摘要
以典型的乳酸菌组成型表达质粒pMG36e为载体,德式乳杆菌保加利亚亚种为受体,采用电转化方法研究受体菌株的生长状态与细胞壁处理方式、质粒浓度、电转化参数、复苏培养基组成与复苏培养时间、受体菌株的限制修饰作用对电转化效率的影响,探讨电转化的最适条件,以提高电转化效率。结果表明:选择含2.5%甘氨酸的SMRS培养基培养受体细胞至对数中期,收获制成感受态细胞,在电场强度12kV/cm、电阻200Ω、电容25μF条件下电击转化,以含20mmol/LMgCl2、CaCl2的SMRS培养基复苏培养2h,涂板筛选得到较高的电转化效率。特别是采用受体菌株修饰的质粒进行电转化,转化效率提高30倍以上,达到6.3×104cfu/μgDNA。本研究结果为进一步构建乳酸菌组成型高效表达系统和食品级基因工程乳酸菌株提供试验依据。
Using the constitutive expression plasmid pMG36e of the lactic acid bacteria as a vector,and the Lactobacilli delbrueckii subsp.bulgaricus as a host,the different factors on the impact of electroporation efficiency was studied by the method of electroporation.Factors include wall treatment,plasmid concentration,electrotransformation parameters,composition of the recovery medium,recovery culture time and the restriction modification of host.Results showed that the optimal values for concentration of glycine and of sucrose and the pulse strength、risistance are 2.5%,0.5 mol/L and 12 kV/cm,200 Ω,respectively.Best electrotransformation efficiency was attained after incubation in 20 mmol/L MgCl2、 CaCl2 containing SMRS broth for another 2 h,combined with the antibiotics in the selective plates.When the receptor-modified strains of plasmid was used to electrotransformation,the transformation efficiency has increased by 30 times,reached 6.3×104 cfu/μg DNA.The research of this paper provided foundation for further study of establish high-efficiency expression system of lactic acid bacteria and the development of genetic engineering technology.
出处
《中国食品学报》
EI
CAS
CSCD
北大核心
2011年第2期8-16,共9页
Journal of Chinese Institute Of Food Science and Technology
基金
国家高技术研究发展计划(863计划)项目(No.2006AA10Z317)
关键词
德式乳杆菌保加利亚亚种
组成型表达载体
质粒pMG36e
电转化
电转化效率
Lactobacillus delbrueckii subsp.bulgaricus
constitutive expression vector
plasmid pMG36e
electrotransformation
electrotransformation efficiency
