One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products
One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products
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DOI：
10.1073/pnas.120163297
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发表时间：
2000-06-06
影响因子：
11.1
通讯作者：
Wanner, BL
中科院分区：
文献类型：
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作者：
Datsenko, KA;Wanner, BL
我们已经开发了一种简单而高效的方法来破坏大肠杆菌中的染色体基因，其中PCR引物提供与靶基因的同源性。在该方法中，重组需要噬菌体λ Red重组酶，其在容易固化的低拷贝数质粒上的诱导型启动子的控制下合成。为了证明该方法的实用性，我们使用具有36至50个碱基的引物产生PCR产物与待失活基因相邻区域同源的nt延伸和携带抗生素抗性基因的模板质粒，侧翼为FRT（FLP识别靶）位点。通过使用各自的PCR产物，我们对染色体基因进行了13种不同的破坏。arcB、cyaA、lacZYA、ompR-envZ、phnR、pstB、pstCA、pstS、pstSCAB-phoU、recA和torSTRCAD基因或操纵子的突变体在引入携带合成的（PCR产生的）DNA的Red表达质粒的细菌中后作为耐药性菌落分离。然后通过使用编码也容易治愈的FLP重组酶的辅助质粒消除抗性基因。这种方法应该是广泛有用的，特别是在大肠杆菌和其他细菌的基因组分析，因为该程序可以在野生型细胞中完成。
We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid, To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA, The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E, coli and other bacteria because the procedure can be done in wild-type cells.