Combined chemical and physical transformation method with RbCl and sepiolite for the transformation of various bacterial species

Abstract

DNA transformation that delivers plasmid DNAs into bacterial cells is fundamental in genetic manipulation to engineer and study bacteria. Developed transformation methods to date are optimized to specific bacterial species for high efficiency. Thus, there is always a demand for simple and species-independent transformation methods. We herein describe the development of a chemico-physical transformation method that combines a rubidium chloride (RbCl)-based chemical method and sepiolite-based physical method, and report its use for the simple and efficient delivery of DNA into various bacterial species. Using this method, the best transformation efficiency for Escherichia coli DH5α was 4.3×10⁶CFU/μg of pUC19 plasmid, which is higher than or comparable to the reported transformation efficiencies to date. This method also allowed the introduction of plasmid DNAs into Bacillus subtilis (5.7×10³CFU/μg of pSEVA3b67Rb), Bacillus megaterium (2.5×10³CFU/μg of pSPAsp-hp), Lactococcus lactis subsp. lactis (1.0×10²CFU/μg of pTRKH3-ermGFP), and Lactococcus lactis subsp. cremoris (2.2×10²CFU/μg of pMSP3535VA). Remarkably, even when the conventional chemical and physical methods failed to generate transformed cells in Bacillus sp. and Enterococcus faecalis, E. malodoratus and E. mundtii, our combined method showed a significant transformation efficiency (2.4×10⁴, 4.5×10², 2×10¹, and 0.5×10¹CFU/μg of plasmid DNA). Based on our results, we anticipate that our simple and efficient transformation method should prove usefulness for introducing DNA into various bacterial species without complicated optimization of parameters affecting DNA entry into the cell.

Keywords: Bacteria; Chemical transformation; Physical transformation; Rubidium chloride; Sepiolite; Transformation.