Red重组系统介导下大肠杆菌改造体系的优化

对Red重组系统介导下大肠杆菌进行遗传改造的转化条件、诱导条件、复苏条件三方面实验参数进行优化研究。结果表明:电转化加入的DNA为20ng,Red重组系统感受态细胞培养浓度为OD600=0.60,所需的电击感受态细胞数为3×108;对大肠杆菌基因做双敲除时,突变第2个基因的阿拉伯糖诱导最适时间比突变首个基因延长,诱导浓度为40μm,复苏时间变化对体系影响不显著。

Optimizing the Conditions for Red-mediated Recombinogenic Engineering of E.coli

Red recombination system,one of important tools for gene replacement,has been applied in Escherichia coli metabolism engineering.Optimization of experimental parameters of this system is necessary for improving the efficiency of mutation.The goal of our research is to optimize the manipulation parameters by comparing the effect of transformation conditions,induction condition and cell recovery conditions on the gene knock-out results of E.coli.The optimized transformation condition was that 20ng DNA is added into 3×108 cells per transformation,culture used for preparation of competent cell is at concentration of 0.60 OD600 value.The induction time required for knocking-out the second gene was relative longer comparing with that for first gene,the ideal concentration for arabinose induction was 40μm.No obvious influence was found when the recovery time was adjusted.