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Team:Paris Bettencourt/Experiments/ComS diffusion
From 2011.igem.org
ComS/ComK switch system
Constructs done
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 where leaded to starvation, in order to make them competent, and then incubated for 30min with the genomic DNA. In this time laps, the strain is randomly making recombination of the outside DNA with its own genome, and make the KO in themself.
Then, the B. Subtilis are selected on Spectinomycine plates, and only the KO clones where selected. At our great surprise, this protocol works very well and we got a huge number of recombinants.
Caracterization of the ComS biobrick
The construct was cloned into our multihost vector and electroporated into Elowitz reporter strain. The YFP expressed in the cell indicates that the ComS gene created is active and the CFP reports that the ComK production has been triggered by overexpression of ComS.
