Revision as of 23:17, 27 October 2010

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Results

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 build. 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. 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 entered the following BioBricks to the partsregistry:

<groupparts>iGEM010 Bielefeld-Germany</groupparts>

@@ Line 23: / Line 23: @@
 On the next pages we are going to present you our BioBrick work.
-One approach of our project was to visualize a read-out by a light emitting system. So a luciferase BioBrick was build. 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 [[Team:Bielefeld-Germany/Results/Characterization/K389004 | here]].
+One approach of our project was to visualize a readout by a light emitting system. So a luciferase BioBrick was build. 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 [[Team:Bielefeld-Germany/Results/Characterization/K389004 | 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 [[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. 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 a phenolic substance like 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. 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.
-Another goal of our project was to have create a light signal that is visible to the human eye. 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 read-out 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 read-out 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 ([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.