Preparation and transformation optimization for supercompetent B. subtilis SCK6 cells.

Xinzhi LI; Zhenghui LU; Yuling Zhou; Shiyu LI; Guimin Zhang

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Preparation and transformation optimization for supercompetent B. subtilis SCK6 cells.

Author: Xinzhi LI ¹ ; Zhenghui LU ¹ ; Yuling ZHOU ¹ ; Shiyu LI ¹ ; Guimin ZHANG ¹
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1. Bioresources Green Transformation Collaborative Innovation Center of Hubei Province, College of Life Sciences, Hubei University, Wuhan 430062, Hubei, China.
Publication Type:Journal Article
Keywords: Bacillus subtilis; ComK gene; competent cells; pDG1730; xylose
From: Chinese Journal of Biotechnology 2017;33(4):692-698
CountryChina
Language:Chinese
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, we optimized the competent cell preparation conditions for conventional plasmid, including the alteration of the medium, the concentration of inducer, the plasmid type, and other parameters. Compared with the original LB medium, YN medium improved the transformation efficiency by about 4 folds. The transformation efficiency enhanced by about 2 folds under induction with 1.5% xylose for 2 h. In addition, with plasmids prepared from E. coli GM272 strain the transformation efficiency increased by about 3 folds. Combining all these findings, the transformation efficiency of pDG1730 plasmid under the optimized conditions could reach 10⁶ CFU/μg, which was 2 orders of magnitude higher than that the original. Our findings provide references for directed evolution of enzymes and metabolic engineering in Bacillus subtilis.