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Team:Marburg SYNMIKRO
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== '''Overall project''' == | == '''Overall project''' == | ||
| - | We aim to establish a system in Escherichia coli that generates a large number of different proteins by combinatorial fusion of few protein coding sub fragments. Our project was inspired by the VDJ-recombination of human immune system using a limited number of protein coding sequences to generate a huge diversity of antibodies. | + | We aim to establish a system in Escherichia coli that generates a large number of different proteins by combinatorial fusion of few protein coding sub fragments. |
| - | We designed an A and B module containing each three different protein coding sub fragments to be recombined. Recombination of one A fragment with one B fragment is achieved by the site-specific Gin recombinase of bacteriophage Mu. Specific Gix sites arranged as direct repeats are causing deletion within the modules and creating | + | Our project was inspired by the VDJ-recombination of human immune system using a limited number of protein coding sequences to generate a huge diversity of antibodies.We designed an A and B module containing each three different protein coding sub fragments to be recombined. Recombination of one A fragment with one B fragment is achieved by the site-specific Gin recombinase of bacteriophage Mu. Specific Gix sites arranged as direct repeats are causing deletion within the modules and creating combined fragments. Other Biobricks like a Gin enhancer and the suicide gene ccdb are located in between the modules and being deleted if an A and B fragment is fused. Those are necessary to ensure proper recombination. To visualize our results we decided to fuse fluorescent proteins of different color with intracellular localization domains. As proof of principle of our recombination machine, we expect E. colis that are different colored as well as showing color at different cell domains. |
(This might also give us the opportunity to quantify whether occurrence of curtain recombination products is related to the position of its sub fragments to the Gin enhancer) | (This might also give us the opportunity to quantify whether occurrence of curtain recombination products is related to the position of its sub fragments to the Gin enhancer) | ||
Revision as of 16:27, 15 July 2012
Overall project
We aim to establish a system in Escherichia coli that generates a large number of different proteins by combinatorial fusion of few protein coding sub fragments. Our project was inspired by the VDJ-recombination of human immune system using a limited number of protein coding sequences to generate a huge diversity of antibodies.We designed an A and B module containing each three different protein coding sub fragments to be recombined. Recombination of one A fragment with one B fragment is achieved by the site-specific Gin recombinase of bacteriophage Mu. Specific Gix sites arranged as direct repeats are causing deletion within the modules and creating combined fragments. Other Biobricks like a Gin enhancer and the suicide gene ccdb are located in between the modules and being deleted if an A and B fragment is fused. Those are necessary to ensure proper recombination. To visualize our results we decided to fuse fluorescent proteins of different color with intracellular localization domains. As proof of principle of our recombination machine, we expect E. colis that are different colored as well as showing color at different cell domains. (This might also give us the opportunity to quantify whether occurrence of curtain recombination products is related to the position of its sub fragments to the Gin enhancer)
