短小芽孢杆菌遗传操作系统的建立及应用
The construction and application of a genetic manipulation system for Bacillus pumilus

摘 要：短小芽孢杆菌SCU11是由本实验室分离得到的野生型菌株BA06经复合变得到，其产生的碱性蛋白酶具有很好的脱毛效果，在生物制革领域有很大的应用前景。但短小芽孢杆菌高效的遗传转化系统还没有建立起来，阻碍了对其进行进一步的遗传改造和基础理论方面的研究。本研究采用高渗透压电转化法，实现了对短小芽孢杆菌的电转化，转化效率可达3.5×103 CFU/μg DNA。进一步构建了温度敏感性E. coli -Bacillus穿梭载体pUCETs，利用该载体成功敲除了短小芽孢杆菌的peptidase C40基因。本研究所建立的电转化系统及基因导入/敲除体系，能够高效的对短小芽孢杆菌的基因组进行编辑。
Abstract: A novel strain of Bacillus pumilus named as SCU11 was screened and mutagenized from the wild strain BA06 in our previous study. The fermentation supernatant of this strain showed efficient dehairing capability, indicating its extracellular proteases has a good application future in leather industry. However, the efficient genetic manipulation system of B. pumilus SCU11 has not yet been established, which greatly hampered the genetic engineering and the basic theory study of this strain. In this study, high osmolarity electroporation method was developed for the efficient transformation of B. pumilus, and the transformation efficiency was obtained up to 3.5×103CFU/μg DNA. Moreover, a temperature sensitive E. coli-Bacillus shuttle vector pUCETs was constructed by making use of a temperature sensitive replication origin, and the peptidase C40 gene of SCU11 was knocked-out based on the pUCETs. The electroporation method and gene manipulation system established in this study could make efficient targeted gene knockout in Bacliius pumilus