Team:Bologna/Wetlab

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<div style="text-align:justify">
<div style="text-align:justify">
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=LAB Experiment =
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__FORCETOC__
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=Introduction=
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[[Image:Collage2.jpg|700px]]
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[[Image:Collage2.jpg|700px|right]]
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To obtain regulated promoters we synthesized four libraries of operator sequences, respectively for LacI, TetR, cI and LexA repressor proteins. Here is explained how we designed the operator libraries and isolated single operators to clone them in the standard format. Moreover, we built and tested: a LacI regulated promoter to tune promoter activation and a lexA based promoter to study the SOS activation (UV and peroxidase).
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[https://2008.igem.org/Team:Bologna/Wetlab ''Up'']
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=At last... Operator sites as BioBricks!=
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Even though transcriptional regulation still plays a pivotal role in synthetic biology, a modular assembly of regulated promoters and the characterization of their properties has not been formalized, yet. At present state, each promoter in the Registry, though complex, is treated as a “standalone” monolithic element. Thus, the choice of a regulated promoter implies a prefixed sigmoid regulation curve. The only option, using the current BioBricks, is to choose a discretional scaling factor in the transfer of the transcription function to the protein through an appropriate RBS. Critically, the choice of one specific transcription factor limits the choice to just one possible promoter. The assembly of regulated promoters as the combination of modular parts, i. e. unregulated promoters and operators, could permit the rapid design of regulatory elements with fixed  characteristics. In fact, promoter transcriptional strength and repressor binding affinity could be independently chosen.
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A first step in the rationalization of promoter design was done in the iGEM 2007 with the introduction in the Registry of a family of constitutive promoters. Each family element differs from the others just for few base pairs in -35 and -10 regions, keeping constant the rest of the sequence and giving rise to a different level of transcription strength. We decided to use this valuable work as a platform for a deeper and more general promoter design strategy. To obtain regulated promoters we decided to combine these parts with short regulatory sequences that operate as a binding site for the transcription factor. To this aim, we synthesized four libraries of operator sequences, respectively for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 LacI], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079046 TetR], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079047 cI] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079048 LexA] repressor proteins ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 see details]). In each library, there are three sequences (see Table below) each with a different repressor binding affinity to the repressor protein. Since the libraries were synthetized on pGA18 and pMA Geneart vectors, we isolated each operator with the intention to clone them into BioBrick standard assembly plasmids. The choice of the operator should give a relative fine-tuning of promoter sensitivity to the repressor. We decided to take the Berkley's costitutive promoter library as a good "collection" from which we could select the unregulated promoter, according with the chosen transcriptional strength.
<br>
<br>
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= Uv Sensitive Trigger =
 
