Strain construction to test the significance of antiactivation in Pseudomonas quorum sensing

The quorum sensing regulatory pathway has been extensively studied for its impact on the virulence of Pseudomonas aeruginosa, an opportunistic pathogen that causes acute and chronic infections in immunocompromised individuals such as those suffering from the genetic disorder cystic fibrosis. Part of the quorum sensing pathway that has not been adequately described is the mechanism behind the accumulation of a threshold concentration of autoinducer. Recently, a protein, QteE, has been shown to inhibit the transcriptional regulator LasR. LasR binds the autoinducer molecule 3-oxo-dodecanoyl-homoserine lactone produced by the synthase LasI. QteE could also have a role in preventing the autoinducer of one cell binding its own LasR and inducing quorum sensing without a threshold concentration of autoinducer, essentially “short-circuiting” the cell. A co-culturing experiment with two QteE-deficient strains of P. aeruginosa, one LasI-proficient and the other LasI-deficient, would determine if this hypothesis is correct. The co-culturing experiment requires a method to differentiate between two different strains of bacteria grown together in the same medium, which in this case is tagging one strain with a red fluorescent protein, mCherry. For this thesis, I integrated a plasmid containing mCherry (pVM3) into the chromosome of the P. aeruginosa strains, and then confirmed their fluorescence with spectroscopy and microscopy. I also attempted to confirm the chromosomal location of the insertion by PCR