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Int. J. Mol. Sci. 2015, 16(4), 7334-7351; doi:10.3390/ijms16047334

Development of an Efficient Electroporation Method for Iturin A-Producing Bacillus subtilis ZK

1
Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, the Chinese Academy of Sciences, No. 9 Section 4, Renmin Nan Road, Chengdu 610041, China
2
University of the Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China
*
Author to whom correspondence should be addressed.
Academic Editor: Ritva Tikkanen
Received: 19 January 2015 / Revised: 9 February 2015 / Accepted: 11 February 2015 / Published: 1 April 2015
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Abstract

In order to efficiently introduce DNA into B. subtilis ZK, which produces iturin A at a high level, we optimized seven electroporation conditions and explored an efficient electroporation method. Using the optimal conditions, the electroporation efficiency was improved to 1.03 × 107 transformants/μg of DNA, an approximately 10,000-fold increase in electroporation efficiency. This efficiency is the highest electroporation efficiency for B. subtilis and enables the construction of a directed evolution library or the knockout of a gene in B. subtilis ZK for molecular genetics studies. In the optimization process, the combined effects of three types of wall-weakening agents were evaluated using a response surface methodology (RSM) design, which led to a two orders of magnitude increase in electroporation efficiency. To the best of our limited knowledge, this study provides the first demonstration of using an RSM design for optimization of the electroporation conditions for B. subtilis. To validate the electroporation efficiency, a case study was performed and a gene (rapC) was inactivated in B. subtilis ZK using a suicide plasmid pMUTIN4. Moreover, we found that the rapC mutants exhibited a marked decrease in iturin A production, suggesting that the rapC gene was closely related to the iturin A production. View Full-Text
Keywords: electroporation; response surface methodology; wall-weakening agent; wild-type B. subtilis; rapC electroporation; response surface methodology; wall-weakening agent; wild-type B. subtilis; rapC
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Zhang, Z.; Ding, Z.-T.; Shu, D.; Luo, D.; Tan, H. Development of an Efficient Electroporation Method for Iturin A-Producing Bacillus subtilis ZK. Int. J. Mol. Sci. 2015, 16, 7334-7351.

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