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In the genetic flip-flop, the amount of LacI to set on the memory is induced by an UV sensitive trigger. Production of LacI molecules by UV induction can be tested replacing the LacI gene by GFP, so it is possible to have a relation of the strength of LacI synthesis measuring the value of fluorescence.
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{| align="left"
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BBa_K079049 and BBa_K079050  are two new constructs submitted to the registry by Bologna’s Igem Team 2008. These UV test circuits can be presented schematically by the following sequences with different promoter (J23118 with 1429 strength and J23100 with 2547 strength).
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|- style="background: #c09a6d; text-align: center;"
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|colspan=3| <font size="+1">LacI</font>
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|- style="background: #c09a6d; text-align: center;"
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|width=100| '''Name'''
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|width=180| '''Sequence'''
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|width=150| '''Affinity'''
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|- style="background:#f6efcd; color:black"
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| [http://partsregistry.org/Part:BBa_K079017 Lac SymL]
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| aattgtgagcgctcacaatt
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| Very Strong
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|- style="background:#f6efcd; color:black"
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| [http://partsregistry.org/Part:BBa_K079018 Lac O1]
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| aattgtgagcggataacaatt
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| Intermediate
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|- style="background:#f6efcd; color:black"
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| [http://partsregistry.org/Part:BBa_K079019 Lac O2]
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| aaatgtgagcgagtaacaacc
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| Weak
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|}
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{| align="right"
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|- style="background: #c09a6d; text-align: center;"
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|colspan=3| <font size="+1">Tet</font>
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|- style="background: #c09a6d; text-align: center;"
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|width=100| '''Name'''
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|width=180| '''Sequence'''
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|width=150| '''Affinity'''
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|- style="background:#f6efcd; color:black"
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| [http://partsregistry.org/Part:BBa_K079036 TeT O]
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| cctatcagtgataga
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| Strong
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|- style="background:#f6efcd; color:black"
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| [http://partsregistry.org/Part:BBa_K079037 TetO-4C]
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| cctgtcagtgacaga
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| Intermediate
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|- style="background:#f6efcd; color:black"
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| [http://partsregistry.org/Part:BBa_K079038 TetO-wt/4C5G]
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| cctatcagtgacgga
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| Weak
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|}
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<br><br><br><br><br><br><br><br><br>
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{| align="left"
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|- style="background: #c09a6d; text-align: center;"
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|colspan=3| <font size="+1">Lambda</font>
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|- style="background: #c09a6d; text-align: center;"
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|width=100| '''Name'''
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|width=180| '''Sequence'''
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|width=150| '''Affinity'''
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|- style="background:#f6efcd; color:black"
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| [http://partsregistry.org/Part:BBa_K079041 Lambda OR1]
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| tatcaccgccagaggta
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| Strong/Intermediate cI/Cro
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|- style="background:#f6efcd; color:black"
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| [http://partsregistry.org/Part:BBa_K079042 Lambda OR2]
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| taacaccgtgcgtgttg
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| Intermediate/Weak cI/Cro
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|- style="background:#f6efcd; color:black"
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| [http://partsregistry.org/Part:BBa_K079043 Lambda OR3]
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| tatcaccgcaagggata
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| Intermediate/Intermediate cI/Cro
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|}
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{| align="right"
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|- style="background: #c09a6d; text-align: center;"
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|colspan=3| <font size="+1">Lex</font>
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|- style="background: #c09a6d; text-align: center;"
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|width=100| '''Name'''
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|width=180| '''Sequence'''
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|width=150| '''Affinity'''
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|- style="background:#f6efcd; color:black"
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| [http://partsregistry.org/Part:BBa_K079039 LexA 1]
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| ctgtatatatatacag
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| Very Strong
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|- style="background:#f6efcd; color:black"
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| [http://partsregistry.org/Part:BBa_K079040 LexA 2]
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| ctgtatgagcatacag
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| Quite Weak
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|}
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<br><br><br><br><br><br><br>
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<br><br><br>
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Single operators or a combination of them, can be also assembled upstream or downstream with respect to a constitutive promoter. In fact, it is known (Cox et al, 2007) that the position of an operator site plays a crucial role in determining repression effects (i.e. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079041 lambda operators]). In particular, the same operator located upstream of the promoter -35 sequence functions as a weaker repressor than one located downstream of the -10 consensus sequence.
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[https://2008.igem.org/Team:Bologna/Wetlab ''Up'']
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= Operator site cloning in standard plasmids =
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Operator sites are Dna sequences very small in length (15 to 20 bp) and a restriction digestion for the religation in standard plasmids is not possible with the existing purification kits. In fact, only Dna sequences down to at least 40 bp can be efficiently purified with the standard kits available.
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Small bands extraction requires laborious condition optimization and hazardous reagents like phenol-chloroform
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So, we decided to set a new protocol up for the isolation and cloning of single operator sites into standard BioBricks plasmids.
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We started from the analysis of an existing custom vector into the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 Registry]. In this vector, a RFP gene was cloned, as a reporter, between the SpeI and PstI restriction sites. Thus, this is the only part in the Registry that can be separated from a plasmid with a S/P digestion.
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Therefore, once the RFP is isolated with S/P, it could be assembled with an operator sequence digested SpeI- PstI, too. This could allow the isolation of the Operator- RFP sequence from the commercial plasmid and the subsequent religation into a standard plasmid. Then, a final extraction of the RFP gene with a SpeI- PstI digestion would leave the operator site inside the standard plasmid.
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[https://2008.igem.org/Team:Bologna/Wetlab ''Up'']
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= Experimental assesment of LacI operator functionality =
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According to the protocol to test operator parts ([https://2008.igem.org/Team:Bologna/Modeling#Procedure_for_Ki-index_identification procedure for K-index identification]),  two circuits were assembled (Figure 1): K079020 is the closed-loop configuration where GFP expression is auto-regulated by the synthesis of LacI repressor protein; K079026 is the open-loop configuration lacking the operator site to determinate the maximum fluorescence due to the constitutive promoter. In the latter construct, GFP was spaced from the promoter inserting the LacI gene sequence, to account for the abortive transcriptions.
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[[Image:fig050.jpg|center]]
 
<br>
<br>
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[[Image:fig049.jpg|center]]
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[[Image:bba020.jpg|center|thumbnail|400 px|Figure 1. Upper panel: Closed-loop configuration (K079020); lower panel Open-loop configuration (K079026)]]
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<br>
<br>
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To test the UV induction we used the UV illuminator.
 
