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

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.

Factors and Conditions That Impact Electroporation of Clostridioides difficile Strains

“…In this study, we used D-sorbitol or sucrose as an osmoprotectant. D-Sorbitol was previously used for electroporating B. subtilis (29,47), and in prior work, our lab used D-sorbitol as an osmoprotectant to show that C. difficile and Paraclostridium bifermentans mechanosensing channels are involved in the release of dipicolinic acid from the spore core during germination (48). Though C. difficile R20291 grown in sorbitol-containing medium could be transformed by electroporation, we could not obtain any transformants from the C. difficile CD630 or JSC10 strain.…”

“…Fragility of the cell wall enhances transformation efficiency (45). Glycine is the most common additive used for electroporating B. subtilis (29), Bacillus mycoides (46), and Clostridium pasteurianum (35). We observed that C. difficile requires glycine to generate transformable cells; the absence of glycine did not produce colonies after electroporation.…”

Optimization and Comparison of Different Methods and Factors for Efficient Transformation of Brucella abortus RB51strain

“…in a study of the transformation of E. coli spheroplasts through the chemical CaCl 2 method[23] and in several other studies with different bacteria. [142425262728] However, some researchers reported that cell-wall-weakening either had no effect on the transformation frequency. [29] It seems that type of bacteria, cell-wall-weakening agents for spheroplast formation, and the transformation method used should be affected on the result.…”

Improvement of iturin A production inBacillus subtilisZK0 by overexpression of thecomAandsigAgenes

“…The overexpression plasmids were introduced into B. subtilis ZK0 by electroporation (Zhang et al . ). Before fermentation, the strain was activated on LB solid medium at 37°C for 24 h and inoculated into 25 ml of LB liquid medium in a shaker at 30°C and 150 g for 18 h. Then, 5 ml of seed culture was added to 45 ml of the fermentation medium (16 g of glucose, 0·1 g of yeast extract, 3·7 g of KH 2 PO 4 , 52·1 g of soy peptone and 7·3 g of MgSO 4 ·7H 2 O per litre of water, pH 8·0) with 1 mmol l −1 IPTG.…”