Team:Paris Bettencourt/Experiments/ComS diffusion

From 2011.igem.org

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<h1>ComS/ComK switch system</h1>
<h1>ComS/ComK switch system</h1>
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<h2>Constructs done</h2>
 
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<p>The ComS/ComK switch device is based on a bistable system known as the 'MeKS' module (interactions among the MecA complex, comK and comS). ComS inhibits the MecA protease and allows the ComK protein to self-amplify. But as ComK inhibits the production of ComS, the system comes back to the original state within a few hours.
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<h2>Abstract</h2>
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<div style="margin-left:50px; margin-right:50px; padding: 5px; border:2px solid black;"><b><p>Results for the ComK/ComS switch system:
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    <li>We successfully BioBricked both the ComS gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K606037">BBa_K606037</a>) and the ComS gene codon optimized (<a href="http://partsregistry.org/Part:BBa_K606038">BBa_K606038</a>) constructs and sent them to the registry</li>
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<li>We successfully knocked-out CodY gene in <i>B.subtilis</i></li>
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<li>We characterized the ∆CodY <i>B.subtilis</i> strain</li>
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</ul></p></b></div>
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<h2>Design  overview</h2>
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<center>
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<img src="https://static.igem.org/mediawiki/2011/7/7f/ComS_summary.jpg" width=700px>
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<p><u><b>Fig1:</b></u> Schematic of the ComS design</p>
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<p>More information on the design <a href="https://2011.igem.org/Team:Paris_Bettencourt/ComS_diffusion">here</a>.</p>
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<h2>Parts and biobrick system construction</h2>
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<h3>ComS constructions</h3>
<p>The ComS gene was synthetized by GeneArt but also insulated by PCR colony, changing the start and the stop codon, and then cloned in pSB1C3. We then cloned in front of a pVeg-SpoVG promoter and behind a RBA RFP double terminator.</p>
<p>The ComS gene was synthetized by GeneArt but also insulated by PCR colony, changing the start and the stop codon, and then cloned in pSB1C3. We then cloned in front of a pVeg-SpoVG promoter and behind a RBA RFP double terminator.</p>
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<p>The next step was to electroporate the construct which is now waiting for caracterization.</p>
<p>The next step was to electroporate the construct which is now waiting for caracterization.</p>
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<h2>Creation of the ∆CodY strain with the reporter</h2>
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<h3>Creation of the ∆CodY strain with the reporter</h3>
<p>The genomic DNA from the CodY::spc strain from Linc Sonensheim laboratory was extracted and purified. The Elowitz's reporter strains were leaded to starvation, in order to make them competent, and then incubated for 30min with the genomic DNA. During this time lapse, the strain is randomly making recombination between the extracellular DNA and its own genome, leading, after selection, to the KO strain.</p>
<p>The genomic DNA from the CodY::spc strain from Linc Sonensheim laboratory was extracted and purified. The Elowitz's reporter strains were leaded to starvation, in order to make them competent, and then incubated for 30min with the genomic DNA. During this time lapse, the strain is randomly making recombination between the extracellular DNA and its own genome, leading, after selection, to the KO strain.</p>

Revision as of 18:55, 28 October 2011

Team IGEM Paris 2011

ComS/ComK switch system

The ComS/ComK switch device is based on a bistable system known as the 'MeKS' module (interactions among the MecA complex, comK and comS). ComS inhibits the MecA protease and allows the ComK protein to self-amplify. But as ComK inhibits the production of ComS, the system comes back to the original state within a few hours.

Abstract

Results for the ComK/ComS switch system:

  • We successfully BioBricked both the ComS gene (BBa_K606037) and the ComS gene codon optimized (BBa_K606038) constructs and sent them to the registry
  • We successfully knocked-out CodY gene in B.subtilis
  • We characterized the ∆CodY B.subtilis strain


Design overview

Fig1: Schematic of the ComS design

More information on the design here.

Parts and biobrick system construction

ComS constructions

The ComS gene was synthetized by GeneArt but also insulated by PCR colony, changing the start and the stop codon, and then cloned in pSB1C3. We then cloned in front of a pVeg-SpoVG promoter and behind a RBA RFP double terminator.

The construct was moved into an integration vector and also a multihost plasmid, called pHM3 from the Harald Putzer laboratory.

The next step was to electroporate the construct which is now waiting for caracterization.

Creation of the ∆CodY strain with the reporter

The genomic DNA from the CodY::spc strain from Linc Sonensheim laboratory was extracted and purified. The Elowitz's reporter strains were leaded to starvation, in order to make them competent, and then incubated for 30min with the genomic DNA. During this time lapse, the strain is randomly making recombination between the extracellular DNA and its own genome, leading, after selection, to the KO strain.

Then, the B. Subtilis are selected on Spectinomycine plates, and only the KO clones were selected. This protocol works very well and we got a huge number of recombinants.

Characterization of the ComS biobrick

The construct was cloned into our multihost vector. Electroporation into Elowitz reporter strain would permit to test the construct. Indeed, YFP expression would indicate that the ComS gene created is active and CFP expression reports that the ComK production has been triggered by overexpression of ComS.



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