Team:ETH Zurich/labblog/20140811

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== Cross talk experiments ==  
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== First cross talk experiments ==  
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= Date :  Friday, 15th August =
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==== Friday, 11th August ====
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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.
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=== Cross talk experiments ===
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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.
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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. All these informations we will use to design a system tailored to our plans.  
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To give an example: we evaluated the sensitivity of the siG0024 strain to different concentrations of 3OC<sub>6</sub>-HSL (LuxI product) and compared its characteristics to  those of other constructs. Additionally, we measured the sensitivity of siG0024 to 3OC<sub>12</sub>-HSL (LasI product) and C<sub>4</sub>-HSL (RhlI product). Indeed, we could observe the induction of sfGFP transcription by comparably low levels of 3OC<sub>12</sub>-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.
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All these informations we will use to design a system tailored to our plans.  
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[[File:ETH_Zurich_crosstalk_luxRR12y.png|center|800px|thumb|Summary of cross-talk experiments results]]
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The figure shows the dose response of xxx to 3OC<sub>12</sub>-HSL, 3OC<sub>6</sub>-HSL and C<sub>4</sub>-HSL. Both 3OC<sub>12</sub>-HSL and 3OC<sub>6</sub>-HSL induce production of GFP, although at different concentrations. This is an example for cross talk between the lux and the las system.
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Latest revision as of 15:24, 11 October 2014