be high in order to turn on Z expression and "OR" logic in which ''either'' X or Y is sufficient.</div>David.bikardhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-25T14:34:39Z<p>David.bikard: </p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
= Summary =<br />
Our project aims at biologically devising a “oscillating FIFO behaviour, synchronized at population level”. Such a setup will trigger periodic events and,therefore, can be considered as a “biological clock”. To completely deserve this appellation, the system has to fulfill the following specifications :<br />
<br />
* Oscillatory system : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer.<br />
* FIFO System : The period of the oscillation is even more interesting if fit allows the sequential switching on and off of several genes. Our setup involves three genes which will get activated and desactivated successively as a “FIFO : First In, First Out”. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).<br />
* Synchronization : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
We will base our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella.<br />
<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer. You would accumulate unprocessed intermediates !</span><br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed [http://www.ncbi.nlm.nih.gov/pubmed/15186773?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum| (Kalir S., Alon U. 2004)] that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO. <br />
<br />
* [[Paris/FFL|Genetic implementation of the FIFO]]: the Feed Forward Loop<br />
* [[Paris/Motivations/E.Coli Flagella| Description of ''E. coli'' flagellum regulatory network]]<br />
<br />
== Oscillations ==<br />
<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology. A master of those systems will surely lead to great scientific breakthroughs in health care and medicine.<br />
<br />
== Oscillating FIFO ==<br />
<br />
<br />
<br />
<br />
<center>[https://static.igem.org/mediawiki/2008/7/78/IGEM_Paris_2008_project.pdf More details [PDF]]</center><br />
<br />
= The 3 Modules =<br />
<br />
*[[Team:Paris/Project/Oscillations|Oscillations]]<br />
*[[Team:Paris/Project/FIFO|FIFO]]<br />
*[[Team:Paris/Project/Synchronisation|Synchronisation]]</div>David.bikardhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-25T14:28:06Z<p>David.bikard: /* FIFO */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
= Summary =<br />
Our project aims at biologically devising a “oscillating FIFO behaviour, synchronized at population level”. Such a setup will trigger periodic events and,therefore, can be considered as a “biological clock”. To completely deserve this appellation, the system has to fulfill the following specifications :<br />
<br />
* Oscillatory system : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer.<br />
* FIFO System : The period of the oscillation is even more interesting if fit allows the sequential switching on and off of several genes. Our setup involves three genes which will get activated and desactivated successively as a “FIFO : First In, First Out”. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).<br />
* Synchronization : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
We will base our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella.<br />
<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer. You would accumulate unprocessed intermediates !</span><br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed [http://www.ncbi.nlm.nih.gov/pubmed/15186773?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum| (Kalir S., Alon U. 2004)] that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO. <br />
<br />
* [[Paris/FFL|Genetic implementation of the FIFO]]: the Feed Forward Loop<br />
* [[Paris/Motivations/E.Coli Flagella| Description of ''E. coli'' flagellum regulatory network]]<br />
<br />
== Oscillations ==<br />
<br />
<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology. A master of those systems will surely lead to great scientific breakthroughs in health care and medicine.<br />
<br />
== Oscillating FIFO ==<br />
<br />
<br />
<br />
<br />
<center>[https://static.igem.org/mediawiki/2008/7/78/IGEM_Paris_2008_project.pdf More details [PDF]]</center><br />
<br />
= The 3 Modules =<br />
<br />
*[[Team:Paris/Project/Oscillations|Oscillations]]<br />
*[[Team:Paris/Project/FIFO|FIFO]]<br />
*[[Team:Paris/Project/Synchronisation|Synchronisation]]</div>David.bikardhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-25T14:27:46Z<p>David.bikard: /* FIFO */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
= Summary =<br />
Our project aims at biologically devising a “oscillating FIFO behaviour, synchronized at population level”. Such a setup will trigger periodic events and,therefore, can be considered as a “biological clock”. To completely deserve this appellation, the system has to fulfill the following specifications :<br />
<br />
* Oscillatory system : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer.<br />
* FIFO System : The period of the oscillation is even more interesting if fit allows the sequential switching on and off of several genes. Our setup involves three genes which will get activated and desactivated successively as a “FIFO : First In, First Out”. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).<br />
* Synchronization : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
We will base our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella.<br />
