大肠杆菌在低Ca~(2 )条件下对外源DNA的摄取

含一定浓度 Ca2 的 L B培养基 ,接种大肠杆菌 HB10 1,37℃振荡培养至 OD6 0 0 为 0 .5左右 ,培养前或培养后加入 p BR32 2质粒 ,4℃放置一段时间后经氨苄青霉素筛选和质粒检测发现 ,大肠杆菌不经过高 Ca2 下的冰浴处理和热激活等过程就能够直接摄取外源 DNA;通过对转化子质粒拷贝数和氨苄青霉素抗性测定发现 ,这种转化方式进入菌体内的质粒 DNA同样能够进行有效的复制和表达 ,与传统的人工转化结果无本质区别 ;在一定范围内 ,大肠杆菌的转化频率与环境中的 Ca2 浓度成正相关 ,并且这种准自然条件下的转化需要一定的时间 .实验结果对揭示大肠杆菌可能具有自然遗传转化的能力以及正确评估基因工程微生物的安全性具有重要意义

Escherichia coli Absorbing External-DNA Under Condition of Lower Ca~( 2 ) Concentration

Escherichia coli was inoculated in Luria Bertani (LB) medium containing lower CaCl2 concentration at 37 ℃ to approximate OD600 0.5.Plasmid pBR322 was added befor or after incubation.The transformants in the culture were screened from LB plates containing ampicillin(1×10－4 g /mL) after deposited at 4 ℃ for a certain time. The result indicated that E.coli could take in external-DNA under the conditions of resemble to the nature and the DNA absorbed can replicate and express regularly. Moreover, the transformation frequency was related to Ca2+ concentration and a proper time was required for accomplishing natural genetic transformation. The result is significant in understanding natural genetic transformation of E.coli and assessing the risk of the application of genetically engineed microorganism. (GEMs)