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Team:Bielefeld-Germany/Project/Approach
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VirA does not work without the help of the VirG protein. The existing virG gene in the iGEM registry contains illegal restrictions site. Moreover it need the help of Chev Protein and sugars to work perfectly with virA. We decided to get some work done by MR. Gene. So we change the sequence manuelly and get the gene synthesized. | VirA does not work without the help of the VirG protein. The existing virG gene in the iGEM registry contains illegal restrictions site. Moreover it need the help of Chev Protein and sugars to work perfectly with virA. We decided to get some work done by MR. Gene. So we change the sequence manuelly and get the gene synthesized. | ||
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=== Creating new biobricks === | === Creating new biobricks === | ||
The applied system in ''E.Coli'' consists of two plasmids. After discussion the possibility of creating only one big plasmid, we unify that one plasmid will minimized the transformation chance and will be difficult to modify via error prone pcr. So we had to change the origin of the psb1C3 plasmid in order to avoid compatibility problems at the transformation. Therefor we cloned the r6K origin into the psb1C3 plasmid. For this purpose we used the r6k sequence and the psb1c3 sequence to create primers for the pcr following the ligation of the pcr products. | The applied system in ''E.Coli'' consists of two plasmids. After discussion the possibility of creating only one big plasmid, we unify that one plasmid will minimized the transformation chance and will be difficult to modify via error prone pcr. So we had to change the origin of the psb1C3 plasmid in order to avoid compatibility problems at the transformation. Therefor we cloned the r6K origin into the psb1C3 plasmid. For this purpose we used the r6k sequence and the psb1c3 sequence to create primers for the pcr following the ligation of the pcr products. | ||
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=== The error prone pcr === | === The error prone pcr === | ||
Revision as of 21:08, 23 August 2010
Contents |
The Approach
First we looked for a sensor system which is able to detect substances of interest. Moreover we needed substances of interest.The system of interest has to be taken out of a differnent bacteria species than E.Coli , in order to avoid any background. Therefor we checked the literature for a well reviewed sensor system, which does not be part of the E.Coli genome. The system of choice is the phenolic sensing system virA of Aggrobacteria tumefaciens. The systems naturally detects acetosyringone, which is a secondary metabolite of plants used as a bacterial attractant. So we hit the literature again to check for any other possible substances, which could be detected by the system. We got a really long list of possibilities and picked 'capcaicin', responsibily for spiciness of a fare.
The biological steps for creating a new sensor system are:
- extracting the virA system out of A. tumefaciens
- create new biobricks out of the exciting environmental parts
- transform the new biobricks into E.Coli
- modify the system for sensibility and specificity bt error prone pcr
- find and select the most promising mutants
Preparing the system
We tried to work with the already existing virA out of the registry. Unfortunately this virA Biobrick did not work at all. The sequenced data of the biobrick did not fit to the published virA sequence. So we had to extract the virA gene via PCR out of the A. tumefaciens genome by ourselves in order to create a new biobrick.
VirA does not work without the help of the VirG protein. The existing virG gene in the iGEM registry contains illegal restrictions site. Moreover it need the help of Chev Protein and sugars to work perfectly with virA. We decided to get some work done by MR. Gene. So we change the sequence manuelly and get the gene synthesized.
Creating new biobricks
The applied system in E.Coli consists of two plasmids. After discussion the possibility of creating only one big plasmid, we unify that one plasmid will minimized the transformation chance and will be difficult to modify via error prone pcr. So we had to change the origin of the psb1C3 plasmid in order to avoid compatibility problems at the transformation. Therefor we cloned the r6K origin into the psb1C3 plasmid. For this purpose we used the r6k sequence and the psb1c3 sequence to create primers for the pcr following the ligation of the pcr products.
The error prone pcr
After cloning the new origin into the psb1-backbone we are able to create the two constructs. The first one will be inside the competent bacteria cells and contains the virG promotor, the virG gene, a terminator, the readout promotor and the luc-readout gene. The second plasmid containing the virA promotor and the virA will be transformed and modified in one step via error prone pcr. The error prone pcr is a pcr under malfunction condition for the polymerase. It is possible to regulate the frequency of the mutagenesis by editing unbalanced concentration of nucleotides and co-factors to the pcr. So we are going to create a new biobrick and transform it into our target by one step. Before we are able to do the error prone pcr we need to get the backbones done.
Survival of the fittest
We use high amounts of the antibiotic kanamycin in order to select the most specific system for our substances. By editing the degree of kanamycin we are able to carefully select the best mutant out of our pcr-tests. The mutated virA system will be induced after the error prone pcr by a mix of possible targets for the system (like capcaicin, dopamin, homovanillic acid etc.). It is important to avoid any acetosyringone, so we will be able to search for systems with new targets.
If we do not handle with a lack of time, we are going to trying to transform a sensitivity into the biobricks. This will increase the sensitivity of our system.
