Elaboration of an electroporation protocol for large plasmids and wild-type strains of Bacillus thuringiensis
Elaboration of an electroporation protocol for large plasmids and wild-type strains of Bacillus thuringiensis
复制标题
苏云金芽孢杆菌大质粒和野生型菌株电穿孔方案的制定
DOI：
10.1111/j.1365-2672.2009.04151.x
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发表时间：
2009-06-01
影响因子：
4
通讯作者：
Sun, M.
中科院分区：
文献类型：
--
作者：
Peng, D.;Luo, Y.;Sun, M.
目的：研究苏云金芽孢杆菌大质粒和野生型菌株的电穿孔方法。方法与结果：通过考察DNA脱盐、弱壁剂、细胞生长条件、电穿孔溶液、电场等因素对电穿孔效率的影响，优化苏云金芽孢杆菌的电穿孔条件。利用这种改进的方法，pHT304的最高效率达到了2×10(10)cfu/g(-1)，是文献报道的10(4)倍。4个大的质粒(29.1、44.9、58和60kb)被成功地转移到苏云金芽孢杆菌无晶体菌株BMB171中，这些结果在以前的方法中是没有得到的。本研究的意义和影响：本研究建立了苏云金芽孢杆菌大质粒和野生型苏云金芽孢杆菌的有效电穿孔方法。这种方法非常适合于苏云金芽孢杆菌基因簇等大片段的克隆和表达。该方法也可作为其他对电穿孔不敏感的芽孢杆菌的参考方法。
Aims: To elaborate an effective electroporation protocol for large plasmids and wild type strains of Bacillus thuringiensis.Methods and Results: The effect of DNA desalting, wall-weakening agency, cell growth conditions, electroporation solutions, and electric fields on electroporation efficiency was evaluated to optimize electroporation conditions for B. thuringiensis. By using this improved method, the greatest efficiency was reached 2 X 10(10) CFU mu g(-1) with pHT304, which is 10(4) times higher than previously reported. Four large plasmids (29.1, 44.9, 58 and 60 kb) were successfully transferred into the acrystalliferous B. thuringiensis strain BMB171; these results have not been achieved with previous protocols. Three wild type B. thuringiensis strains which could not be transformed previously were also transferred successfully.Conclusions: This improved method is more efficient for small plasmids; it is also appropriate for large plasmids and wild type B. thuringiensis strains which were not transformed by previous procedures.Significance and Impact of the Study: The present study established an effective electroporation protocol for large plasmids and wild type strains of B. thuringiensis. This method is well suited for the cloning and expression of huge DNA fragments such as gene Clusters in B. thuringiensis. It also can be used as a reference method for other Bacillus strains that are refractory to electroporate.