High efficiency and steady transformation of Leuconostoc by electroporation

Because of the poor reproducibility and the slightly low efficiency of transformation of Leuconostoc, we re-optimized the method for electrotransformation. Using plasmid pCW4 as a vector and Leuconostoc as a host, we improved pH of PBS buffer, time of pre-treatment with lithium acetate(LiAc) and dithiothreitol(DTT), concentrations of lysozyme and sucrose in the LiAc-DTT to increase the transformation efficiency. To obtain a steady efficiency, precise inoculation amount and concentration of ampicillin were applied. An overnight culture of Leuconostoc was diluted with MRS to an optical density at 600nm wavelength(OD600) of 0.048 and incubated until an OD600 of 0.5 was reached. The cells were pre-treated with LiAc-DTT(0.5 mol·L-1sucrose) supplemented with 100 U·mL-1lysozyme for 20min. After pre-treatment, PBS of pH 6.9 was used to wash and resuspend the cells. Using this protocol, we achieved a higher transformation efficiency of 2.47×105μg-1 DNA.