Guan Zheng-Bing, Wang Kai-Qiang, Shui Yan, Liao Xiang-Ru
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, P. R. China.
The Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, P. R. China.
J Basic Microbiol. 2017 Dec;57(12):1065-1068. doi: 10.1002/jobm.201700370. Epub 2017 Oct 20.
In this study, we established a Cre/loxP mutant recombination system (Cre/lox71-66 system) for markerless gene deletion to facilitate our follow-up rational genetic engineering to the strain Bacillus pumilus W3. This modified method uses two mutant loxP sites, which after recombination creates a double-mutant loxP site that is poorly recognized by Cre recombinase, facilitating multiple gene deletions in a single genetic background. Two selected genes, cotA and sigF, were continuously knocked out and verified at different levels using this method. This method is simple and efficient and can be easily implemented for multiple gene deletions in B. pumilus.
在本研究中，我们建立了一种用于无标记基因缺失的Cre/loxP突变重组系统（Cre/lox71-66系统），以促进我们对短小芽孢杆菌W3菌株进行后续合理的基因工程操作。这种改良方法使用了两个突变的loxP位点，重组后会产生一个Cre重组酶识别能力较差的双突变loxP位点，便于在单一遗传背景下进行多个基因的缺失。利用该方法对两个选定的基因cotA和sigF进行了连续敲除，并在不同水平上进行了验证。该方法简单高效，可轻松应用于短小芽孢杆菌的多个基因缺失操作。