<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer. You would accumulate unprocessed intermediates !</span><br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed [http://www.ncbi.nlm.nih.gov/pubmed/15186773?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum| (Kalir S., Alon U. 2004)] that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO. <br />
<br />
* [[Paris/FFL|Genetic implementation of the FIFO]]: the Feed Forward Loop<br />
* Description of ''E. coli'' flagellum regulatory network [[Paris/Motivations/E.Coli Flagella| here]]<br />
<br />
== Oscillations ==<br />
<br />
<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology. A master of those systems will surely lead to great scientific breakthroughs in health care and medicine.<br />
<br />
== Oscillating FIFO ==<br />
<br />
<br />
<br />
<br />
<center>[https://static.igem.org/mediawiki/2008/7/78/IGEM_Paris_2008_project.pdf More details [PDF]]</center><br />
<br />
= The 3 Modules =<br />
<br />
*[[Team:Paris/Project/Oscillations|Oscillations]]<br />
*[[Team:Paris/Project/FIFO|FIFO]]<br />
*[[Team:Paris/Project/Synchronisation|Synchronisation]]</div>David.bikardhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-25T14:20:22Z<p>David.bikard: /* FIFO */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
= Summary =<br />
Our project aims at biologically devising a “oscillating FIFO behaviour, synchronized at population level”. Such a setup will trigger periodic events and,therefore, can be considered as a “biological clock”. To completely deserve this appellation, the system has to fulfill the following specifications :<br />
<br />
* Oscillatory system : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer.<br />
* FIFO System : The period of the oscillation is even more interesting if fit allows the sequential switching on and off of several genes. Our setup involves three genes which will get activated and desactivated successively as a “FIFO : First In, First Out”. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).<br />
* Synchronization : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
We will base our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella.<br />
<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer. You would accumulate unprocessed intermediates !</span><br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed [http://www.ncbi.nlm.nih.gov/pubmed/15186773?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum| (Kalir S., Alon U. 2004)] that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO. <br />
<br />
For a detailed description of ''E. coli'' flagellum regulatory network, please go [[Paris/Motivations/E.Coli Flagella| here]]<br />
<br />
== Oscillations ==<br />
<br />
<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology. A master of those systems will surely lead to great scientific breakthroughs in health care and medicine.<br />
<br />
== Oscillating FIFO ==<br />
<br />
<br />
<br />
<br />
<center>[https://static.igem.org/mediawiki/2008/7/78/IGEM_Paris_2008_project.pdf More details [PDF]]</center><br />
<br />
= The 3 Modules =<br />
<br />
*[[Team:Paris/Project/Oscillations|Oscillations]]<br />
*[[Team:Paris/Project/FIFO|FIFO]]<br />
*[[Team:Paris/Project/Synchronisation|Synchronisation]]</div>David.bikardhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-25T14:20:03Z<p>David.bikard: /* FIFO */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
= Summary =<br />
Our project aims at biologically devising a “oscillating FIFO behaviour, synchronized at population level”. Such a setup will trigger periodic events and,therefore, can be considered as a “biological clock”. To completely deserve this appellation, the system has to fulfill the following specifications :<br />
<br />
* Oscillatory system : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer.<br />
* FIFO System : The period of the oscillation is even more interesting if fit allows the sequential switching on and off of several genes. Our setup involves three genes which will get activated and desactivated successively as a “FIFO : First In, First Out”. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).<br />
* Synchronization : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
We will base our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella.<br />
<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer :-) You would accumulate unprocessed intermediates!</span><br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed [http://www.ncbi.nlm.nih.gov/pubmed/15186773?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum| (Kalir S., Alon U. 2004)] that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO. <br />
<br />
For a detailed description of ''E. coli'' flagellum regulatory network, please go [[Paris/Motivations/E.Coli Flagella| here]]<br />
<br />
== Oscillations ==<br />
<br />
<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology. A master of those systems will surely lead to great scientific breakthroughs in health care and medicine.<br />
<br />
== Oscillating FIFO ==<br />
<br />
<br />
<br />
<br />
<center>[https://static.igem.org/mediawiki/2008/7/78/IGEM_Paris_2008_project.pdf More details [PDF]]</center><br />
<br />
= The 3 Modules =<br />
<br />
*[[Team:Paris/Project/Oscillations|Oscillations]]<br />
*[[Team:Paris/Project/FIFO|FIFO]]<br />
*[[Team:Paris/Project/Synchronisation|Synchronisation]]</div>David.bikardhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-25T14:13:20Z<p>David.bikard: /* FIFO */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
