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 polymyxa JSa-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200Ω,电容25μF的电击条件下,加入1.0μg/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0.8μg/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。
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.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2014年第11期89-94,共6页
Food Science
基金
国家自然科学基金面上项目(31271828)
"十二五"国家科技支撑计划项目(2011BAD23B05)
关键词
多黏类芽孢杆菌JSa-9
电转化
转化效率
Paenibacillus polymyxa JSa-9
electroporation
transformation efficiency
