Developing an Improved Electrocompetent E. coli Host

描述（由申请人提供）：将DNA有效递送到E.大肠杆菌中的表达水平仍然是构建高度复杂和代表性基因文库的主要限制。迄今为止开发的最有效的方法是使用电穿孔-将放电传递到细胞，从而创建DNA可以进入的通道。我们创造了一个变种E大肠杆菌菌株通过一种新的进化过程，其导致能够在电穿孔过程中存活的显著更健壮的菌株，并且其伴随地表现出效率的提高。然而，连接的DNA的效率-必须引入到E.大肠杆菌中的表达，仍然是非常低效的。我们建议扩展这一进化过程，以创造更适合接受更大的连接DNA分子的变体。了解这些突变体的分子基础将为如何创造其他细菌物种的改良宿主提供模板。此外，突变的鉴定 负责抵抗电击，它可以提供洞察细菌和高等生物的生存途径。

DESCRIPTION (provided by applicant): 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.