Laboratory of Protein Engineering and Biopharmaceutical Sciences, Key Laboratory of Industrial Fermentation and Cooperative Innovation Center of Industrial Fermentation, Hubei University of Technology, Wuhan, Hubei, China.
Methods Mol Biol. 2023;2704:85-96. doi: 10.1007/978-1-0716-3385-4_5.
Targeted mutagenesis by homologous recombination (TMHR) is an efficient allelic exchange mutagenesis for bacterial genome engineering in synthetic biology. Unlike other allelic exchange methods, TMHR does not require a heterologous recombinase to insert or excise a selectable marker from the genome. In contrast, positive and negative selection is achieved solely by suicide vector-encoded functional and host cell proteins. Here we describe a concise protocol to knock out and knock in a 3-ketosteroid-1,2-dehydrogenase gene (kstd) in Mycobacterium neoaurum HGMS2 using TMHR approach. The homology arms flanking the kstd gene are amplified by PCR in vitro and then subcloned into a common homologous recombination vector. The vector is then electroporated into the HGMS2 competent cells. The replacement of the kstd gene by homologous recombination produces antibiotic-resistant single-crossover recombination via the first allelic exchange. Double-crossover markerless mutants are directly separated using sucrose-mediated counterselection. These two steps can generate seamless mutations down to a single DNA base pair. The whole process takes less than 2 weeks.
同源重组靶向诱变（TMHR）是合成生物学中用于细菌基因组工程的一种高效等位基因交换诱变方法。与其他等位基因交换方法不同，TMHR 不需要异源重组酶将选择性标记从基因组中插入或切除。相反，正选择和负选择仅通过自杀载体编码的功能和宿主细胞蛋白来实现。在这里，我们描述了一种使用 TMHR 方法敲除和敲入分枝杆菌新亚种 HGMS2 中 3-酮类固醇-1,2-脱氢酶基因（kstd）的简洁方案。侧翼 kstd 基因的同源臂通过体外 PCR 扩增，然后亚克隆到一个常见的同源重组载体中。然后将该载体电穿孔到 HGMS2 感受态细胞中。同源重组替换 kstd 基因会通过第一次等位基因交换产生抗生素抗性单交叉重组。使用蔗糖介导的反选择直接分离无标记的双交叉突变体。这两个步骤可以产生无缝突变，甚至可以达到单个 DNA 碱基对。整个过程不到 2 周。
