High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus coagulans P4-102B

Abstract:

[Objective] One of the genetic manipulation restriction factors in Bacillus coagulansis the absence of an efficient and reproducible transformation method. This study optimized the concentrations of the osmoticums, sorbitol and mannitol, in the growth, electroporationand recovery media and other conditions to improved the transformation efficiency of B. coagulans P4-102B. [Methods] To optimize the conditions for electroproation of B. coagulans P4-102B, a shuttle vector for E. coli and B. coagulans pNW33N was used. Factors including growth phase, competent cell density, electroporation buffer, re-growth medium were investigated. [Results] Results showed that the electro-transformation efficiency under high osmolarity was higher than that under the low osmolarity and the stability of electro-transformation efficiency was enhance. The highest transformation efficiency of 2.7×102 cfu/μg DNA in strain P4-102B was obtained under the optimized conditions: growth OD600 0.8, electro-transformation buffer SMG [(0.5 mol/L sorbitol, 0.5 mol/L mannitol and glycerol 10% (w/v)], 1 mm cuvette, electro-transformation field strength 14 kV/cm and pulse constant for 5 ms, re-growth medium RGM (LB with 0.5 mol/L sorbitol and 0.38 mol/L mannitol). [Conclusion] High osmolarity improved the stabitity and reproducibility of electro-transformation and obtained a high electro-transformation efficiency.