Team:Marburg SYNMIKRO

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“The Recombinator”: an intelligent Genetically Engineered Slot Machine (iGESM)

The vertebrate immune system produces billions of different antibodies. This diversity is generated by random VDJ-recombination of a limited number of antibody subfragments. This inspired us to construct an automatic recombination system in E. coli that generates large numbers of novel proteins by combinatorial fusion of functional domains. The site-specific DNA recombinase Gin of bacteriophage Mu depends on the presence of a DNA enhancer element for efficient recombination. This allowed us to construct a system, called “The Recombinator”, which automatically shuts down after successful recombination. We visualized the randomizing function of our genetically engineered slot machine by combining colors with cellular localization domains. By scaling up the number of recombination modules and functional domains our system will be able to generate a multitude of new proteins. We envision that “The Recombinator” will serve as a tool to create novel enzymatic activities for innovative drug design, environmental detoxification and metabolic engineering.

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-== '''Overall project''' ==
+== “The Recombinator”: an intelligent Genetically Engineered Slot Machine (iGESM) ==
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-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.
+The vertebrate immune system produces billions of different antibodies. This diversity is generated by random VDJ-recombination of a limited number of antibody subfragments. This inspired us to construct an automatic recombination system in E. coli that generates large numbers of novel proteins by combinatorial fusion of functional domains. The site-specific DNA recombinase Gin of bacteriophage Mu depends on the presence of a DNA enhancer element for efficient recombination. This allowed us to construct a system, called “The Recombinator”, which automatically shuts down after successful recombination. We visualized the randomizing function of our genetically engineered slot machine by combining colors with cellular localization domains. By scaling up the number of recombination modules and functional domains our system will be able to generate a multitude of new proteins. We envision that “The Recombinator” will serve as a tool to create novel enzymatic activities for innovative drug design, environmental detoxification and metabolic engineering.
-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 create 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 to fluoresce in different colors 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)
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