Experimental results overview

We started experiments using LuxI producing sender cells and OHHL inducible GFP expressing receiver cells. We used these to characterize OHHL dose response as well as the OHHL diffusion in agar plates. The main goal of the experiments was to find the conditions regarding time and distance for later experiments. Having set those parameters we tried to construct a library of pLux promoters with sensitivities differing from those of the wild type pLux. Through site-saturation promoter mutagenesis we changed the LuxR binding sites. The promoter variants were characterized doing OHHL dose response curves and we were able to select for two interesting variants. In parallel we characterized our hydrolase reporter system by testing different substrates and investigating kinetics. During all of these experiments we encountered problems with the leakiness of the pLux promoter meaning that there was basal expression of reporter even in the absence of OHHL. By testing different LuxR generating constructs we tried to minimize the leakiness of the system, because we expect our enzymatic reporter system to be even more sensitive than the fluorescent protein reporters. So far we have successfully set up the game structure with the wild type pLux promoter and a GFP reporter. We have characterized one possible promoter variant with a much lower sensitivity for OHHL and we successfully used five different hydrolases without observing crosstalk. Using a positive feedback loop we could also reduce the basal expression of LuxR and therefore the leakiness of the system.

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