苏云金芽胞杆菌高效电转化方法的建立

Elaboration of an Electroporation Protocol for Bacillus Thuringiensis

电转化已经成为苏云金芽胞杆菌工程菌构建过程中非常重要的一种外源基因导入方法.高效率的电转化方法将加速这类研究进展.通过对培养基、培养时间、感受态细胞制备、电转化缓冲液、电压参数的优化,建立一种高效的苏云金杆菌电转化方法.苏云金杆菌生长在含有0.5 mol/L山梨醇的BHI培养基中,30℃振荡培养至OD600值达到0.6～0.8,细菌细胞经1 g/L蛋白酶K在37℃处理20 min,然后用电转化缓冲液洗涤2遍,设定电压为1 800 V,电击1次.利用该方法,pHT315质粒在苏云金杆菌中的转化效率大幅提高.

Electroporation has already become an important approach to introduce heterologous genes into Bacillus thuringiensis. An highperformance electroporation protocol will undoubtedly accelerate its research progress. Several factors associated with electroporation efficiency were optimized, including medium, culturingtime, preparation of competent cells, electroporation buffer, voltage. For preparation of competent cells, Bacillus thuringiensis strain was grown in BHI medium supplemented with 0.5 mol/L sorbitol and shaked at 30 ℃ until the OD600  value reached 0.6-0.8. Then cell was harvested and treated by 1 g/L proteinase K for 20 min and washed twice in electroporation solution. After mixed with plasmid, the competent cells and plasmid mixture were pulsed once at 1 800 V. By using this protocol, the greatest efficiency was achieved with plasmid pHT315.