Recombineering with tolC as a selectable/counter-selectable marker: remodeling the rRNA operons of Escherichia coli.-猫眼课题宝

DOI：

10.1093/nar/gkm1084

发表时间：

2008-01

期刊：

Nucleic acids research

影响因子：

14.9

通讯作者：

中科院分区：

生物学2区

文献类型：

作者：

DeVito JA

来源链接：

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2248734/?report=classic

这项工作描述了tolC作为大肠杆菌DNA易于修饰的选择性/反选择性标记的新用途。表达TolC（一种外膜蛋白）对有毒小分子具有相对抗性，而缺乏TolC则使细胞对colicin E1具有耐受性。这些特征，加上λredgam重组系统，允许选择大肠杆菌染色体或质粒DNA上的任何地方的tolC插入/缺失。该方法避免了其他已发表的重组方法所需的最小生长介质、专门的洗涤方案和冗长的孵育时间。作为TolC选择系统的严格测试，7个23S rRNA基因中有6个连续无缝地从大肠杆菌染色体上去除，而不影响复杂rrn操纵子内邻近基因的表达。由此产生的无质粒菌株在染色体上的自然位置保留了一个23S rRNA基因（rrlC），是该类型的第一个突变体。这些新的rRNA突变体将有助于研究rRNA基因调控和核糖体功能。tolC选择具有效率高、背景低、富集介质方便等优点，是一种应用广泛的DNA重组修饰方法。

This work describes the novel use of tolC as a selectable/counter-selectable marker for the facile modification of DNA in Escherichia coli. Expression of TolC (an outer membrane protein) confers relative resistance to toxic small molecules, while its absence renders the cell tolerant to colicin E1. These features, coupled with the λredgam recombination system, allow for selection of tolC insertions/deletions anywhere on the E. coli chromosome or on plasmid DNA. This methodology obviates the need for minimal growth media, specialized wash protocols and the lengthy incubation times required by other published recombineering methods. As a rigorous test of the TolC selection system, six out of seven 23S rRNA genes were consecutively and seamlessly removed from the E. coli chromosome without affecting expression of neighboring genes within the complex rrn operons. The resulting plasmid-free strain retains one 23S rRNA gene (rrlC) in its natural location on the chromosome and is the first mutant of its kind. These new rRNA mutants will be useful in the study of rRNA gene regulation and ribosome function. Given its high efficiency, low background and facility in rich media, tolC selection is a broadly applicable method for the modification of DNA by recombineering.