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= UV Induction =
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K079020 and K079026 were transformed in HL1Blue bacterial cells according to the standard protocol. One colony from each plate was picked up and let grow overnight in LB medium at 37°C. One milliliter for each of the two samples was collected by O/N cultures and spinned  at 6000-8000 rpm for three minutes. The supernatant was harvested and the pellet resuspended. Slides were prepared for the fluorescence bacteria image acquisition. For each slide five different views were acquired.  Finally, images were elaborated with the Visual Fluo Bacteria Software. Examples of fluorescence bacteria image are shown in Figure 2a-2b. The fluorescence images reveal the repression due to the presence of the Lac operator.
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Plasmids with the constructs have been transformed in DH5alfa bacteria by standard protocol and one colony from the plate was picked up and cultured overnight in  5ml LB medium broth with ampicillin. The day after the culture  was diluted in 5ml LB and antibiotic in order to have OD = 0.1 and let it grown for another one hour; after that the culture was divided in five 15ml tubes (1ml of bacteria per tube), in order to be induced.
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<br>
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Using 1ml of culture is a choice done in order to have a thickness as thick as possible to perform an irradiation as uniform as possible .
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Tests with difference distances from the lamp with different exposition times were done to respect the maximum lethal UV dose of bacteria and avoid mutagenesis factors in the E. coli bacteria cells.
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{|align="center"
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The following table illustrates the test setup used and OD after one hour:
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|[[Image:closed.jpg|thumbnail|350 px|Figure 2a. Open-Loop configuration (K079026)]]
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|[[Image:open.jpg|thumbnail|350 px|Figure 2b. Closed-Loop configuration (K079020)]]
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|}
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[[Image:tabuv.jpg|center]]
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<br>
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After induction by UV the samples were kept for 2 hours in dark by silver paper to increase the RecA and LexA response.  The OD samples was measured and the sample transferred in a 1ml tube, spinned at 6000-8000rpm for three minutes and the supernatant was discard and the pellet  resuspended for the slide preparation in order to acquire and elaborate the bacterial fluorescence view by microscope and Bacteria Visual Fluo Software.
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Bacterial fluorescence distribution is shown in Figure.3
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Unfortunately, using 15ml tube and maybe the not the correct time/distance induction, the experimental tests didn’t shown GFP expression. It could be depended because of not uniform irradiation of the sample. In the Fig. is possible to see a picture of leak answer by bacteria stimulated by UV at the distance of 4cm using less than one second time as exposure.
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<br>
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[[Image:colonia1.jpg|400px|thumbnail|Fig.3|center]]
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[[Image:Box.jpg|thumbnail|center|500 px|Figure 3. Box Plot of bacterium fluorescence (n=683). Max and minimum values are indicated by the horizontal bars. The median and lower and upper quartiles are also indicated.]]
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= Hydrogen Peroxide Induction =
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<br>
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[[Image:schema.jpg|right]]
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Results of fluorescence bacteria analysis by software are reported in tab.
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The reaction of hydrogen peroxide with transition metals imposes on cells an oxidative stress condition that can result in damage to cell components such as proteins, lipids and principally to DNA. Escherichia Coli cells are able to deal with these adverse events via DNA repair mechanisms or OxyR and SosRS anti-oxidant inducible pathways which are elicited by cells to avoid the introduction of oxidative lesions by hydrogen peroxide.
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Among the systems the OxyR gene interacts with, there is the SOS response.<br><br>
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Since we have not success with UV induction, to test the correct functionality of K079049 and K079050 constructs, they were induced by peroxide hydrogen. Low concentration of  (1-3mM) results in SOS gene induction in wild-type cells [Imlay and Linn, 1987; Goerlich et alt. 1989].
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The plasmid contained the LexA Operator was transformed into DH5alfa bacteria according to the standard protocol and one colony was picked up from the plate and let it grown overnight in 15ml tube with 5ml LB broth and ampicillin. Cultured colony was diluted in 5ml LB broth with ampicillin and 1ul H2O2 (11M) to have medium with 2.2mM concentration of H202 and 0.1 starting OD.
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<br>
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Testing BBa_K079050 construct, two negative controls were used to prove that H202 induction works correctly and that can be used in flip-flop without interferences between Lac Operon and SOS System:<br>
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* BBa_K079050 grown in LB medium without H202 <br>
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[[Image:tab2.jpg|center]]
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* BBa_K079029 grown in LB medium with H202 (Fig. )
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[[Image:immag2.jpg|center|thumbnail| 500 px| Figure 1 BBa_K079029]]
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<br>
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Tubes were kept to the dark by using silver paper and let them growing for 2 hours at 37°C. 1 ml of bacteria sample has been collected and transferred inside 1ml tube and  spinned at 6000-8000 rpm for 3 minutes. The supernatant was thrown away and the pellet  resuspended for the slide preparation.
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The open loop and closed loop circuit fluorescence ratio ([https://2008.igem.org/Team:Bologna/Modeling#Procedure_for_Ki-index_identification h factor]) is
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[[Image:immag3.jpg|center]]
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<br>
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In addition to prove that K079050 construct can be used in the flip-flop circuit without interference between Lac Operon and SOS System a test IPTG and H202 of two previously constructs were done.  Fig.  shows the results.
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[[Image:h2.jpg|center]]
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[[Image:immag4.jpg|center]]
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As expected the construct with Lex A operator induced by IPTG and Lac-Operon-based-circuit induced by H2O2 didn’t produced GFP synthesis, instead the other two sample correctly induced presented good level of fluorescence.
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<br>
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We conclude that H2O2 induction gives an uniform activity of the regulator promoter (J23100) and the level of promoter activity can be used to set on the memory.
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The Ki-index corresponding to this h factor is equal to 4.43 ( [https://2008.igem.org/Team:Bologna/Modeling#Procedure_for_Ki-index_identification see Figure 6 in Modeling 1.5]). The kr-range for the bistability is from 4 to 6  ([https://2008.igem.org/Team:Bologna/Modeling#Bifurcation_analysis see Figure 4 in Modeling 1.4 ])
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[https://2008.igem.org/Team:Bologna/Wetlab ''Up'']
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= Uv-Sensitive Trigger =
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In the genetic Flip-Flop, the amount of LacI to set ON the memory is induced by an [https://2008.igem.org/Team:Bologna/Modeling#The_genetic_Flip-Flop UV-sensitive trigger ]. Production of LacI molecules by UV induction can be tested by replacing the LacI gene with GFP in the UV trigger, so it is possible to have a relation of the strength of LacI synthesis measuring the value of fluorescence.<br> We realized two new constructs (K079049 and K079050) to test the UV induction. We used two different promoters (J23118 with 1429 strength and J23100 with 2547 strength). These UV test circuits are presented schematically in Figure 4-5.
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<br>
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[[Image:fig050.jpg|center|thumbnail|388 px| Figure 4. K079050 GFP reporter protein under the control of the J23100      constitutive promoter and LexA 2 operator ]]
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<br>
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[[Image:fig049.jpg|center|thumbnail|388 px| Figure 5. K079049 GFP reporter protein under the control of the J23118 constitutive promoter and LexA 2 operator ]]
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<br>
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To test the UV induction we used the [https://2008.igem.org/Team:Bologna/Wetlab#Homemade_UV_Illuminator UV illuminator].
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[https://2008.igem.org/Team:Bologna/Wetlab ''Up'']
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= UV Induction =
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Plasmids with the K079049 and K079050 constructs have been transformed in DH5alfa bacteria by standard protocol and one colony from the plate was picked up and cultured overnight in  5ml LB medium broth with ampicillin. The day after the culture  was diluted in 5ml LB and antibiotic in order to have OD = 0.1 and let it grown for another one hour; after that the culture was divided in five 15ml tubes (1ml of bacteria per tube).<br>
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The 1ml volume was used to have a layer of medium enough thin to perform an uniform irradiation.<br>