= Summary =<br />
Our project aims at biologically devising a “oscillating FIFO behaviour, synchronized at population level”. Such a setup will trigger periodic events and,therefore, can be considered as a “biological clock”. To completely deserve this appellation, the system has to fulfill the following specifications :<br />
<br />
* Oscillatory system : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer.<br />
* FIFO System : The period of the oscillation is even more interesting if fit allows the sequential switching on and off of several genes. Our setup involves three genes which will get activated and desactivated successively as a “FIFO : First In, First Out”. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).<br />
* Synchronization : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
We will base our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella.<br />
<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer :-) You would accumulate unprocessed intermediates!</span><br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed [http://www.ncbi.nlm.nih.gov/pubmed/15186773?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum| (Kalir S., Alon U. 2004)] that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO. <br />
<br />
For a detailed description of E.Coli flagellum regulatory network, please go [[Paris/Motivations/E.Coli Flagella| here]]<br />
<br />
== Oscillations ==<br />
<br />
<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology. A master of those systems will surely lead to great scientific breakthroughs in health care and medicine.<br />
<br />
== Oscillating FIFO ==<br />
<br />
<br />
<br />
<br />
<center>[https://static.igem.org/mediawiki/2008/7/78/IGEM_Paris_2008_project.pdf More details [PDF]]</center><br />
<br />
= The 3 Modules =<br />
<br />
*[[Team:Paris/Project/Oscillations|Oscillations]]<br />
*[[Team:Paris/Project/FIFO|FIFO]]<br />
*[[Team:Paris/Project/Synchronisation|Synchronisation]]</div>David.bikardhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-25T14:07:36Z<p>David.bikard: /* FIFO */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
= Summary =<br />
Our project aims at biologically devising a “oscillating FIFO behaviour, synchronized at population level”. Such a setup will trigger periodic events and,therefore, can be considered as a “biological clock”. To completely deserve this appellation, the system has to fulfill the following specifications :<br />
<br />
* Oscillatory system : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer.<br />
* FIFO System : The period of the oscillation is even more interesting if fit allows the sequential switching on and off of several genes. Our setup involves three genes which will get activated and desactivated successively as a “FIFO : First In, First Out”. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).<br />
* Synchronization : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
We will base our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella.<br />
<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
[[Image:fries.jpg|thumb|The preparation of French fries is a good example of FIFO behavior]]<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer :-) You would accumulate unprocessed intermediates!</span><br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed [http://www.ncbi.nlm.nih.gov/pubmed/15186773?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum| (Kalir S., Alon U. 2004)] that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO. <br />
<br />
For a detailed description of E.Coli flagellum regulatory network, please go [[Paris/Motivations/E.Coli Flagella| here]]<br />
<br />
== Oscillations ==<br />
<br />
<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology. A master of those systems will surely lead to great scientific breakthroughs in health care and medicine.<br />
<br />
== Oscillating FIFO ==<br />
<br />
<br />
<br />
<br />
<center>[https://static.igem.org/mediawiki/2008/7/78/IGEM_Paris_2008_project.pdf More details [PDF]]</center><br />
<br />
= The 3 Modules =<br />
<br />
*[[Team:Paris/Project/Oscillations|Oscillations]]<br />
*[[Team:Paris/Project/FIFO|FIFO]]<br />
*[[Team:Paris/Project/Synchronisation|Synchronisation]]</div>David.bikardhttp://2008.igem.org/File:Fries.jpgFile:Fries.jpg2008-10-25T14:06:54Z<p>David.bikard: </p>
<hr />
<div></div>David.bikardhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-25T14:06:24Z<p>David.bikard: /* FIFO */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
= Summary =<br />
Our project aims at biologically devising a “oscillating FIFO behaviour, synchronized at population level”. Such a setup will trigger periodic events and,therefore, can be considered as a “biological clock”. To completely deserve this appellation, the system has to fulfill the following specifications :<br />
<br />
* Oscillatory system : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer.<br />
* FIFO System : The period of the oscillation is even more interesting if fit allows the sequential switching on and off of several genes. Our setup involves three genes which will get activated and desactivated successively as a “FIFO : First In, First Out”. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).<br />
* Synchronization : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
We will base our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella.<br />
