乳酸乳球菌高效电转化方法的建立

Optimization of High-Efficiency Electrotransformation Conditions for Lactococcus lactis

关键词:  乳酸乳球菌  电转化  转化效率  感受态细胞  诱导型表达载体

Key words:  Lactococcus lactis  electroporation  electroporation efficiency  competent cells  inducible expression plasmid

摘要:

以乳酸菌诱导型表达质粒pNZ8149为载体,Lactococcus lactis NZ3900为受体,采用电转化方法研究电场强度、电击杯规格、受体菌株的生长状态、感受态细胞浓度及分装体积、质粒浓度、电击脉冲时间以及电击杯使用次数等因素对电转化效率的影响。结果表明：在固定电阻200Ω、电容25 μF条件下,收获对数生长前期的乳球菌制作感受态细胞,并以80 μL体积分装,采用2mm规格的电击杯,加入浓度为1.2 μg /μL的质粒,在电场强度和脉冲时间分别为10 kV/cm和5 ms的条件下完成电击转化。转化后的菌液恢复培养2 h后涂板计数,转化效率最高可以达到2.6?104 CFU/μg DNA。本研究结果为进一步构建乳酸菌高效表达系统和食品级基因工程乳酸菌株奠定了方法基础。

Abstract:

Using the inducible expression plasmid pNZ8149 of the lactic acid bacteria as a vector, and Lactococcus lactis NZ3900 as a host, the different factors on the impact of electroporation efficiency were studied by the method of electroporation. Factors include electric field strength, cuvette model, cell growth phase, competent cells concentration, volume of competent cells, plasmid concentration, pulse constant and usage counter of cuvette. Results showed that when several parameters in improved protocol were resistance 200Ω, capacitance 25 μF, the optimum factors were the cell harvested at the early exponential growth phase, 80μL competent cells, 2 mm cuvette, 1.2 μg /μL plasmid DNA, electrotransformation at the 10 kV/cm and pulse constant for 5 ms. After incubation in GM17MC broth for another 2 h, the transformation efficiency went up to 2.6?104 CFU/μg DNA with those optimum factors above. The present research provided fundamental method for further study of establish high-efficiency expression system of lactic acid bacteria and the development of genetic engineering technology.