Our objective was to analyze the of our system for chitin detection and activation of the chininolytic metabolism of Vibrio fisherii ES144, we proposed two different strategies: First, we needed to characterize the expression of the two constitutive promoters of the two-component system (Sensor Promoter and CBP Promoter). We made a construct where the CBP promoter leads the expression of the mCherry reporter. The characterization was made using fluorometry and using as control the constitutive promoter of E. coli (BBa_J23119).
The characterization of the chitin sensor on E. coli will be made on the future using the same strategy.
Aditionally, we needed to made the characterization of the inducible promoter of the two component system (ChiP) as a response of the extracellular chitin concentration. In order to do that we realized a construct where ChiP was Upstream from the mCherry reporter. Without a termination codon, mCherry expression is controlled by the ChiP promoter. This construct will then be introduced into bacteria that contain plasmids that have the sequence for the Sensor and for CBP, where the level of fluorescence will be a measure of the promoter activation inducible to different extracellular concentrations of chitin. For this characterization the vectors have already been constructed and the transformation will be done soon. We´ll be sure to show you our results at the Jamboree!!
The characterization was performed using the same experimental conditions with a fluorometer. The strain with the pCPB-mCherry vector was grown in LB media with the antibiotic Kanamycin and the strain with the BBa_J23199 was grown in LB media with Ampicillin. The appropriate media with antibiotic was used as blank and for each strain. The intensities were normalized to delete the blank autofluorescence and also the difference between the brightness of each fluorescence protein.