植物乳杆菌G63电转化方法的优化

Optimization of Electroporation Conditions for Lactobacillus plantarum G63

为获得植物乳杆菌G63高效的电转化方法,本研究从细胞生长状态、细胞弱化剂质量浓度、洗涤液、质粒添加量和电击参数等方面对菌株G63的电转化效率进行优化。结果表明:取菌株G63对数生长中期的细胞制备感受态,以1 g/100 m L甘氨酸作为细胞弱化剂,分别用1 mmol/L Mg Cl2和30 g/100 m L聚乙二醇1000洗涤细胞,并用30 g/100 m L聚乙二醇1000作为电击液,加入20μg穿梭质粒,在1.5 k V和400Ω条件下进行电击,可以获得最高的电转化效率,转化效率达到1.18×103 CFU/μg DNA,满足后续遗传学实验要求。

The objective of this work was to explore an efficient method for Lactobacillus plantarum G63 electrotransformation. We evaluated the effects of the growth phase, weakening cells, washing buffer, plasmid DNA concentration and electrical parameters on electroporation efficiency. The results showed that after culture in MRS medium containing 1 g/100 m L glycine to mid-logarithmic phase, the cells were harvested and washed with 1 mmol/L Mg Cl2 and 30 g/100 m L PEG1000. The optimal electroporation conditions could be achieved by using 30 g/100 m L PEG1000 as the electroporation buffer with 20 μg of plasmid DNA added in 80 μL of the cell suspension, and pulsing under specifi c conditions（fi eld strength, 1.5 k V; resistance, 400 Ω）. The transformation effi ciency was 1.18 × 103 CFU/μg DNA under the optimal conditions, which could meet the requirements for subsequent genetic experiments.