<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
[[Image:fries.jpg|thumb|The fryer is ON.]]<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer :-) You would accumulate unprocessed intermediates!</span><br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed [http://www.ncbi.nlm.nih.gov/pubmed/15186773?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum| (Kalir S., Alon U. 2004)] that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO. <br />
<br />
For a detailed description of E.Coli flagellum regulatory network, please go [[Paris/Motivations/E.Coli Flagella| here]]<br />
<br />
== Oscillations ==<br />
<br />
<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology. A master of those systems will surely lead to great scientific breakthroughs in health care and medicine.<br />
<br />
== Oscillating FIFO ==<br />
<br />
<br />
<br />
<br />
<center>[https://static.igem.org/mediawiki/2008/7/78/IGEM_Paris_2008_project.pdf More details [PDF]]</center><br />
<br />
= The 3 Modules =<br />
<br />
*[[Team:Paris/Project/Oscillations|Oscillations]]<br />
*[[Team:Paris/Project/FIFO|FIFO]]<br />
*[[Team:Paris/Project/Synchronisation|Synchronisation]]</div>David.bikardhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-25T14:06:06Z<p>David.bikard: /* FIFO */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
= Summary =<br />
Our project aims at biologically devising a “oscillating FIFO behaviour, synchronized at population level”. Such a setup will trigger periodic events and,therefore, can be considered as a “biological clock”. To completely deserve this appellation, the system has to fulfill the following specifications :<br />
<br />
* Oscillatory system : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer.<br />
* FIFO System : The period of the oscillation is even more interesting if fit allows the sequential switching on and off of several genes. Our setup involves three genes which will get activated and desactivated successively as a “FIFO : First In, First Out”. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).<br />
* Synchronization : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
We will base our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella.<br />
<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
[[Image:Fries.jpg|thumb|The fryer is ON.]]<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer :-) You would accumulate unprocessed intermediates!</span><br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed [http://www.ncbi.nlm.nih.gov/pubmed/15186773?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum| (Kalir S., Alon U. 2004)] that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO. <br />
<br />
For a detailed description of E.Coli flagellum regulatory network, please go [[Paris/Motivations/E.Coli Flagella| here]]<br />
<br />
== Oscillations ==<br />
<br />
<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology. A master of those systems will surely lead to great scientific breakthroughs in health care and medicine.<br />
<br />
== Oscillating FIFO ==<br />
<br />
<br />
<br />
<br />
<center>[https://static.igem.org/mediawiki/2008/7/78/IGEM_Paris_2008_project.pdf More details [PDF]]</center><br />
<br />
= The 3 Modules =<br />
<br />
*[[Team:Paris/Project/Oscillations|Oscillations]]<br />
*[[Team:Paris/Project/FIFO|FIFO]]<br />
*[[Team:Paris/Project/Synchronisation|Synchronisation]]</div>David.bikardhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-25T14:04:15Z<p>David.bikard: /* FIFO */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
= Summary =<br />
Our project aims at biologically devising a “oscillating FIFO behaviour, synchronized at population level”. Such a setup will trigger periodic events and,therefore, can be considered as a “biological clock”. To completely deserve this appellation, the system has to fulfill the following specifications :<br />
<br />
* Oscillatory system : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer.<br />
* FIFO System : The period of the oscillation is even more interesting if fit allows the sequential switching on and off of several genes. Our setup involves three genes which will get activated and desactivated successively as a “FIFO : First In, First Out”. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).<br />
* Synchronization : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
We will base our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella.<br />
<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
[[Image:Fryer.jpg|thumb|The fryer is ON.]]<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer :-) You would accumulate unprocessed intermediates!</span><br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed [http://www.ncbi.nlm.nih.gov/pubmed/15186773?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum| (Kalir S., Alon U. 2004)] that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO. <br />
<br />
For a detailed description of E.Coli flagellum regulatory network, please go [[Paris/Motivations/E.Coli Flagella| here]]<br />
<br />
== Oscillations ==<br />
<br />
<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology. A master of those systems will surely lead to great scientific breakthroughs in health care and medicine.<br />
<br />
== Oscillating FIFO ==<br />
<br />
<br />
<br />
<br />
<center>[https://static.igem.org/mediawiki/2008/7/78/IGEM_Paris_2008_project.pdf More details [PDF]]</center><br />
<br />
= The 3 Modules =<br />
<br />
