Team:ETH Zurich/labblog/20140811

From 2014.igem.org

(Difference between revisions)
(Cross talk experiments)
(Cross talk experiments)
Line 2: Line 2:
<html><article class="quorum sensing experiments" id="20140815"></html>
<html><article class="quorum sensing experiments" id="20140815"></html>
 +
 +
<html><article class="Quorum sensing experiments" id="20140815"></html>
== Cross talk experiments ==  
== Cross talk experiments ==  

Revision as of 14:05, 18 August 2014

Cross talk experiments

Friday, 15th 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 xxxxx Thus we evaluated for example the sensitivity of the piG0051 construct to different concentrations of 3OC6-HSL (LuxI product), but also its sensitivity towards 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. Therefore 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 want to increase AHL production. All these informations we will use to design a system tailored to our plans.

ETH Zurich crosstalk luxRR12y.png

Top