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Tests with difference distances from the lamp (3 and 4 cm) with different exposition times (1, 10 and 15 s) were done. Minimum distance and maximum time were fixed to avoid lethal UV dose and mutagenesis.<br>
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After induction by UV the samples were kept for 2 hours in dark by silver paper to increase the RecA and LexA response.  The OD was measured and the sample transferred in a 1ml tube, spinned at 6000-8000rpm for three minutes, the supernatant was harvested and the pellet resuspended. Slides were prepared for the fluorescence bacteria image acquisition that were elaborated with the  [https://2008.igem.org/Team:Bologna/Software#Visual_Fluo_Bacteria:_a_software_for_the_analysis_of_fluorescence_bacteria_image Visual Fluo Bacteria Software]. <br>
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OD was not altered by UV irradiation but we didn't observe GFP. Probably we were not able to find the right time/distance setting. Moreover we are not sure that an uniform irradiation  takes place.
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[https://2008.igem.org/Team:Bologna/Wetlab ''Up'']
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= Hydrogen Peroxide Induction =
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Since we have not success with UV induction, we tested the correct functionality of K079049 and K079050 constructs by using  hydrogen peroxide induction. It is well known [''Assad et al. 2004''] that hydrogen peroxide is an important reactive oxygen species (ROS). The reaction of hydrogen peroxide with transition metals imposes on cells an oxidative stress that can result in damage to cell components such as proteins, lipids and principally DNA. In E.coli low concentration of H2O2 (1-3mM) results in SOS gene induction [''Imlay and Linn, 1987; Goerlich et alt. 1989''].
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The plasmid contained the LexA Operator was transformed into DH5alfa bacteria according to the standard protocol and one colony was picked up from the plate and let it grown overnight in 15ml tube with 5ml LB medium and ampicillin. Cultured colony was diluted in 5ml LB with antibiotic and 1ul H2O2 (11M) to have medium with 2.2mM concentration of H202 and 0.1 starting OD.
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As control, to prove that H2O2 has effect on the K079050, one sample of the same culture was diluted in 5ml LB medium with antibiotic without H2O2.
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Additionally, to demonstrate that H202 induction has not interferences with the LacI regulated promoter we also tested the K079029 (Figure 6) that was grown in the LB medium with 2.2mM of H2O2.
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<br>
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[[Image:immag2.jpg|center|thumbnail| 500 px| Figure 6. K079029 GFP expression controlled by LacI repressor under the J23114 promoter]]
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<br>
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Tubes were kept to the dark by using silver paper and let them growing for 2 hours at 37°C.  1 ml of bacteria sample has been collected and transferred inside 1ml tube and  spinned at 6000-8000 rpm for 3 minutes. The supernatant was discard and the pellet  resuspended. Slides were prepared for the fluorescence bacteria image acquisition that were elaborated with the  [https://2008.igem.org/Team:Bologna/Software#Visual_Fluo_Bacteria:_a_software_for_the_analysis_of_fluorescence_bacteria_image Visual Fluo Bacteria Software].
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<br>
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{|align="center"
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|[[Image:imm4.jpg|center|thumbnail|300 px|Figure 7. K079050 with H2O2]]
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|[[Image:immag5.jpg|center|thumbnail|300 px|Figure 8. K079050 without H2O2]]
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|[[Image:immag6.jpg|center|thumbnail|300 px|Figure 9. K079029 with H2O2]]
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|}
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<br>
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It is possible to see that GFP is sinthetized only in the construct with Lex A operator.
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In addition to prove that K079050 construct can be used in the Flip-Flop circuit without interference with IPTG,  we induced both K079050 and K079029 as positive control for 2 hours with 1mM of IPTG.
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<br>
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{|align="center"
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|[[Image:imm7.jpg|center|thumbnail|300 px| Figure 10. K079029 induced by IPTG]]
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|[[Image:imm8.jpg|center|thumbnail|300 px| Figure 11. K079050 induced by IPTG]]
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|}
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<br>
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As expected IPTG was not able to induce the construct with Lex A operator but it induced  the circuit  with LacI repressed promoter. We conclude that H2O2 induction gives an uniform activity of the regulator promoter (J23100) and the level of promoter activity can be used to set ON the memory.
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[https://2008.igem.org/Team:Bologna/Wetlab ''Up'']
[https://2008.igem.org/Team:Bologna/Wetlab ''Up'']
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= Homemade UV Illuminator =
= Homemade UV Illuminator =
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The UV source that we use is a GW6 Sylvania, a lamp that emitt UVC at 253,7nm near the absorption's peak of DNA with an optical output power of 1,6W; to use it [[Team:Bologna/Biosafety#Ethidium_Bromide_and_UV_Rays|'''safely''']] we built a box of mdf(medium density-fibreboard) that surrounds the lamp and prevent the UVc leakeage, moreover we use a diaphragm that allows passage only to the light absorbing the remainder. We insert in that box two pierced brackets, that permits to choose the distance between the lamp and the sample and then in accord with the Lambert-Beer law's the exposure time.
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The UV source that we use is a GW6 Sylvania, a lamp that emitt UVC at 253,7nm near the absorption's peak of DNA with an optical output power of 1,6W; to use it [[Team:Bologna/Biosafety#Ethidium_Bromide_and_UV_Rays|safely]] we built a box of mdf(medium density-fibreboard) that surrounds the lamp and prevent the UVc leakeage, moreover we use a diaphragm that allows passage only to part of the light, absorbing the remainder. We insert in that box two pierced brackets, that permits to choose the distance between the lamp and the sample and then in accord with the Lambert-Beer law's the exposure time.
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Finally we embedded this structure in a polistyrene box for its handling and greater safety (Figure 2-3).
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Finally we embedded this structure in a polistyrene box for its handling and greater safety (Figure 12-13).
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<br>
{|align="center"
{|align="center"
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|[[Image:scatola1.jpg|center|thumbnail|300 px|Figure 2]]                 
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|[[Image:scatola1.jpg|center|thumbnail|300 px|Figure 12]]                 
|width=70|               
|width=70|               
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|[[Image:scatola2.jpg|center|thumbnail|300 px| Figure 3]]
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|[[Image:scatola2.jpg|center|thumbnail|300 px| Figure 13]]
|}
|}
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Our project use UV light for its space selectivity, that gives the possibility to irradiate a target zone
Our project use UV light for its space selectivity, that gives the possibility to irradiate a target zone
without interfering with the other. To do that we build a mold to make a matrix of agarose gel;this
without interfering with the other. To do that we build a mold to make a matrix of agarose gel;this
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give us a square of 25mm side's with 16 cells inside where we can locate bacteria( Figure 4 )
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give us a square of 25mm side's with 16 cells inside where we can locate bacteria( Figure 14 ).
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[[Image:matrice.jpg|center|thumbnail|300 px| Figure 4]]
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<br>
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[[Image:matrice.jpg|center|thumbnail|300 px| Figure 14]]
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<br>
Once do that is easy with an optical mask, like those used in photolithography for electronics
Once do that is easy with an optical mask, like those used in photolithography for electronics
circuits, stimulate only the selected bacteria.To realize this device we use palsticard because it is
circuits, stimulate only the selected bacteria.To realize this device we use palsticard because it is
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very easy to shape and clean( Figure 5 ).
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very easy to shape and clean( Figure 15 ).
The mold is made of two parts that will form a sandwich with the slide: one will create the shape  
The mold is made of two parts that will form a sandwich with the slide: one will create the shape  
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of the gel matrix the other is a cover that that will give the picture into gel .<br>
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of the gel matrix( Figure 16 ) the other is a cover that that will give the picture into gel.
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<br>
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{|align="center"
{|align="center"
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|[[Image:imm1.jpg|center|thumbnail|300 px|Figure 5]]
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|[[Image:imm1.jpg|center|thumbnail|300 px|Figure 15]]
|width=70|  
|width=70|  
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|[[Image:imm2.jpg|center|thumbnail|300 px|Figure 6]]
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|[[Image:imm2.jpg|center|thumbnail|300 px|Figure 16]]
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The matrix is obtained through the pressure of the cover part over the mold part( Figure 17 )
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[[Image:pinze.jpg|center|thumbnail|300 px|Figure 17]]
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The matrix is obtained through the pressure of the cover part over the mold part
 