*[[Team:Paris/Project/Oscillations|Oscillations]]<br />
*[[Team:Paris/Project/FIFO|FIFO]]<br />
*[[Team:Paris/Project/Synchronisation|Synchronisation]]</div>David.bikardhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-25T14:03:50Z<p>David.bikard: /* FIFO */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
= Summary =<br />
Our project aims at biologically devising a “oscillating FIFO behaviour, synchronized at population level”. Such a setup will trigger periodic events and,therefore, can be considered as a “biological clock”. To completely deserve this appellation, the system has to fulfill the following specifications :<br />
<br />
* Oscillatory system : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer.<br />
* FIFO System : The period of the oscillation is even more interesting if fit allows the sequential switching on and off of several genes. Our setup involves three genes which will get activated and desactivated successively as a “FIFO : First In, First Out”. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).<br />
* Synchronization : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
We will base our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella.<br />
<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
[[Image:Fryer.jpg|thumb|The fryer is ON.]]<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer :-) You would accumulate unprocessed intermediates!</span><br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed [http://www.ncbi.nlm.nih.gov/pubmed/15186773?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum| (Kalir S, Alon U. 2004)] that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO. <br />
<br />
For a detailed description of E.Coli flagellum regulatory network, please go [[Paris/Motivations/E.Coli Flagella| here]]<br />
<br />
== Oscillations ==<br />
<br />
<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology. A master of those systems will surely lead to great scientific breakthroughs in health care and medicine.<br />
<br />
== Oscillating FIFO ==<br />
<br />
<br />
<br />
<br />
<center>[https://static.igem.org/mediawiki/2008/7/78/IGEM_Paris_2008_project.pdf More details [PDF]]</center><br />
<br />
= The 3 Modules =<br />
<br />
*[[Team:Paris/Project/Oscillations|Oscillations]]<br />
*[[Team:Paris/Project/FIFO|FIFO]]<br />
*[[Team:Paris/Project/Synchronisation|Synchronisation]]</div>David.bikardhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-25T14:03:27Z<p>David.bikard: /* FIFO */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
= Summary =<br />
Our project aims at biologically devising a “oscillating FIFO behaviour, synchronized at population level”. Such a setup will trigger periodic events and,therefore, can be considered as a “biological clock”. To completely deserve this appellation, the system has to fulfill the following specifications :<br />
<br />
* Oscillatory system : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer.<br />
* FIFO System : The period of the oscillation is even more interesting if fit allows the sequential switching on and off of several genes. Our setup involves three genes which will get activated and desactivated successively as a “FIFO : First In, First Out”. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).<br />
* Synchronization : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
We will base our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella.<br />
<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
[[Image:Fryer.jpg|thumb|The fryer is ON.]]<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer :-) You would accumulate unprocessed intermediates!</span><br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed [http://www.ncbi.nlm.nih.gov/pubmed/15186773?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum|(Kalir S, Alon U. 2004)] that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO. <br />
<br />
For a detailed description of E.Coli flagellum regulatory network, please go [[Paris/Motivations/E.Coli Flagella| here]]<br />
<br />
== Oscillations ==<br />
<br />
<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology. A master of those systems will surely lead to great scientific breakthroughs in health care and medicine.<br />
<br />
== Oscillating FIFO ==<br />
<br />
<br />
<br />
<br />
<center>[https://static.igem.org/mediawiki/2008/7/78/IGEM_Paris_2008_project.pdf More details [PDF]]</center><br />
<br />
= The 3 Modules =<br />
<br />
*[[Team:Paris/Project/Oscillations|Oscillations]]<br />
*[[Team:Paris/Project/FIFO|FIFO]]<br />
*[[Team:Paris/Project/Synchronisation|Synchronisation]]</div>David.bikardhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-25T13:56:33Z<p>David.bikard: /* FIFO */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
= Summary =<br />
Our project aims at biologically devising a “oscillating FIFO behaviour, synchronized at population level”. Such a setup will trigger periodic events and,therefore, can be considered as a “biological clock”. To completely deserve this appellation, the system has to fulfill the following specifications :<br />
<br />
* Oscillatory system : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer.<br />
* FIFO System : The period of the oscillation is even more interesting if fit allows the sequential switching on and off of several genes. Our setup involves three genes which will get activated and desactivated successively as a “FIFO : First In, First Out”. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).<br />
