高渗提高凝结芽孢杆菌P4-102B菌株的电击转化效率

High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus coagulans P4-102B

摘要:

【目的】凝结芽孢杆菌(Bacillus coagulans)是一种非常有工业应用前景的微生物，但遗传转化的困难是限制对其进行代谢工程改造的关键因素。本研究主要考察生长培养基、电击缓冲液、复苏培养基中添加高渗剂如山梨醇、甘露醇等对凝结芽孢杆菌转化效率稳定性的影响，并对凝结芽孢杆菌电击转化条件进行优化。【方法】用穿梭质粒pNW33N电转化凝结芽孢杆菌P4-102B，系统地考察高渗条件下细胞生长阶段、感受态菌体浓度、电击缓冲液组分和复苏培养基组成等因素对转化效率的影响。【结果】同一电场压力下高渗体系转化效率较低渗体系明显提高且稳定性较好，菌体在含有0.5 mol/L山梨醇的LB培养基中生长到OD600为0.8时收集，用SMG [0.5 mol/L山梨醇，0.5 mol/L甘露醇，10% (质量体积比)甘油]电击缓冲液洗涤菌体4次制备感受态，在固定电场强度14 kV/cm、脉冲时间5 ms、1 mm规格的电转杯进行电击转化，电转化后立即加入含有0.5 mol/L山梨醇和0.38 mol/L甘露醇的LB复苏培养基培养，能够获得最佳的转化效率2.7×102 cfu/μg DNA。【结论】使用高渗电击转化法能够提高电击转化的稳定性和重复性，并且可以获得较高的转化效率。

Abstract:

[Objective] One of the genetic manipulation restriction factors in Bacillus coagulansis the absence of an efficient and reproducible transformation method. This study optimized the concentrations of the osmoticums, sorbitol and mannitol, in the growth, electroporationand recovery media and other conditions to improved the transformation efficiency of B. coagulans P4-102B. [Methods] To optimize the conditions for electroproation of B. coagulans P4-102B, a shuttle vector for E. coli and B. coagulans pNW33N was used. Factors including growth phase, competent cell density, electroporation buffer, re-growth medium were investigated. [Results] Results showed that the electro-transformation efficiency under high osmolarity was higher than that under the low osmolarity and the stability of electro-transformation efficiency was enhance. The highest transformation efficiency of 2.7×102 cfu/μg DNA in strain P4-102B was obtained under the optimized conditions: growth OD600 0.8, electro-transformation buffer SMG [(0.5 mol/L sorbitol, 0.5 mol/L mannitol and glycerol 10% (w/v)], 1 mm cuvette, electro-transformation field strength 14 kV/cm and pulse constant for 5 ms, re-growth medium RGM (LB with 0.5 mol/L sorbitol and 0.38 mol/L mannitol). [Conclusion] High osmolarity improved the stabitity and reproducibility of electro-transformation and obtained a high electro-transformation efficiency.