Cross talk experiments

After having constructed the necessary regulator and sensor plasmids we were able to start first cross talk experiments. Therefor we transformed competent cells with different combinations of regulator and sensor plasmids. One example would be the strain siG0024 which contains the regulator plasmid piG0041 producing LuxR under a constitutive promoter and the sensor plasmid piG0051 that encodes for GFP. The latter is under control of a pluxR promoter, hence it will be transcribed in the presence of LuxR and Lux. To assess cross talk and leakiness we conducted multiple experiments: after adding one of the three AHLs to the cultures we analyzed the raise in GFP production using a microplate reader. Thus we evaluated for example the sensitivity of the piG0051 construct to different concentrations of Lux, but also its sensitivity towards Rhl and Las. Actually, we could observe the induction of GFP transcription by comparably low levels of Las; cross talk can be observed for the Las system and the Lux system. Additionally, we tested the influence of different ribosomal binding sites (RBSs) on the GFP production rates. All these informations we will use to design a system suitable for our plans.