基于Red重组系统和Xer重组系统的大肠杆菌多基因删除方法

快速、高效删除大肠杆菌染色体DNA的目的基因是大肠杆菌代谢工程研究的前提和基础。利用Red重组系统结合Xer重组系统删除了野生型大肠杆菌CICIM B0013的ackA-pta基因和pps基因。实验证明了可重复应用dif位点实现大肠杆菌染色体上多基因突变的叠加,同时,在染色体上并未留下抗生素标记,借此能够高效地实现多基因缺失突变株的构建。此外,本方法重组效率高,实验步骤较简便。

Multiple Gene Inactivation Approach in Escherichia coli Mediated by a Combination of Red Recombination and Xer Recombination

Rapid and efficient inactivation of the target gene on Escherichia coli chromosome is the precondition and groundwork of researches on metabolic engineering. In the present study, we demonstrated a multiple gene inactivation approach in E. coli mediated by Red recombination and Xer recombination. The chromosomal genes, ackA-pta and pps, in a wild type strain E. coli CICIM B0013 were inactivated by this method indicating that dif sites can be reused to inactivate multiple chromosomal genes with no antibiotic resistance selectable marker remain. Furthermore, this method has high recombination efficiency and simplified steps.