大肠埃希菌TG_1电穿孔法转化条件优化研究  

Optimization of Electroporation Conditions for Escherichia coli Strain TG_1

作　　者：蒋华波[1] 夏梓元 张琼阁[2] 王超群[2] 郑骄阳[2] 陆斌[1] 

机构地区：[1]第二军医大学药学院,生化药学教研室,上海200433 [2]上海市长征医院内分泌科,上海200003

出　　处：《生物技术进展》2017年第1期72-76,共5页Current Biotechnology

基　　金：国家自然科学基金项目(81472283;81270890)资助

摘　　要：为了确立稳定的高效率电转化方案,提高构建文库的库容,分别对细菌培养温度、生长状态、电场强度、感受态细胞浓度和体积、外源基因的质量、甘油/甘露醇缓冲液体积等条件进行优化,分析了各因素对转化效率的影响。结果显示:细菌培养温度为16℃,细菌生长的OD_(600)值为0.5,密度梯度离心洗涤法,感受态细胞浓度高于1×10^(11)/m L,并设定电场强度14.25 k V/cm时进行电穿孔,转化效率最高,可达到9×10~9CFU/μg DNA。研究获得的电转化优化条件为高库容文库的构建提供了一个重要的途径。To build up a stable protocol of electroporation transformation method with high efficiency and improve storage capacity of genomie library, we investigated the factors affecting the transformation efficiency, including culture temperature, growth state, voltage, the density and volume of competent cells, the amount of the exogenous gene and the volume of glycerol/mannitol buffer solution, which may contribute to construct highly diverse library. Results showed that when cells growing under 16℃ and harvested in mid-exponential phase （OD600 of 0. 5 ）, using glyeerol/mannitol density gradient centrifugation techniques and adjusting the cell density up to 1 × 10^11/mL, the electroporation voltage of 14.25 kV/cm, the highest transformation efficiency could reach to 9 × 10^9 CFU/μg DNA. The results showed that the optimal conditions for electroporation transformation would provide an important access for high-complexity library construction.

关 键 词：大肠埃希菌TG1 电穿孔转化 转化效率 

分 类 号：Q78[生物学—分子生物学]