响应面法优化嗜热链球菌AR333电转化条件

Optimization of electroporation method of Streptococcus thermophilus AR333 by response surface methodology

摘要:

【背景】嗜热链球菌AR333是本实验室从发酵乳中筛选出的一株高产活性胞外多糖乳酸菌。【目的】建立嗜热链球菌AR333高效电转化体系。【方法】通过单因素试验和Box-Behnken响应面法优化电转化条件。【结果】嗜热链球菌AR333最优电转化条件为甘氨酸浓度8.3 g/L，OD600为0.8，10%甘油(体积比)和0.5 mol/L蔗糖的电转缓冲液，pIB184质粒80 ng，电场强度14 kV/cm，0.4 mol/L山梨醇、2 mmol/L CaCl2和20 mmol/L MgCl2的LM17复苏培养基，复苏时间5 h。【结论】在最优电转化条件下，嗜热链球菌AR333电转化效率达到3.68×105 CFU/μg-DNA，比优化前提高了14倍，实现了嗜热链球菌AR333的高效遗传转化，为其功能解析和基因工程改造奠定基础。

Abstract:

[Background] Streptococcus thermophilus AR333, isolated from fermented dairy products, could produce a high-yield active exopolysaccharide. [Objective] To establish a high-efficiency electroporation method of S. thermophilus AR333. [Methods] Electroporation conditions were optimized by single factor experiment and Box-Behnken response surface methodology. [Results] The optimum condition was glycine content 8.3 g/L, cell density of 0.8 (OD600), buffer composed of 10% glycerol (V/V) and 0.5 mol/L sucrose, 80 ng pIB184 plasmid, electric field strength 14 kV/cm, the recovery LM17 media composed of 0.4 mol/L sorbitol, 2 mmol/L CaCl2 and 20 mmol/L MgCl2 and recovery time of 5 h. [Conclusion] The transformants reached 3.68×105 CFU/μg-DNA under the optimal condition, which was improved about 14-fold in comparison with that of initial condition. The optimized condition was used to achieve a high electro-transformation efficiency, which lays a foundation for genetic engineering of AR333.