Latest revision as of 02:19, 28 October 2010

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Results

Figure 1: Visualization of induced (from left to right) <partinfo>K389421</partinfo>, <partinfo>K389422</partinfo> and <partinfo>K389423</partinfo> sensitivity tuner amplified vir-system.

On the next pages we are going to present you our BioBrick work.

One approach of our project was to visualize a readout by a light emitting system. So a luciferase BioBrick was built. This BioBrick is a highly sensitive reporter gene which was successfully applied in different devices with a PoPS output. The characterization of this BioBrick can be found here.

The second step was to establish a complete VirA/G signaling system from Agrobacterium tumefaciens in Escherichia coli. For this purpose three new BioBricks were created - virA, virG and a vir promoter. The VirA receptor recognizes the phenolic substance acetosyringone and transmits this information to the VirG response regulator - a bacterial transcription factor which activates vir promoters. This natural VirA/G signaling system works in E. coli and was measured and characterized by using the reporter genes mRFP and luciferase.

In the third step we tried to modify the VirA receptor by directed mutagenesis using error-prone PCR, in order to detect substances other than acetosyringone (e.g. capsaicin). For that purpose we designed a high-throughput screening system with a selective system based on the expression of a kanamycin resistance. We tried to modify the receptor by different modification strategies like error prone (random mutagenesis) or specific amino acid changes (mut1 / mut2). The constructs mut1 and mut2 were done by an in silico approach. Unfortunately they did not work as expected. In a short period of time we found many positive clones, but further analysis indicated that the VirA receptor was changed to constitutive activity. Thereby, due to the short time available, we did not manage to construct a receptor that was specific for other phenolic substance, e.g. capsaicin.

We tried to create a light signal that is visible to the human eye as another part of our project. So genetic amplifiers (Cambridge, iGEM 2007, amplifier project) were added to our luciferase BioBrick. This amplified luciferase readout was coupled with the VirA/G signaling system in order to visualize the induction behaviour. The amplifiers worked so far and gave a visible luciferase readout but the basal expression of the vir promoter was also amplified so the induced status of the VirA/G system was not discriminable properly from the uninduced status.

We entered the following BioBricks to the partsregistry:

<groupparts>iGEM010 Bielefeld-Germany</groupparts>

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 The second step was to establish a complete VirA/G signaling system from ''Agrobacterium tumefaciens'' in ''Escherichia coli''. For this purpose three new BioBricks were created - ''virA'', ''virG'' and a ''vir'' promoter. The VirA receptor recognizes the phenolic substance acetosyringone and transmits this information to the VirG response regulator - a bacterial transcription factor which activates ''vir'' promoters. This natural VirA/G signaling system works in ''E. coli'' and was measured and characterized by using the reporter genes [[Team:Bielefeld-Germany/Results/Characterization/K389016 | mRFP]] and [[Team:Bielefeld-Germany/Results/Characterization/K389015 | luciferase]].
-In the third step we tried to modify the VirA receptor by directed mutagenesis using error-prone PCR, in order to detect substances other than acetosyringone (e.g. capsaicin). For that purpose we designed a [[Team:Bielefeld-Germany/Project/Theory#Screening_system | high-throughput screening system]] with a selective system based on the expression of a kanamycin resistance. We tried to modify the receptor by different modification strategies like error prone (random mutagenesis) or specific amino acid changes ([[Team:Bielefeld-Germany/Project/Approach#Directed_mutagenesis | mut1 / mut2]]). The constructs mut1 and mut2 were done by an ''in silico'' approach. Unfortunately they did not work as expected. In a short period of time we found many positive clones, but further analysis indicated that the VirA receptor was changed to constitutive activity. Thereby, due to the short time available, we did not manage to construct a receptor that was specific for other phenolic substance, e.g. capsaicin.
+In the third step we tried to modify the VirA receptor by directed mutagenesis using error-prone PCR, in order to detect substances other than acetosyringone (e.g. capsaicin). For that purpose we designed a [[Team:Bielefeld-Germany/Project/Theory#Screening_system | high-throughput screening system]] with a selective system based on the expression of a kanamycin resistance. We tried to modify the receptor by different modification strategies like error prone (random mutagenesis) or specific amino acid changes ([[Team:Bielefeld-Germany/Project/Approach#Directed_mutagenesis |mut1 / mut2]]). The constructs mut1 and mut2 were done by an ''in silico'' approach. Unfortunately they did not work as expected. In a short period of time we found many positive clones, but further analysis indicated that the VirA receptor was changed to constitutive activity. Thereby, due to the short time available, we did not manage to construct a receptor that was specific for other phenolic substance, e.g. capsaicin.
-We tried to create a light signal that is visible to the human eye as another part of our project. So genetic amplifiers ([http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project#Results Cambridge, iGEM 2007, amplifier project]) were added to our luciferase BioBrick. This amplified luciferase readout was coupled with the VirA/G signaling system in order to visualize the induction behaviour. The amplifiers worked so far and gave a visible luciferase readout but the basal expression of the ''vir'' promoter was also amplified so the induced status of the VirA/G system was not discriminable properly from the uninduced status.
+We tried to create a light signal that is visible to the human eye as another part of our project. So genetic amplifiers ([https://2007.igem.org/Cambridge/Amplifier_project#Results Cambridge, iGEM 2007, amplifier project]) were added to our luciferase BioBrick. This amplified luciferase readout was coupled with the VirA/G signaling system in order to visualize the induction behaviour. The amplifiers worked so far and gave a visible luciferase readout but the basal expression of the ''vir'' promoter was also amplified so the induced status of the VirA/G system was not discriminable properly from the uninduced status.