[Objective] Gene knock out technique is very important for gene function study.We developed a simple and efficient method to knock out chromosomal genes in Escherichia coli.[Methods] Using the Escherichia coli Keio singlemutant library,we combined Red homology recombination with P1 phage transduction and developed a method to knock out the genes in Escherichia coli MG1655.[Results] We obtained β-oxidation mutants △fadD,△ fadE and △fadD△ fadE and fatty acid synthesis mutants △fabH,△fabF and △fabH-△fabF.There were no obvious growth changes between △fadD or △ fadE mutant strains and MG1655.However,△fabH and △fabH-△fabF mutant strains grew much slower than the wild type strain.The fatty acid contents in △fadD,△ fadE and △fadD-△fadE were 18.2 mg/L,20.0 mg/L and 19.2 mg/L respectively,higher than 17.5 mg/L in wild type.The fatty acid contents in △fabH,△fabF and △fabH-△fabF were 12.6 mg/L,15.2 mg/L and 11.2mg/L respectively,lower than that in wild type.[Conclusion] Using Keio mutant library,P1 phage transduction and resistant gene elimination,we have established a simple and efficient method for gene knock-out in Escherichia coli.%[目的]基因敲除技术是研究基因功能的重要手段.我们试图建立一种快速、高效的大肠杆菌基因敲除方法.[方法]利用大肠杆菌(Escherichia coli) BW25113单基因缺失体Keio文库,将经典的Red同源重组技术与P1噬菌体转导技术相结合,对E.coli MG1655脂肪酸代谢基因进行快速敲除.[结果]获得了大肠杆菌β-氧化途径的缺失菌株△fadD、△fadE和△fadD-△fadE；脂肪酸合成途径缺失菌株△fabH、△fabF和△fabH-△fabF.敲除fadD和fadE对生长情况没有影响；敲除fabH后,生长速度明显减慢；敲除fabF对生长几乎没有影响.FadD、FadE及双敲缺失体的脂肪酸含量18.2 mg/L、20.0mg/L和19.2 mg/L,略高于野生型17.5 mg/L;FabH、FabF及双敲缺失体的含量分别为12.6 mg/L、15.2 mg/L和11.2 mg/L,明显低于野生型.[结论]在单基因突变体文库基础上,利用P1噬菌体转导、Red同源重组和抗性基因消除进行基因敲除,简化了构建大肠杆菌单基因和多重突变体的方法.
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