【摘要】 为获得宿主菌 ,研究了十二烷基硫酸钠 (SDS)对枯草芽孢杆菌中质粒的消除 .将过夜培养的枯草芽孢杆菌2 4/pMX45接种于含SDS( 0— 0 .0 0 8% )的LB培养基中 ,当SDS浓度 (w /v)大于或等于 0 .0 0 6 %时 ,菌体不能生长 .SDS的亚致死浓度为 0 .0 0 5 % ,其致死率达 99% .菌液稀释后涂LB平板 ,再随机挑选单菌落至抗性平板 ,检测由质粒编码的红霉素抗性是否丢失 .氯化铯 溴化乙锭梯度离心及质粒DNA的电泳图像证实了 2 4/pMX45的衍生菌株A7中的质粒已完全被消除 .A7延迟期较短 ,并且细胞浓度高于 2 4/pMX45 .用SDS处理 8h后 ,2 4/pMX45的遗传标记开始丢失 ,消除率持续增高至 2 2h ,随之消除质粒的菌体量保持恒定 .在未经SDS处理的对照实验中 ,相同条件下培养 2 4h及 48h后 ,没有发现质粒自然丢失的现象 ,因此SDS能消除枯草芽孢杆菌中的质粒更多还原

【Abstract】 Curing of Bacillus subtilis plasmid using sodium dodecyl sulfate (SDS)was studied in order to obtain a host strain. An overnight culture of Bacillus subtilis 24/pMX45 was used to inoculate fresh LB containing SDS (0-0.008%). No growth of 24/pMX45 was observed when LB contained an SDS concentration of 0.006% or greater, and the sublethal concentration (w/v) of SDS was 0.005% with a killing rate of 99%. Samples were diluted and plated on LB agar, individual colonies were randomly picked to a selective agar medium by tooth to screen for loss of plasmid-encoded erythomycin resistance. CsCl-EtBr gradient centrifugation and plasmid DNA profile demonstrated that plasmid-cured derivative A7 has completely lost its plasmid. A7 had a shorter lag, and its cell concentration was consistently higher than that of the 24/pMX45. Elimination of the plasmid was first observed after 24/pMX45 had been treated with SDS for 8 h. The percent elimination then continued to increase until about 22 h, after which the fraction of cured cell in the population remained constant. Plasmid cured cell numbers were measured in a separate control culture of 24/pMX45 untreated by SDS. No spontaneous loss of pMX45 was observed after 24/pMX45 were incubated for 24 h and 48 h with shaking at 37 ℃.These results suggested that SDS can be used as curing agent to eliminate the plasmid of Bacillus subtilis.更多还原