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[[Image:pinze.jpg|center|thumbnail|300 px|Figure 7]]
 
and that is the result:
and that is the result:
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[[Image:imm3.jpg|center|thumbnail|300 px|Figure 8]]
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<br>
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[[Image:imm3.jpg|center|thumbnail|300 px|Figure 18]]
[https://2008.igem.org/Team:Bologna/Wetlab ''Up'']
[https://2008.igem.org/Team:Bologna/Wetlab ''Up'']
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= Bibliography =
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Operator sites are Dna sequences very small in length (15 to 20 bp) and a restriction digestion for the religation in standard plasmids is not possible with the existing purification kits. In fact, only Dna sequences down to at least 40 bp can be efficiently purified with the standard kits available.  
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*Friedberg EC, Walker GC and Siede W (1995) DNA Repair and Mutagenesis. Academic Press, New York, 698 pp<br>
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Small bands extraction requires laborious condition optimization and hazardous reagents like phenol-chloroform
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*Courcelle J, Khoudursky A, Peter B, Brown PO and Hanawalt PC (2001) Comparative gene expression profiles  following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics 158:41-64<br>
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So, we decided to set a new protocol up for the isolation and cloning of single operator sites into standard BioBricks plasmids.  
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*Wagner J, Cruz P, Kim SR, Yamada M, Matsui K, Fuchs RP and Nohmi T (1999) The dinB gene encodes a novel E.  coli DNA polymerase, DNA pol IV, involved in mutagenesis. Mol Cell 4:281-286<br>
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*Mustard JA and Little JW (2000) Analysis of Escherichia coli RecA interactions with LexA, lambda CI, and UmuD by site-directed mutagenesis of rec<br>
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*Fernandez De Henestrosa AR, Ogi T, Aoyagi S, Chafin D, Hayes JJ, Ohmori H and Woodgate R (2000)<br> Identification of additional genes belonging to the lexA regulon in Escherichia coli. Mol Microbiol 35:1560-1572<br>
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*Imlay JA and Linn S (1987) Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide. J Bateriol 169:2967-2976<br>
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*Goerlich O, Quillardet P and Hofnung M (1989) Induction of the SOS response by hydrogen peroxide in various Escherichia coli mutants with altered protection against oxidative DNA damage. J Bacteriol 171:6141-6147<br>
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*Imlay JA and Linn S (1987) Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide<br>
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We started from the analysis of an existing promoter library into the Registry. As it can be seen in figure below, these plasmids were meant for the construction of promoter basic parts and their derivatives. They  can be used two ways:
 
