食品科学

• 生物工程 • 上一篇    下一篇

多黏类芽孢杆菌JSa-9电转化方法的优化

高 玲,邓 阳,陆兆新,吕凤霞,别小妹   

  1. 南京农业大学食品科技学院,江苏 南京 210095
  • 出版日期:2014-06-15 发布日期:2014-07-03

Optimization of Electroporation Conditions for Paenibacillus polymyxa JSa-9

GAO Ling, DENG Yang, LU Zhao-xin, LÜ Feng-xia, BIE Xiao-mei   

  1. College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
  • Online:2014-06-15 Published:2014-07-03

摘要:

为建立多黏类芽孢杆菌JSa-9菌株的高效电击转化体系,本实验研究菌体培养时间、电场强度、电击缓冲液、质粒用量以及电击后复苏培养时间等因素对JSa-9菌株电转化效率的影响。结果表明:Paenibacillus polymyxaJSa-9培养至OD595 nm为0.3时,电转化效率最高,为0.12×103 CFU/μg DNA;用0.5 mol/L甘露醇、0.5 mol/L山梨醇以及10%甘油的电击缓冲液洗涤细胞,在电场强度17.5 kV/cm、电阻200 Ω,电容25 μF的电击条件下,加入1.0 μg/100 mL质粒DNA,复苏培养时间18 h,电转化效率达到约为0.36×103 CFU/μg DNA;制备P. polymyxa JSa-9原生质体,在电场强度5.0 kV/cm的电击条件下,加入0.8 μg/100 mL质粒DNA,电转化效率较感受态电转提高到约1.0×103 CFU/μg DNA。

关键词: 多黏类芽孢杆菌JSa-9, 电转化, 转化效率

Abstract:

To establish an electrotransformation method for Paenibacillus polymyxa strain JSa-9, the effects of different
factors on the transformation efficiency of strain JSa-9 by electroporation were explored by evaluating cell growth phase,
electric field intensity, electroporation buffer, plasmid DNA contentation and recovery time. The results showed that a
transformation efficiency of 0.12 × 103 CFU transformants per μg DNA was achieved when P. polymyxa JSa-9 cell culture
reached an OD595 nm of 0.3. Under the conditions of 17.5 kV/cm, 200 Ω and 25 μF for electric field strength, electric resistance
and capacitance, respectively, the electroporation efficiency was roughly 0.5 × 103 transformants per μg DNA when adding
1.0 μg/100 mL of plasmid DNA to an electroporation buffer containing 0.5 mol/L sorbitol, 0.5 mol/L mannitol, and 10%
glycerol and recovering for 17 h. Moreover, the transformation efficiency of P. polymyxa JSa-9 protoplasts was increased
to approximately 1.0 × 103 CFU transformants per μg DNA under electric field strength of 5.0 kV/cm upon addition of
0.8 μg/100 mL plasmid DNA.

Key words: Paenibacillus polymyxa JSa-9, electroporation, transformation efficiency