利用Red重组系统对大肠杆菌ClpP基因的敲除

利用含有质粒pKD4 6的菌株BW2 5 113,在阿拉伯糖诱导后 ,表达λ噬菌体的 3个重组蛋白 ,宿主菌就具有了同源重组的能力 .设计的引物 5′端有 5 0bp的拟敲除基因的同源臂 ,3′端为扩增引物 ,以pKD3为模板 ,扩增两侧含FRT位点的氯霉素抗性基因 ,将此线性片段电转入具重组功能的感受态细胞 ,利用氯霉素平板就可以筛选到阳性转化体 .再利用表达Flp重组酶的质粒pCP2 0 ,可将FRT位点之间的氯霉素抗性基因删除 .利用该重组系统 ,构建了ClpP蛋白酶缺失的大肠杆菌工程菌株 ,可望在减少外源蛋白的降解方面发挥一定的作用 .

Deletion of clpP in Chromosome of E.coli by Red Recombination

Plasmid pKD46 can express three proteins: Gam, Bet and Exo. Gam inhibits the host RecBCD exonuclease V, so that Bet and Exo can gain access to DNA ends to promote recombination. BW25113 with pKD46 has the function of recombination when induced by L arabinose. PCR products were obtained by using primers with 50 bp extension which were homologous to clpP and by using template plasmid named pKD3 carrying chloramphenicol resistance gene flanked by FRT sites. The PCR products were introduced into BW25113 by electroporation. The strains expressing chloramphenicol resistance gene were selected by chloramphenicol agar. The chloramphenicol resistance gene was then eliminated by using a helper plasmid, pCP20, encoding the Flp recombinase.Using this system, ClpP gene in chromosome of Escherichia coli was deleted.