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1. Insertion of a promoter element between XbaI and SpeI sites results in a RFP reporter while retaining the ability to do BioBrick assembly. Part J61002 is the tet promoter variant of the plasmid.
 
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2. Insertion of a protein generating device or RNA gene (cutting the part with XbaI/PstI, inserting into  SpeI/PstI of J61002) results in a standard pSB1A2 plasmid containing an r0040.yourpart composite.
 
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[[Image:PromotoriBerkley.jpg |center|thumbnail|300px]]
 
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Thus, we decided to isolate the RFP protein from one of the promoter family member with a SpeI- PstI enzymatic digestion. Then, this part could be assemble with an operator sequence digested SpeI- PstI, too. This could allow the isolation of the Operator- RFP sequence from the commercial plasmid and the subsequent religation into a standard plasmid. Then, a final extraction of the RFP gene with a SpeI- PstI digestion would leave the operator site inside the standard plasmid.
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[https://2008.igem.org/Team:Bologna/Wetlab ''Up'']

Latest revision as of 12:57, 4 December 2008

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HOME PROJECT TEAM SOFTWARE MODELING WET LAB LAB-BOOK SUBMITTED PARTS BIOSAFETY AND PROTOCOLS


Contents

Introduction

Collage2.jpg

To obtain regulated promoters we synthesized four libraries of operator sequences, respectively for LacI, TetR, cI and LexA repressor proteins. Here is explained how we designed the operator libraries and isolated single operators to clone them in the standard format. Moreover, we built and tested: a LacI regulated promoter to tune promoter activation and a lexA based promoter to study the SOS activation (UV and peroxidase).


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At last... Operator sites as BioBricks!

Even though transcriptional regulation still plays a pivotal role in synthetic biology, a modular assembly of regulated promoters and the characterization of their properties has not been formalized, yet. At present state, each promoter in the Registry, though complex, is treated as a “standalone” monolithic element. Thus, the choice of a regulated promoter implies a prefixed sigmoid regulation curve. The only option, using the current BioBricks, is to choose a discretional scaling factor in the transfer of the transcription function to the protein through an appropriate RBS. Critically, the choice of one specific transcription factor limits the choice to just one possible promoter. The assembly of regulated promoters as the combination of modular parts, i. e. unregulated promoters and operators, could permit the rapid design of regulatory elements with fixed characteristics. In fact, promoter transcriptional strength and repressor binding affinity could be independently chosen.

A first step in the rationalization of promoter design was done in the iGEM 2007 with the introduction in the Registry of a family of constitutive promoters. Each family element differs from the others just for few base pairs in -35 and -10 regions, keeping constant the rest of the sequence and giving rise to a different level of transcription strength. We decided to use this valuable work as a platform for a deeper and more general promoter design strategy. To obtain regulated promoters we decided to combine these parts with short regulatory sequences that operate as a binding site for the transcription factor. To this aim, we synthesized four libraries of operator sequences, respectively for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 LacI], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079046 TetR], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079047 cI] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079048 LexA] repressor proteins ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K079045 see details]). In each library, there are three sequences (see Table below) each with a different repressor binding affinity to the repressor protein. Since the libraries were synthetized on pGA18 and pMA Geneart vectors, we isolated each operator with the intention to clone them into BioBrick standard assembly plasmids. The choice of the operator should give a relative fine-tuning of promoter sensitivity to the repressor. We decided to take the Berkley's costitutive promoter library as a good "collection" from which we could select the unregulated promoter, according with the chosen transcriptional strength.


LacI
Name Sequence Affinity
[http://partsregistry.org/Part:BBa_K079017 Lac SymL] aattgtgagcgctcacaatt Very Strong
[http://partsregistry.org/Part:BBa_K079018 Lac O1] aattgtgagcggataacaatt Intermediate
[http://partsregistry.org/Part:BBa_K079019 Lac O2] aaatgtgagcgagtaacaacc Weak
Tet
Name Sequence Affinity
[http://partsregistry.org/Part:BBa_K079036 TeT O] cctatcagtgataga Strong
[http://partsregistry.org/Part:BBa_K079037 TetO-4C] cctgtcagtgacaga Intermediate
[http://partsregistry.org/Part:BBa_K079038 TetO-wt/4C5G] cctatcagtgacgga Weak










Lambda
Name Sequence Affinity
[http://partsregistry.org/Part:BBa_K079041 Lambda OR1] tatcaccgccagaggta Strong/Intermediate cI/Cro
[http://partsregistry.org/Part:BBa_K079042 Lambda OR2] taacaccgtgcgtgttg Intermediate/Weak cI/Cro
[http://partsregistry.org/Part:BBa_K079043 Lambda OR3] tatcaccgcaagggata Intermediate/Intermediate cI/Cro
Lex
Name Sequence Affinity
[http://partsregistry.org/Part:BBa_K079039 LexA 1] ctgtatatatatacag Very Strong
[http://partsregistry.org/Part:BBa_K079040 LexA 2] ctgtatgagcatacag Quite Weak












Single operators or a combination of them, can be also assembled upstream or downstream with respect to a constitutive promoter. In fact, it is known (Cox et al, 2007) that the position of an operator site plays a crucial role in determining repression effects (i.e. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K079041 lambda operators]). In particular, the same operator located upstream of the promoter -35 sequence functions as a weaker repressor than one located downstream of the -10 consensus sequence.


