明串珠菌的稳定高效电转化

High efficiency and steady transformation of Leuconostoc by electroporation

明串珠菌电转化的再现性差、效率稍低,因此重新优化了电转化方法。以pCW4为载体,明串珠菌为受体菌,固定接菌量和氨苄浓度,优化了PBS溶液的pH值、LiAc-DTT孵育时间、溶菌酶的量和孵育液中蔗糖浓度。利用MRS(含质量浓度0.48 mg·L-1氨苄)培养明串珠菌(初始OD600为0.048),OD600为0.5左右时收集菌体,用LiAc-DTT溶液(含100 U·mL-1溶菌酶、浓度0.5 mol·L-1蔗糖)孵育20 min,再用pH值为6.9的PBS洗涤和电转化,得到较高的转化率,为2.47×105μg-1DNA。

Because of the poor reproducibility and the slightly low efficiency of transformation of Leuconostoc, we re-optimized the method for electrotransformation. Using plasmJd 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 effi-ciency. To obtain a steady efficiency, precise inoculation amount and concentration of ampicillin were applied. An overnight culture of Leu- conostoc 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^-1 sucrose） supplemented with 100 U·L^-1 lysozyme 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 2.47×10^5μg^-1 DNA.