National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, 100101, Beijing, China,
World J Microbiol Biotechnol. 2010 Jul;26(7):1323-9. doi: 10.1007/s11274-009-0305-y. Epub 2010 Jan 8.
A simple and general method for disrupting chromosomal genes and introducing insertions is described. This procedure involves eliminating wild-type bacterial genes and introducing mutant alleles or other insertions at the original locus of the wild-type gene. To demonstrate the utility of this approach, the tig gene of Escherichia coli was replaced by homologous recombination with a cassette containing the chloramphenicol resistance gene and the sacB gene. The cassette was then removed and the tig mutant alleles were moved into the native tig location. Sequencing and Western blotting results demonstrated that insertions or deletions can be introduced precisely in E. coli using our approach. Our system does not require extra in vitro manipulations such as restriction digestion or ligation, and does not require use of specific plasmids or strains which are used to prevent false positive transformants caused by template plasmid transformation. This technique can be used widely in bacterial genome analysis.
描述了一种简单而通用的破坏染色体基因并引入插入的方法。该方法涉及消除野生型细菌基因，并在野生型基因的原始基因座处引入突变等位基因或其他插入物。为了证明该方法的实用性，用含有氯霉素抗性基因和 sacB 基因的盒通过同源重组替换了大肠杆菌中的 tig 基因。然后去除盒，并将 tig 突变等位基因转移到天然 tig 位置。测序和 Western blot 结果表明，我们的方法可以在大肠杆菌中精确引入插入或缺失。我们的系统不需要额外的体外操作，如限制消化或连接，也不需要使用特定的质粒或菌株，这些质粒或菌株用于防止由于模板质粒转化而导致的假阳性转化体。该技术可广泛用于细菌基因组分析。
