Study on the electro-transformation conditions of improving transformation efficiency for Bacillus subtilis

Abstract

Aims: To optimize the transformation conditions and improve the transformation efficiency of Bacillus subtilis WB800 and DB104. Methods and Results: Trehalose, which could decrease the damage of electric shock to the cells, was added to the electroporation medium containing sorbitol and mannitol. The factors affecting the transformation efficiency, such as the growth phase of bacteria, cell concentration, electric field strength and plasmid variety, were examined and improved. The new method increased the transformation efficiency of B. subtilis by nearly 100‐fold compared with the conventional one. Conclusions: With the optimized method, the transformation efficiency came up to 3·64 × 105 transformants μg−1 DNA for WB800, and 2·10 × 105 transformants μg−1 DNA for DB104. Significance and Impact of the Study: This improvement in transformation efficiency will be largely attributed to the research of expression of exogenous genes in B. subtilis, gene library construction for directed evolution and transformation of wild‐type B. subtilis strains.

Genetic engineering of Geobacillus spp.

“…It is quite complicated to determine and optimize the conditions for effective electrotransformation, as each species and strain can be influenced by many different factors. However, once this method is optimized, it results in high transformation efficiencies (up to 10 9 -10 10 CFU of transformed E. coli and 10 5 -10 6 CFU of transformed B. subtilis per μg DNA) and is highly reproducible (Aune and Aachmann, 2010;Lu et al, 2012;Xue et al, 1999).…”

Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain

“…By adding sorbitol and mannitol into the electroporation medium, Xue et al [27] increased the transformation efficiency of B. subtilis by an approximately 5000-fold. Recently, trehalose [15] and GLYB [25] were also tested for their protection on the cells under stress conditions, and generated improved transformation efficiencies of B. subtilis by 100-fold and 700-fold, respectively. In this study, different concentrations of GLYB and their impacts on survival rate were studied.…”

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.…”