Key Laboratory of Molecular Animal Nutrition of Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, People's Republic of China.
J Microbiol Methods. 2013 Nov;95(2):207-14. doi: 10.1016/j.mimet.2013.07.020. Epub 2013 Jul 31.
There are some drawbacks for MazF-cassette constructed in previous reports for marker-free genetic manipulation in Bacillus subtilis, including cloning-dependent methodology and non-strictly controlled expression system. In our study, the modifications on mazF-cassette are carried out, such as using mini Zeocin resistance gene as positive-selectable marker and strictly controlled xyl promoter from the B. subtilis to replace non-strictly controlled IPTG-inducible Pspac or xyl promoter from Bacillus megaterium. Then the mini-mazF-cassette was successfully applied to knock-out the amyE gene, to delete a 90-kb gene cluster, and to knock-in a green fluorescent protein expression cassette employing a cloning-independent methodology, without introducing undesirable redundant sequences at the modified locus in the B. subtilis 1A751. Besides, the mini-mazF-cassette could be used repeatedly to delete multiple genes or gene clusters with only a 2- to 2.5-kb PCR-fused fragment, which largely reduced the frequency of nucleic acid mutations generated by PCR compared to previous reports. We further demonstrated that the frequency of spontaneous mazF-resistant mutants was lower, and the frequency of generating desired clones was nearly 100%. The entire procedure for marker-free genetic manipulation using the mini-mazF-cassette can be finished in about 3days. This modified cassette has remarkable improvement compared to existing approaches and is applicable for available manipulating Bacillus species chromosomes.
在之前关于枯草芽孢杆菌无标记遗传操作的 MazF 盒构建的报告中，存在一些缺点，包括依赖克隆的方法和非严格控制的表达系统。在我们的研究中，对 mazF 盒进行了修改，例如使用 mini Zeocin 抗性基因作为阳性选择标记，并用严格控制的来自枯草芽孢杆菌的 xyl 启动子取代非严格控制的 IPTG 诱导型 Pspac 或来自巨大芽孢杆菌的 xyl 启动子。然后，成功地将 mini-mazF 盒应用于敲除 amyE 基因、删除 90-kb 基因簇以及敲入绿色荧光蛋白表达盒，采用非克隆依赖的方法，而不在枯草芽孢杆菌 1A751 的修饰位点引入不需要的冗余序列。此外，mini-mazF 盒可重复用于删除多个基因或基因簇，只需 2-2.5kb 的 PCR 融合片段，与之前的报告相比，大大降低了 PCR 产生核酸突变的频率。我们进一步证明，自发 mazF 抗性突变体的频率较低，并且产生所需克隆的频率接近 100%。使用 mini-mazF 盒进行无标记遗传操作的整个过程可以在大约 3 天内完成。与现有方法相比，该改良的盒具有显著的改进，适用于现有的枯草芽孢杆菌属染色体的操作。
