PCR-mediated gene replacement in Escherichia coli
PCR-mediated gene replacement in Escherichia coli
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DOI：
10.1016/s0378-1119(00)00071-8
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发表时间：
2000-04-04
期刊：
影响因子：
3.5
通讯作者：
Poteete, AR
中科院分区：
文献类型：
--
作者：
Murphy, KC;Campellone, KG;Poteete, AR
大肠杆菌菌株的超重组特性（其中recBCD基因已被lambda red重组功能替代）被用于开发通用PCR介导的大肠杆菌基因替代方案。通过电穿孔将重组 PCR 产生的线性 DNA 底物引入含有 recBCD Delta::red 取代的菌株中。该技术允许在大肠杆菌中进行基因替换，而无需事先克隆感兴趣的基因。此外，反选择标记 sacB 已用于构建未标记的精确基因删除，而不需要形成包含 sacB 的质粒整合体。在其他实验中，使用高浓度线性 DNA 片段（源自质粒消化物）对 reBCD Delta::red 菌株进行电穿孔，得到的线性转化率接近电穿孔幸存者的 1%。将lambda led 和gain 放置在染色体中除recBCD (galK) 之外的基因座上，产生的菌株与包含recBCD 替换的lambda I ed 的菌株一样hyperrec。这里描述的基因替换技术已用于构建 lacZ 和 sulA 的缺失替换等位基因，以及对大肠杆菌中一般同源重组重要的六个基因。其中三个替换是在没有事先克隆基因的情况下进行的。 (C) 2000 Elsevier Science B.V. 保留所有权利。
The hyper-recombinogenic properties of an E. coli strain in which the recBCD genes have been replaced by lambda red recombination functions were exploited in the development of a general PCR-mediated gene replacement scheme for Escherichia coli. Linear DNA substrates generated by recombinant PCR are introduced by electroporation into strains containing the recBCD Delta::red substitution. This technique allows for gene replacement in E. coli without prior cloning of the gene of interest. In addition, the counter-selectable marker sacB has been used to construct unmarked precise gene deletions without the need to form sacB-containing plasmid integrates. In other experiments, electroporation of recBCD Delta::red strains with high concentrations of linear DNA fragments (derived from plasmid digests) gave linear transformation rates approaching 1% of the survivors of electroporation. The placement of lambda led and gain at a locus in the chromosome other than recBCD (galK) resulted in a strain that was as hyperrec as one containing the lambda I ed for recBCD substitution. The gene replacement technique described here has been used for the construction of deletion-substitution alleles of lacZ and sulA, as well as six genes important for general homologous recombination in E, coli. Three of these replacements were performed without prior cloning of the genes. (C) 2000 Elsevier Science B.V. All rights reserved.