Team:ETH Zurich/labblog/20140811

Cross talk experiments

Friday, 11th August

After having constructed the necessary regulator and sensor plasmids we were able to start first cross talk experiments. Therefore 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 sfGFP. The latter is under control of a pluxR promoter, hence it will be transcribed in the presence of LuxR and Lux. To assess crosstalk and leakiness we conducted multiple experiments: after adding one of the three AHLs to the cultures we analyzed the raise in sfGFP production using a microtiterplate reader. In a first step we measured the dose response of constructs with different ribosomal binding sites (RBSs) and with or without riboregulator. We could observe differences not only in sensitivity, but also in leakiness; some strains expressed GFP even without AHL induction. To give an example: we evaluated the sensitivity of the siG0024 strain to different concentrations of 3OC₆-HSL (LuxI product) and compared its characteristics to those of other constructs. Additionally, we measured the sensitivity of siG0024 to 3OC₁₂-HSL (LasI product) and C₄-HSL (RhlI product). Indeed, we could observe the induction of sfGFP transcription by comparably low levels of 3OC₁₂-HSL. Crosstalk can be observed between the Las and the Lux system. To simulate a situation most similar to the final experiment, we added the diluted supernatant of AHL producer cells to the test cultures. However, we could not detect any GFP production as reaction to the supernatant. We assume that the AHL concentration is not high enough to induce transcription. By constructing stronger RBSs on the producer plasmids we aim at increasing the AHL production. All these informations we will use to design a system tailored to our plans.

Summary of cross-talk experiments results

The figure shows the dose response of xxx to 3OC₁₂-HSL, 3OC₆-HSL and C₄-HSL. Both 3OC₁₂-HSL and 3OC₆-HSL induce production of GFP, although at different concentrations. This is an example for cross talk between the lux and the las system.