低温培养对大肠杆菌电穿孔转化效率的影响

Effect of Low-temperature Culture on the Transformation Efficiency of Escherichia coli by Electroporation

在18℃和37℃振荡培养大肠杆菌DH5α并制备感受态细胞,用Eppendorf公司的Multiporator电转移仪进行电穿孔转化,研究了培养温度对转化效率的影响.实验结果表明,低温(18℃)培养的细菌,回收时的OD600值在0.5～1.0时,转化效率都维持在高水平;而在37℃培养的细菌,转化效率仅在OD600 0.6～0.7时出现一个锐利的峰值,此OD600值前后转化效率显著下降.在不影响细胞生长的情况下,18℃培养细菌的转化效率随电场强度增加而升高;而37℃培养的细菌在电场强度超过20.5 kV/cm时转化效率将大幅度下降.另外,还对影响转化效率的其他因素(培养基、振荡培养时的转速、细胞密度、质粒浓度、电击前后冰浴时间等)进行了优化,建立了一个便利而高效的质粒DNA电转化方法,获得1.02×1010cfu/μg DNA的高转化效率.

E. coli DH5α was cultured at 18℃ and 37℃ respectively to prepare competent cells for electroporation with Eppendorf Multiporator to investigate the effect of low-temperature cultivation on the transformation efficiency. Our data proved high competence of electroporation in a wide range of cell concentrations (OD600 0. 5-1.0) at harvest when DH5α was grown at 18℃ . When cultured at 37℃ , there was a sharp peak of transformation efficiency at OD600 0.6-0.7. Transformation efficiency increased with increased field strength at 18℃ of cultivation and decreased significantly when field strength reached 20. 5 kV/cm at 37℃ of cultivation. And some influencing factors during cell culture and transformation, such as medium, cell density, concentration of plasmid DNA, pre- and post-shock incubation, concentration of ampicillin, etc, were also optimized. In conclusion, we have established a simple and efficient method for plasmid electro-transformation with high yield of transformation of 1.02 ×1010cfu/μg of DNA, which facilitated experiment schedule, and was efficient for several E. coli strains.