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Operator site cloning in standard plasmids

Operator sites are Dna sequences very small in length (15 to 20 bp) and a restriction digestion for the religation in standard plasmids is not possible with the existing purification kits. In fact, only Dna sequences down to at least 40 bp can be efficiently purified with the standard kits available. Small bands extraction requires laborious condition optimization and hazardous reagents like phenol-chloroform So, we decided to set a new protocol up for the isolation and cloning of single operator sites into standard BioBricks plasmids.

We started from the analysis of an existing custom vector into the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 Registry]. In this vector, a RFP gene was cloned, as a reporter, between the SpeI and PstI restriction sites. Thus, this is the only part in the Registry that can be separated from a plasmid with a S/P digestion.

Therefore, once the RFP is isolated with S/P, it could be assembled with an operator sequence digested SpeI- PstI, too. This could allow the isolation of the Operator- RFP sequence from the commercial plasmid and the subsequent religation into a standard plasmid. Then, a final extraction of the RFP gene with a SpeI- PstI digestion would leave the operator site inside the standard plasmid.


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Experimental assesment of LacI operator functionality

According to the protocol to test operator parts (procedure for K-index identification), two circuits were assembled (Figure 1): K079020 is the closed-loop configuration where GFP expression is auto-regulated by the synthesis of LacI repressor protein; K079026 is the open-loop configuration lacking the operator site to determinate the maximum fluorescence due to the constitutive promoter. In the latter construct, GFP was spaced from the promoter inserting the LacI gene sequence, to account for the abortive transcriptions.


Figure 1. Upper panel: Closed-loop configuration (K079020); lower panel Open-loop configuration (K079026)


K079020 and K079026 were transformed in HL1Blue bacterial cells according to the standard protocol. One colony from each plate was picked up and let grow overnight in LB medium at 37°C. One milliliter for each of the two samples was collected by O/N cultures and spinned at 6000-8000 rpm for three minutes. The supernatant was harvested and the pellet resuspended. Slides were prepared for the fluorescence bacteria image acquisition. For each slide five different views were acquired. Finally, images were elaborated with the Visual Fluo Bacteria Software. Examples of fluorescence bacteria image are shown in Figure 2a-2b. The fluorescence images reveal the repression due to the presence of the Lac operator.


Figure 2a. Open-Loop configuration (K079026)
Figure 2b. Closed-Loop configuration (K079020)


Bacterial fluorescence distribution is shown in Figure.3


Figure 3. Box Plot of bacterium fluorescence (n=683). Max and minimum values are indicated by the horizontal bars. The median and lower and upper quartiles are also indicated.


Results of fluorescence bacteria analysis by software are reported in tab.


Tab2.jpg


The open loop and closed loop circuit fluorescence ratio (h factor) is


H2.jpg


The Ki-index corresponding to this h factor is equal to 4.43 ( see Figure 6 in Modeling 1.5). The kr-range for the bistability is from 4 to 6 (see Figure 4 in Modeling 1.4 )


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Uv-Sensitive Trigger

In the genetic Flip-Flop, the amount of LacI to set ON the memory is induced by an UV-sensitive trigger . Production of LacI molecules by UV induction can be tested by replacing the LacI gene with GFP in the UV trigger, so it is possible to have a relation of the strength of LacI synthesis measuring the value of fluorescence.
We realized two new constructs (K079049 and K079050) to test the UV induction. We used two different promoters (J23118 with 1429 strength and J23100 with 2547 strength). These UV test circuits are presented schematically in Figure 4-5.


Figure 4. K079050 GFP reporter protein under the control of the J23100 constitutive promoter and LexA 2 operator


Figure 5. K079049 GFP reporter protein under the control of the J23118 constitutive promoter and LexA 2 operator


To test the UV induction we used the UV illuminator.


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UV Induction

Plasmids with the K079049 and K079050 constructs have been transformed in DH5alfa bacteria by standard protocol and one colony from the plate was picked up and cultured overnight in 5ml LB medium broth with ampicillin. The day after the culture was diluted in 5ml LB and antibiotic in order to have OD = 0.1 and let it grown for another one hour; after that the culture was divided in five 15ml tubes (1ml of bacteria per tube).
The 1ml volume was used to have a layer of medium enough thin to perform an uniform irradiation.

Tests with difference distances from the lamp (3 and 4 cm) with different exposition times (1, 10 and 15 s) were done. Minimum distance and maximum time were fixed to avoid lethal UV dose and mutagenesis.

After induction by UV the samples were kept for 2 hours in dark by silver paper to increase the RecA and LexA response. The OD was measured and the sample transferred in a 1ml tube, spinned at 6000-8000rpm for three minutes, the supernatant was harvested and the pellet resuspended. Slides were prepared for the fluorescence bacteria image acquisition that were elaborated with the Visual Fluo Bacteria Software.
OD was not altered by UV irradiation but we didn't observe GFP. Probably we were not able to find the right time/distance setting. Moreover we are not sure that an uniform irradiation takes place.


