Team:Paris Bettencourt/Experiments/ComS diffusion

From 2011.igem.org

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<p>CodY is known to repress many genes of the steady state phase during the exponential phase. In order to avoid this problem, we have created a B.subtilis strain ∆CodY. This will allow the MeKS system to be active during the exponential growth phase, as well as thousands of stationnary phase genes. This mutation is not lethal.</p>
<p>CodY is known to repress many genes of the steady state phase during the exponential phase. In order to avoid this problem, we have created a B.subtilis strain ∆CodY. This will allow the MeKS system to be active during the exponential growth phase, as well as thousands of stationnary phase genes. This mutation is not lethal.</p>
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<p>During the exponential phase, the ∆CodY strain ComK and ComS genes are expressed almost one after the other and MecA is active. Hence, the YFP and the CFP genes can be expressed in a stochastic manner and with a time lag between them. In the strain of Elowitz (PY79 B. subtilis with pHP13::PcomG-cfp-PcomS-yfp), CodY is expressed, which blocks the system MeKS in exponential phase. Thus, that strain can not express neither CFP nor YFP genes.</p>
+
<p>During the exponential phase, in the ∆CodY strain, ComK and ComS genes are expressed almost one after the other and MecA is active. Hence, the YFP and the CFP genes can be expressed in a stochastic manner and with a time lag between them. In the strain of Elowitz (PY79 B. subtilis with pHP13::PcomG-cfp-PcomS-yfp), CodY is expressed, which blocks the system MeKS in exponential phase. Thus, that strain can not express neither CFP nor YFP genes.</p>
-
<p>To compare the two strains above, we first focus on YFP images. We see that ∆CodY strain do not express YFP and Elowitz' strain express YFP.  Secondly, we focus on CFP images. We clearly see that the ∆CodY strain do not express CFP apart from a bit of leakage. In the Elowitz' strain, we can not observe any fluorescence (CFP).</p>
+
<p>To compare the two strains above, we first focus on YFP images. We see that ∆CodY strain does not express YFP and Elowitz' strain expresses YFP.  Secondly, we focus on CFP images. We clearly see that the ∆CodY strain does not express CFP apart from a bit of leakage. In the Elowitz' strain, we can not observe any fluorescence (CFP).</p>
-
<p>The results are not conclusive but we have also many hypotheses. First, the knock out of the strain failed but we get the resistant strain to the expected antibiotic. Second, we do not observe any fluorescence in the ∆CodY strain in the same time although we were expecting at least YFP or CFP, we could wonder if the bistability of the system in exponential phase works.</p>
+
<p>The results are not conclusive but we have also many hypotheses. First, the knock out of the strain failed but we get the resistant strain to the expected antibiotic. Second, we do not observe any fluorescence in the ∆CodY strain although we were expecting at least YFP or CFP. We could wonder if the bistability of the system in exponential phase works.</p>
<p>To solve the troubles, we should put ComS in both strains.</p>
<p>To solve the troubles, we should put ComS in both strains.</p>
<br/>
<br/>

Revision as of 03:00, 29 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
  • We saw that the system does not behave as our model predicted, with a YFP-noise level lower than expected


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.

Here is the cloning plan we followed. Unfortunately, we didn't have the time to go until the B. Subtilis plasmid.





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 40 min 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.

Characterization of the ∆CodY strain

We characterized ∆CodY B. subtilis strain.

∆CodY in B.subtilis at 37°C in exponential phase
B.subtilis ∆CodY at 37°C (trans image)
B.subtilis ∆CodY at 37°C (yfp image)
B.subtilis ∆CodY at 37°C (cfp image)


Py79 control strain at 37°C in exponential phase (see the paper: [2])
B.subtilis PY79 at 37°C (trans image)
B.subtilis PY79 at 37°C (yfp image)
B.subtilis PY79 at 37°C (cfp image)


CodY is known to repress many genes of the steady state phase during the exponential phase. In order to avoid this problem, we have created a B.subtilis strain ∆CodY. This will allow the MeKS system to be active during the exponential growth phase, as well as thousands of stationnary phase genes. This mutation is not lethal.

During the exponential phase, in the ∆CodY strain, ComK and ComS genes are expressed almost one after the other and MecA is active. Hence, the YFP and the CFP genes can be expressed in a stochastic manner and with a time lag between them. In the strain of Elowitz (PY79 B. subtilis with pHP13::PcomG-cfp-PcomS-yfp), CodY is expressed, which blocks the system MeKS in exponential phase. Thus, that strain can not express neither CFP nor YFP genes.

To compare the two strains above, we first focus on YFP images. We see that ∆CodY strain does not express YFP and Elowitz' strain expresses YFP. Secondly, we focus on CFP images. We clearly see that the ∆CodY strain does not express CFP apart from a bit of leakage. In the Elowitz' strain, we can not observe any fluorescence (CFP).

The results are not conclusive but we have also many hypotheses. First, the knock out of the strain failed but we get the resistant strain to the expected antibiotic. Second, we do not observe any fluorescence in the ∆CodY strain although we were expecting at least YFP or CFP. We could wonder if the bistability of the system in exponential phase works.

To solve the troubles, we should put ComS in both strains.



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