Team:INSA-Lyon/Project/Stage3/Strategy

From 2010.igem.org

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<h3>Stage 3 : Strategy</h3>
 
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<p>First, we decided to pursue a computer-based approach using databases such that NCBI. We compared FAS I enzyme function for different organisms (mammals and bacteria). We therefore managed to show a high-level of similarity between the domains of the FAS I. </p>
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<h2>Strategy</h2>
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<p>Second, we wanted to analyze these collected data to design a more evolved structure of the operon gene PHA. This is based on the hypothesis that a multi-functional enzyme would increase the yield of lipid production by E.coli.</p>
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<p> We chose to follow two differents strategies to obtain our systems of regulation.<br /></p>
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<h3><font color="purple">Thermoregulation</font></h3>
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<p>For this system of regulation, we decided to synthesized directly the sequence of the curli promoter with the igem restriction sites. In the same time a part with the gene ompR234 is created by PCR, from the genome of the chassi PHL818. You can find <a href="https://2010.igem.org/Team:INSA-Lyon/Project/Stage3/Strategy/Designcurli">here</a> some details about the design of these sequences.<br /></p>
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<p>Then we ligate an igem reporter gene with the curli promoter and we wanted to make a double transformation with ompR234 to see if our parts work as we hoped. <br/></p><br/>
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<a href="http://lh5.ggpht.com/_Uc3bmii-yi0/TMg58qKi8EI/AAAAAAAAAms/Fmtlk3nJ144/s912/Curli_GFP.png">
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<div style="text-align:center;">
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<img class="image"src="http://lh3.ggpht.com/_zZap34AU7o8/TMfUhgHZylI/AAAAAAAAABo/Uuvk6X8bV8I/s800/curli%2014K.png"
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alt="curli 14K ou curli 22B" title="Curli promoter and reporter gene ligation" />
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</a>
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<img class="image2"src="http://lh5.ggpht.com/_Uc3bmii-yi0/TMg59gvCO0I/AAAAAAAAAm4/EWBiy2sfqwk/Curli%20GFP_finale.png" alt="construction finale" title="final contruction">
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<p style="font-size:0.9em; text-indent:0px; margin-left: 300px;text-align:left"> <br/><em>Curli promoter and reporter gene ligation </em><br/><br/></p>
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<a href="http://lh4.ggpht.com/_zZap34AU7o8/TMfUg5CxIeI/AAAAAAAAABg/GEPJb3PV4z0/s800/18A%20OmpR234.png">
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<img class="image"src="http://lh4.ggpht.com/_zZap34AU7o8/TMfUg5CxIeI/AAAAAAAAABg/GEPJb3PV4z0/s800/18A%20OmpR234.png"
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alt="Constitutive promoter and ompR234 gene ligation" title="Constitutive promoter and ompR234 gene ligation" />
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<img src="http://lh6.ggpht.com/_Uc3bmii-yi0/TMg9-ve_0mI/AAAAAAAAAnE/QsHQjo9Giz0/18A%20OmpR234.png" alt="construction finale" title="final construction"><br />
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<p style="font-size:0.9em; text-indent:0px; margin-left: 300px;text-align:left"><br /><br /><em>Constitutive promoter and ompR234 gene ligation </em><br/><br/></p><br /><br />
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<p>The final plasmid construction of the thermometer RNA will contain a constitutive promoter(BBa_J23119), the thermometer RNA, the PPI-protein fused and a terminator.
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<br/><br/></p><br/><br/>
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<a href="http://lh3.ggpht.com/_Uc3bmii-yi0/TMg5863f6QI/AAAAAAAAAmw/h_PnBEphSx0/s512/18A%201N%20PPI.png">
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<img class="image"src="http://lh3.ggpht.com/_Uc3bmii-yi0/TMg5863f6QI/AAAAAAAAAmw/h_PnBEphSx0/s512/18A%201N%20PPI.png"
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alt="Phasin-intein construction" title="Phasin-intein construction" />
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</a>
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<img class="image2"src="http://lh4.ggpht.com/_Uc3bmii-yi0/TMhFUQhiSLI/AAAAAAAAAnY/a75XZT3PTdM/18A%201N%20PPi_finale.png" alt="construction finale" title="final contruction">
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<p style="font-size:0.9em; text-indent:0px; margin-left: 300px;text-align:left"> <br/><em>Phasin-intein construction</em><br/><br/></p><br/><br/>
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<h3><font style="color:purple">Control under Arabinose</font></h3><br>
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<p>In this part we decided to realize three different constructions to control the regulation.
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The first one consists in the constitutive production of LuxR and LuxI proteins to control the promoter LuxR/HSL.<br /></p> <br/>
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<p>The second one is the construction with the promoter LuxR/HSL to control the expression of the operon PhaCAB.
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To finish a construction with Pbad/araC, which is a promoter whose activity depends on addition of arabinose in medium. In this case, the promoter influences the synthesis of a cI repressor and a phasin-intein construction. The cI repressor is isolated from bacteriophage λ and negatively regulates the activity of the LuxR/HSL regulated promoter.
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You can find more details on the three constructions <a href="https://2010.igem.org/wiki/index.php?title=Team:INSA-Lyon/Project/Stage3/Strategy/construction1">here</a>.
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<br/></p>
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<p style="text-align:center;"><a href="#top">Top of Page</a></p>
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Latest revision as of 20:47, 27 October 2010





Strategy


We chose to follow two differents strategies to obtain our systems of regulation.



Thermoregulation


For this system of regulation, we decided to synthesized directly the sequence of the curli promoter with the igem restriction sites. In the same time a part with the gene ompR234 is created by PCR, from the genome of the chassi PHL818. You can find here some details about the design of these sequences.

Then we ligate an igem reporter gene with the curli promoter and we wanted to make a double transformation with ompR234 to see if our parts work as we hoped.


curli 14K ou curli 22B construction finale


Curli promoter and reporter gene ligation



Constitutive promoter and ompR234 gene ligation construction finale



Constitutive promoter and ompR234 gene ligation



The final plasmid construction of the thermometer RNA will contain a constitutive promoter(BBa_J23119), the thermometer RNA, the PPI-protein fused and a terminator.



Phasin-intein construction construction finale


Phasin-intein construction





Control under Arabinose



In this part we decided to realize three different constructions to control the regulation. The first one consists in the constitutive production of LuxR and LuxI proteins to control the promoter LuxR/HSL.


The second one is the construction with the promoter LuxR/HSL to control the expression of the operon PhaCAB. To finish a construction with Pbad/araC, which is a promoter whose activity depends on addition of arabinose in medium. In this case, the promoter influences the synthesis of a cI repressor and a phasin-intein construction. The cI repressor is isolated from bacteriophage λ and negatively regulates the activity of the LuxR/HSL regulated promoter. You can find more details on the three constructions here.


Top of Page

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