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Hydrogen Peroxide Induction

Since we have not success with UV induction, we tested the correct functionality of K079049 and K079050 constructs by using hydrogen peroxide induction. It is well known [Assad et al. 2004] that hydrogen peroxide is an important reactive oxygen species (ROS). The reaction of hydrogen peroxide with transition metals imposes on cells an oxidative stress that can result in damage to cell components such as proteins, lipids and principally DNA. In E.coli low concentration of H2O2 (1-3mM) results in SOS gene induction [Imlay and Linn, 1987; Goerlich et alt. 1989].

The plasmid contained the LexA Operator was transformed into DH5alfa bacteria according to the standard protocol and one colony was picked up from the plate and let it grown overnight in 15ml tube with 5ml LB medium and ampicillin. Cultured colony was diluted in 5ml LB with antibiotic and 1ul H2O2 (11M) to have medium with 2.2mM concentration of H202 and 0.1 starting OD.

As control, to prove that H2O2 has effect on the K079050, one sample of the same culture was diluted in 5ml LB medium with antibiotic without H2O2. Additionally, to demonstrate that H202 induction has not interferences with the LacI regulated promoter we also tested the K079029 (Figure 6) that was grown in the LB medium with 2.2mM of H2O2.


Figure 6. K079029 GFP expression controlled by LacI repressor under the J23114 promoter


Tubes were kept to the dark by using silver paper and let them growing for 2 hours at 37°C. 1 ml of bacteria sample has been collected and transferred inside 1ml tube and spinned at 6000-8000 rpm for 3 minutes. The supernatant was discard and the pellet resuspended. Slides were prepared for the fluorescence bacteria image acquisition that were elaborated with the Visual Fluo Bacteria Software.


Figure 7. K079050 with H2O2
Figure 8. K079050 without H2O2
Figure 9. K079029 with H2O2


It is possible to see that GFP is sinthetized only in the construct with Lex A operator. In addition to prove that K079050 construct can be used in the Flip-Flop circuit without interference with IPTG, we induced both K079050 and K079029 as positive control for 2 hours with 1mM of IPTG.


Figure 10. K079029 induced by IPTG
Figure 11. K079050 induced by IPTG


As expected IPTG was not able to induce the construct with Lex A operator but it induced the circuit with LacI repressed promoter. We conclude that H2O2 induction gives an uniform activity of the regulator promoter (J23100) and the level of promoter activity can be used to set ON the memory.


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Homemade UV Illuminator

The UV source that we use is a GW6 Sylvania, a lamp that emitt UVC at 253,7nm near the absorption's peak of DNA with an optical output power of 1,6W; to use it safely we built a box of mdf(medium density-fibreboard) that surrounds the lamp and prevent the UVc leakeage, moreover we use a diaphragm that allows passage only to part of the light, absorbing the remainder. We insert in that box two pierced brackets, that permits to choose the distance between the lamp and the sample and then in accord with the Lambert-Beer law's the exposure time. Finally we embedded this structure in a polistyrene box for its handling and greater safety (Figure 12-13).


Figure 12
Figure 13


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Gel matrix

Our project use UV light for its space selectivity, that gives the possibility to irradiate a target zone without interfering with the other. To do that we build a mold to make a matrix of agarose gel;this give us a square of 25mm side's with 16 cells inside where we can locate bacteria( Figure 14 ).


Figure 14


Once do that is easy with an optical mask, like those used in photolithography for electronics circuits, stimulate only the selected bacteria.To realize this device we use palsticard because it is very easy to shape and clean( Figure 15 ). The mold is made of two parts that will form a sandwich with the slide: one will create the shape of the gel matrix( Figure 16 ) the other is a cover that that will give the picture into gel.


Figure 15
Figure 16


The matrix is obtained through the pressure of the cover part over the mold part( Figure 17 )


Figure 17


and that is the result:


Figure 18


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Bibliography

  • Friedberg EC, Walker GC and Siede W (1995) DNA Repair and Mutagenesis. Academic Press, New York, 698 pp
  • Courcelle J, Khoudursky A, Peter B, Brown PO and Hanawalt PC (2001) Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics 158:41-64
  • Wagner J, Cruz P, Kim SR, Yamada M, Matsui K, Fuchs RP and Nohmi T (1999) The dinB gene encodes a novel E. coli DNA polymerase, DNA pol IV, involved in mutagenesis. Mol Cell 4:281-286
  • Mustard JA and Little JW (2000) Analysis of Escherichia coli RecA interactions with LexA, lambda CI, and UmuD by site-directed mutagenesis of rec
  • Fernandez De Henestrosa AR, Ogi T, Aoyagi S, Chafin D, Hayes JJ, Ohmori H and Woodgate R (2000)
    Identification of additional genes belonging to the lexA regulon in Escherichia coli. Mol Microbiol 35:1560-1572
  • Imlay JA and Linn S (1987) Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide. J Bateriol 169:2967-2976
  • Goerlich O, Quillardet P and Hofnung M (1989) Induction of the SOS response by hydrogen peroxide in various Escherichia coli mutants with altered protection against oxidative DNA damage. J Bacteriol 171:6141-6147
  • Imlay JA and Linn S (1987) Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide


Up