Experimental results overview

In our game, we use the LuxI-LuxR quorum sensing system to transport the signal from the sender (mine) to the receiver (non-mine) cells. We started experiments using LuxI producing sender cells and AHL inducible GFP expressing receiver cells. We used this sender-receive module to characterize AHL diffusion in agar plates. The main goal of these experiments was to find conditions regarding time and distance for the design of the game grid. Having set these parameters, we tried to construct a library of P_LuxR promoters with AHL sensitivities differing from those of the wild type P_LuxR. Through site-saturation promoter mutagenesis we changed the LuxR binding sites. The promoter variants were characterized studying AHL dose response curves and we were able to select for several mutant variants. We than moved on to a set_up using hydrolases as output in order to have a color code reading visible by eye and simple to play. THe experiments with the hydrolases show that our system is leaky and we reviewed the circuit and optimized it by introduction of a negative feedback loop. In the end we set-up a preliminary game.

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 AHL. 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 P_Lux promoter and a GFP reporter. We have characterized one possible promoter variant with a much lower sensitivity for AHL and we successfully used five different hydrolases without observing crosstalk.

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