Team:Marburg SYNMIKRO/Results

Summary

We aimed to establish a system in Escherichia coli cells that generates a large number of novel proteins by combinatorial fusion of subfragments. Our project was inspired by the VDJ-recombination of the vertebrate immune system. There, a limited number of protein coding sequences is used to generate the enormous diversity of antibodies. We designed A and B modules containing different protein coding subfragments to be recombined. Fusion of one A fragment with one B fragment is achieved by the site-specific DNA recombinase Gin of bacteriophage Mu. Recombination sites were arranged as direct repeats thus causing deletions upon recombination. Gin catalyzed recombination depends on the presence of an enhancer element.This enhancer was placed together with the suicide gene sacB between the A and B modules. This design guarantees that recombination automatically stops when the central fragment is deleted. To test our system and to visualize the combinatorial activity we fused fluorescent proteins of different color to different intracellular localization domains. We expect that after successful recombination E. coli cells will glow in different colors located at different positions within the cell.