Efficient delivery of DNA into E. coli remains a major limitation in the construction of high complex and representative gene libraries. The most efficient way developed to date is using electroporation - delivering an electric discharge to cell creating channels through which DNA can enter. We have created a mutant E. coli strain through a novel evolution process that results in a significantly more robust strain capable of surviving the electroporation process, and it concomitantly exhibits an improvement in efficiency. However, the efficiency with ligated DNA - the substrate that must be introduced into E. coli during library construction - remains highly inefficient. We propose to extend this evolutionary process to create variants that are better suited to accept larger, ligated DNA molecules. Understanding the molecular basis of these mutants will provide a template for how to create improved hosts of other bacterial species. In addition, identification of the mutation(s) responsible for resistance to electric shock it may provide insights into survival pathways in bacteria and higher organisms.
Preparing large and representative gene libraries is an important component for both basic science and in the evolution of proteins as therapeutics and diagnostics. A major limitation to the construction of these libraries is the ability to introduce he material efficiently into E. coli. We propose to create an E. coli host to overcome this limitation