Optimization of chemico-physical transformation methods for various bacterial species using diverse chemical compounds and nanomaterials

Abstract

Bacterial transformation is a fundamental technology to deliver engineered plasmids into bacterial cells, which is essential in industrial protein production, chemical production, etc. Previously, we developed a simple chemico-physical transformation method that can be applied to various bacterial species. Here, to accelerate the advance of bacteria biotechnology we optimize our method by combinatorially evaluating chemical compounds (rubidium chloride, lithium acetate, cesium chloride, dimethyl sulfoxide, and magnesium chloride) for increasing membrane permeability and nanomaterials (sepiolite, gold(III) chloride, multiwalled carbon nanotube, and chitosan) for piercing the membranes. The best transformation efficiencies were achieved as follows; 2.84 × 104 CFU/μg DNA in Lactococcus lactis subsp. lactics (0.1 M CsCl and gold(III) chloride), 3.60 × 104 CFU/μg DNA in Enterococcus faecalis (1 M Li-acetate and MWCNT), 2.41 × 104 CFU/μg DNA in Bacillus sp. (0.01 M RbCl and sepiolite), 3.49 × 104 CFU/μg DNA (0.1 M RbCl and gold(III) chloride) in Ralstonia eutropha (also known as Cupriavidus necator) and 8.78 × 104 CFU/μg DNA (1 M RbCl and chitosan) in Methylomonas sp. DH-1. The efficiencies are up to 100-fold higher than those without optimization. Accordingly, our fast and simple chemico-physical transformation with chemical–nanomaterial optimization allows for the efficient DNA entry into various bacterial cells with high efficiency.