枯草芽孢杆菌SCK6超级感受态的制备和转化条件优化.PDF

692 生 物 工 程 学 报 李信志 等/枯草芽孢杆菌SCK6 超级感受态的制备和转化条件优化 Chinese Journal of Biotechnology /cjbcn April 25, 2017, 33(4): 692−698 DOI: 10.13345/j.cjb.160362 ©2017 Chin J Biotech, All rights reserved ·生物育种与工艺优化· SCK6 李信志，卢争辉，周玉玲，李世宇，张桂敏 430062 , , , . SCK6 . , 2017, 33(4): 692–698. Li XZ, Lu ZH, Zhou YL, et al. Preparation and transformation optimization for supercompetent B. subtilis SCK6 cells. Chin J Biotech, 2017, 33(4): 692–698. 摘 要: 枯草芽孢杆菌是一种革兰氏阳性好氧细菌，因其安全性和高分泌特性，已被广泛用作异源蛋白的表 达宿主。然而，相比大肠杆菌，枯草芽孢杆菌的转化效率低，限制了其作为宿主的异源蛋白的定向进化。本文 通过改变培养基、诱导剂浓度、质粒类型等参数优化感受态制备的条件，利用常规质粒进行转化，结果表明， 用营养丰富的YN 培养基替代常规LB 培养基制备感受态，可以使转化效率提高4 倍左右；加入 1.5%的木糖诱 导2 h ，感受态的转化效率又提高2 倍左右；用大肠杆菌Escherichia coli GM272 来源的质粒又可进一步提高转 化效率3 倍左右。综合最优条件制备 SCK6 的感受态，转化整合型质粒pDG1730 ，效率可以达到106 CFU/µg ， 相对未优化的条件提高了2 个数量级，为基于枯草芽孢杆菌的酶的定向进化和代谢工程奠定了基础。 : 枯草芽孢杆菌，ComK 基因，木糖，感受态，pDG1730 Preparation and transformation optimization for supercompetent B. subtilis SCK6 cells Xinzhi Li, Zhenghui Lu, Yuling Zhou, Shiyu Li, and Guimin Zhang Bioresources Green Transformation Collaborative Innovation Center of Hubei Province, College of Life Sciences, Hubei University, Wuhan 430062, Hubei, China Abstract: Bacillus subtilis is Gram-positive aerobic bacterium and widely used as a heterologous protein expression host because of its safety and high protein secretion property. However, comparing to Escherichia coli , the low transformation efficiency limits the application of B. subtilis as a host cell for directed evolution of heterologous enzymes. Therefore