* Synchronization : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
We will base our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella.<br />
<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
[[Image:Fryer.jpg|thumb|The fryer is ON.]]<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer :-) You would accumulate unprocessed intermediates!</span><br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed (Alon, ...) that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO. <br />
<br />
For a detailed description of E.Coli flagellum regulatory network, please go [[Paris/Motivations/E.Coli Flagella| here]]<br />
<br />
== Oscillations ==<br />
<br />
<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology. A master of those systems will surely lead to great scientific breakthroughs in health care and medicine.<br />
<br />
== Oscillating FIFO ==<br />
<br />
<br />
<br />
<br />
<center>[https://static.igem.org/mediawiki/2008/7/78/IGEM_Paris_2008_project.pdf More details [PDF]]</center><br />
<br />
= The 3 Modules =<br />
<br />
*[[Team:Paris/Project/Oscillations|Oscillations]]<br />
*[[Team:Paris/Project/FIFO|FIFO]]<br />
*[[Team:Paris/Project/Synchronisation|Synchronisation]]</div>David.bikardhttp://2008.igem.org/File:Fryer.jpgFile:Fryer.jpg2008-10-25T13:55:45Z<p>David.bikard: </p>
<hr />
<div></div>David.bikardhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-25T13:53:01Z<p>David.bikard: /* Oscillations */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
= Summary =<br />
Our project aims at biologically devising a “oscillating FIFO behaviour, synchronized at population level”. Such a setup will trigger periodic events and,therefore, can be considered as a “biological clock”. To completely deserve this appellation, the system has to fulfill the following specifications :<br />
<br />
* Oscillatory system : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer.<br />
* FIFO System : The period of the oscillation is even more interesting if fit allows the sequential switching on and off of several genes. Our setup involves three genes which will get activated and desactivated successively as a “FIFO : First In, First Out”. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).<br />
* Synchronization : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
We will base our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella.<br />
<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer :-) You would accumulate unprocessed intermediates!</span><br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed (Alon, ...) that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO. <br />
<br />
For a detailed description of E.Coli flagellum regulatory network, please go [[Paris/Motivations/E.Coli Flagella| here]]<br />
<br />
== Oscillations ==<br />
<br />
<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology. A master of those systems will surely lead to great scientific breakthroughs in health care and medicine.<br />
<br />
== Oscillating FIFO ==<br />
<br />
<br />
<br />
<br />
<center>[https://static.igem.org/mediawiki/2008/7/78/IGEM_Paris_2008_project.pdf More details [PDF]]</center><br />
<br />
= The 3 Modules =<br />
<br />
*[[Team:Paris/Project/Oscillations|Oscillations]]<br />
*[[Team:Paris/Project/FIFO|FIFO]]<br />
*[[Team:Paris/Project/Synchronisation|Synchronisation]]</div>David.bikardhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-25T13:44:47Z<p>David.bikard: /* Challenge for synthetic biology */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
= Summary =<br />
Our project aims at biologically devising a “oscillating FIFO behaviour, synchronized at population level”. Such a setup will trigger periodic events and,therefore, can be considered as a “biological clock”. To completely deserve this appellation, the system has to fulfill the following specifications :<br />
<br />
* Oscillatory system : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer.<br />
* FIFO System : The period of the oscillation is even more interesting if fit allows the sequential switching on and off of several genes. Our setup involves three genes which will get activated and desactivated successively as a “FIFO : First In, First Out”. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).<br />
* Synchronization : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
We will base our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella.<br />
<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer :-) You would accumulate unprocessed intermediates!</span><br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed (Alon, ...) that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO. <br />
<br />
For a detailed description of E.Coli flagellum regulatory network, please go [[Paris/Motivations/E.Coli Flagella| here]]<br />
<br />
== Oscillations ==<br />
<br />
<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead to this complex behavior with different properties : number of cycles, oscillating period and robustness. <br />
<br />
Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology. A success will probably lead<br />
<br />
== Oscillating FIFO ==<br />
<br />
<br />
<br />
<br />
<center>[https://static.igem.org/mediawiki/2008/7/78/IGEM_Paris_2008_project.pdf More details [PDF]]</center><br />
<br />
= The 3 Modules =<br />
<br />
*[[Team:Paris/Project/Oscillations|Oscillations]]<br />
*[[Team:Paris/Project/FIFO|FIFO]]<br />
*[[Team:Paris/Project/Synchronisation|Synchronisation]]</div>David.bikard