鳗弧菌Vibro anguillarum MVM425sh高效电转化条件的优化

目的建立高效的鳗弧菌电转化系统。方法分别从感受态细胞制备的洗涤缓冲液与浓度、复苏培养基、二级细胞收获时间、电转化的电压与脉冲时间等方面,对鳗弧菌的电转化条件进行优化。结果获得的鳗弧菌最佳转化条件为二级培养3h左右收获细胞,用272mmol/L蔗糖洗涤缓冲液制备电转化细胞,在2.0kV3ms的脉冲条件下,pUC18的转化率达106个转化子/μgDNA。结论采用优化的电转化方法,可获得较高的转化率,为鳗弧菌中的常规遗传操作奠定了良好的基础。

Optimization of Technical Condition for Transformation of Vibro anguillarum MVM425sh Strain by Electroporation

Objective To optimize the technical condition for transformation of Vibro anguillarum MVM425sh strain by electroporation.Methods Explore the influence of technical conditions such as washing solution of competent cells,medium for cell resuscitation,recovery time of secondary cells,voltage for transformation and pulse time on transformation efficay of Vibro anguillarum MVM425sh strain.Results The condition for transformation was optimized as follows:recover cells 3 h after secondary culture,prepare electrotransformed cells using 272 mmol/L sucrose as washing solution and transform competent cells at 2.0 kV for 3 ms.The transformation efficacy under this condition reached 106 transformants/μg DNA.Conclusion High transformation efficacy was obtained under the optimized condition.The study laid a foundation of routine genetic manipulation of Vibro anguillarum.