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Team:INSA-Lyon/Project/Stage3/Strategy
From 2010.igem.org
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| - | <p>For this system of regulation, we decided to synthesized directly the | + | <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> |
| - | 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. </p><br/> | + | <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/> |
<a href="http://lh5.ggpht.com/_Uc3bmii-yi0/TMg58qKi8EI/AAAAAAAAAms/Fmtlk3nJ144/s912/Curli_GFP.png"> | <a href="http://lh5.ggpht.com/_Uc3bmii-yi0/TMg58qKi8EI/AAAAAAAAAms/Fmtlk3nJ144/s912/Curli_GFP.png"> | ||
<div style="text-align:center;"> | <div style="text-align:center;"> | ||
<img class="image"src="http://lh3.ggpht.com/_zZap34AU7o8/TMfUhgHZylI/AAAAAAAAABo/Uuvk6X8bV8I/s800/curli%2014K.png" | <img class="image"src="http://lh3.ggpht.com/_zZap34AU7o8/TMfUhgHZylI/AAAAAAAAABo/Uuvk6X8bV8I/s800/curli%2014K.png" | ||
| - | alt="curli 14K ou curli 22B" title="Curli and reporter gene ligation" /> | + | alt="curli 14K ou curli 22B" title="Curli promoter and reporter gene ligation" /> |
</a> | </a> | ||
| - | <img class="image2"src="http://lh5.ggpht.com/_Uc3bmii-yi0/TMg59gvCO0I/AAAAAAAAAm4/EWBiy2sfqwk/Curli%20GFP_finale.png" alt="construction finale" title="contruction | + | <img class="image2"src="http://lh5.ggpht.com/_Uc3bmii-yi0/TMg59gvCO0I/AAAAAAAAAm4/EWBiy2sfqwk/Curli%20GFP_finale.png" alt="construction finale" title="final contruction"> |
| - | <p style="font-size:0.9em; text-indent:0px; margin-left: 300px;text-align:left"> <br/><em>Curli and reporter gene ligation </em></p> | + | <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> |
</div><br/><br/> | </div><br/><br/> | ||
<a href="http://lh4.ggpht.com/_zZap34AU7o8/TMfUg5CxIeI/AAAAAAAAABg/GEPJb3PV4z0/s800/18A%20OmpR234.png"> | <a href="http://lh4.ggpht.com/_zZap34AU7o8/TMfUg5CxIeI/AAAAAAAAABg/GEPJb3PV4z0/s800/18A%20OmpR234.png"> | ||
<div style="text-align:center"> | <div style="text-align:center"> | ||
<img class="image"src="http://lh4.ggpht.com/_zZap34AU7o8/TMfUg5CxIeI/AAAAAAAAABg/GEPJb3PV4z0/s800/18A%20OmpR234.png" | <img class="image"src="http://lh4.ggpht.com/_zZap34AU7o8/TMfUg5CxIeI/AAAAAAAAABg/GEPJb3PV4z0/s800/18A%20OmpR234.png" | ||
| - | alt="Constitutive | + | alt="Constitutive promoter and ompR234 gene ligation" title="Constitutive promoter and ompR234 gene ligation" /> |
</a> | </a> | ||
| - | <img src="http://lh6.ggpht.com/_Uc3bmii-yi0/TMg9-ve_0mI/AAAAAAAAAnE/QsHQjo9Giz0/18A%20OmpR234.png" alt="construction finale" title="construction | + | <img src="http://lh6.ggpht.com/_Uc3bmii-yi0/TMg9-ve_0mI/AAAAAAAAAnE/QsHQjo9Giz0/18A%20OmpR234.png" alt="construction finale" title="final construction"><br /> |
| - | <p style="font-size:0.9em; text-indent:0px; margin-left: 300px;text-align:left"> <br/><em>Constitutive | + | <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 /> |
<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. | <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. | ||
| - | </p> | + | <br/><br/></p><br/><br/> |
| - | + | <a href="http://lh3.ggpht.com/_Uc3bmii-yi0/TMg5863f6QI/AAAAAAAAAmw/h_PnBEphSx0/s512/18A%201N%20PPI.png"> | |
| + | <div style="text-align:center;"> | ||
| + | <img class="image"src="http://lh3.ggpht.com/_Uc3bmii-yi0/TMg5863f6QI/AAAAAAAAAmw/h_PnBEphSx0/s512/18A%201N%20PPI.png" | ||
| + | alt="Phasin-intein construction" title="Phasin-intein construction" /> | ||
| + | </a> | ||
| + | <img class="image2"src="http://lh4.ggpht.com/_Uc3bmii-yi0/TMhFUQhiSLI/AAAAAAAAAnY/a75XZT3PTdM/18A%201N%20PPi_finale.png" alt="construction finale" title="final contruction"> | ||
| + | <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/> | ||
| + | </div> | ||
<br /> | <br /> | ||
<br/> | <br/> | ||
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<br> | <br> | ||
<p>In this part we decided to realize three different constructions to control the regulation. | <p>In this part we decided to realize three different constructions to control the regulation. | ||
| - | The first one consists in the | + | The first one consists in the constitutive production of LuxR and LuxI proteins to control the promoter LuxR/HSL.<br /></p> <br/> |
| - | + | <p>The second one is the construction with the promoter LuxR/HSL to control the expression of the operon PhaCAB. | |
| - | The second one is the construction with the | + | 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. |
| - | To finish a construction with | + | 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>. |
| - | </p> | + | <br/></p> |
<br /> | <br /> | ||
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 promoter and reporter gene ligation
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
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.
