Qu Jingqiu, Liu Cuihua, Liu Weifeng, Tao Yong, Zhu Kun
Institute of Microbiology Chinese Academy of Sciences, Beijing 100190, China.
Wei Sheng Wu Xue Bao. 2013 Jun 4;53(6):608-14.
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.
Using the Escherichia coli Keio single-mutant library, we combined Red homology recombination with P1 phage transduction and developed a method to knock out the genes in Escherichia coli MG1655.
We obtained beta-oxidation mutants deltafadD, deltafadE and deltafadD-deltafadE and fatty acid synthesis mutants deltafabH, deltafabF and deltafabH-deltafabF. There were no obvious growth changes between deltafadD or deltafadE mutant strains and MG1655. However, deltafabH and deltafabH-deltafabF mutant strains grew much slower than the wild type strain. The fatty acid contents in deltafadD, deltafadE and deltafadD-deltafadE 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 deltafabH, deltafabF and deltafabH-deltafabF were 12.6 mg/L, 15.2 mg/L and 11.2mg/L respectively, lower than that in wild type.
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.
基因敲除技术对于基因功能研究非常重要。我们开发了一种简单高效的方法来敲除大肠杆菌中的染色体基因。
利用大肠杆菌Keio单突变体文库，我们将Red同源重组与P1噬菌体转导相结合，开发了一种敲除大肠杆菌MG1655中基因的方法。
我们获得了β-氧化突变体ΔfadD、ΔfadE和ΔfadD-ΔfadE以及脂肪酸合成突变体ΔfabH、ΔfabF和ΔfabH-ΔfabF。ΔfadD或ΔfadE突变株与MG1655之间没有明显的生长变化。然而，ΔfabH和ΔfabH-ΔfabF突变株的生长比野生型菌株慢得多。ΔfadD、ΔfadE和ΔfadD-ΔfadE中的脂肪酸含量分别为18.2mg/L、20.0mg/L和19.2mg/L，高于野生型的17.5mg/L。ΔfabH、ΔfabF和ΔfabH-ΔfabF中的脂肪酸含量分别为12.6mg/L、15.2mg/L和11.2mg/L，低于野生型。
利用Keio突变体文库、P1噬菌体转导和抗性基因消除，我们建立了一种简单高效的大肠杆菌基因敲除方法。
