Team:ETH Zurich/labblog/20140811

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== Cross talk experiments ==  
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== First cross talk experiments ==  
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==== Friday, 15th August ====
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==== 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.
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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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.
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.
All these informations we will use to design a system tailored to our plans.  
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|]]
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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.  
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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