摘要
在现有电转化和聚乙二醇/醋酸锂(PEG/LiAc)诱导的化学转化的基础上,进一步研究和探索了酿酒酵母感受态细胞不同预处理及相应的转化条件对转化效率的影响。结果表明:处于对数生长早期(OD600=0.6)的酵母细胞的转化效果最佳;电转化中分别以120 mmol/L的DTT和300 mmol/L的LiAc预处理感受态酵母菌10 min,可以得到较高的转化效果;联合使用120 mmol/L DTT和300 mmol/L LiAc能大幅提升酵母转化效率,达到7.15×105cfu/μg,PEG/LiAc诱导的化学转化中使用300 mmol/L LiAc能大幅提升酵母转化效率,达到2.75×105cfu/μg,远高于文献报道的水平。同时证明了改进后的转化方法同样能够提高外源DNA同源重组转化效率。
Electroporation and transformation induced by PEG/LiAc（polyethylene glycol/lithium acetate）is widely used for the transformation of Saccharomy cescerevisiae.However,it can not meet the highly efficient transformation requirement（more than 105）of yeast-based pool screening.Based on the existing yeast transformation,the effects of different reagents and/or conditions on the transformation efficiency were further studied.The results show that the optimum period of Saccharomy cescerevisiae for transformation is the early stage of cell growth（OD600=0.6）.In electroporation of Saccharomy cescerevisiae,the optimum concentration of DTT is 120 mmol/L,and that of LiAc is 300 mmol/L.Combination of DTT and LiAc will greatly promote the transformation efficiency to 7.15×105 cfu/μg.In transformation induced by PEG/LiAc,the transformation efficiency is increased to 2.75×105 cfu/μg when the concentration of LiAc is 300 mmol/L,which is much higher than the efficiency reported by other researchers.At the same time,it is confirmed that the optimum method is very useful for promoting the transformation efficiency of homologous recombination.This new optimized transformation system facilitates the research related to saccharomy cescerevisiae.
出处
《实验室研究与探索》
CAS
北大核心
2012年第4期5-8,78,共5页
Research and Exploration In Laboratory
基金
国家自然科学基金资助项目(31100549)
上海浦江基金项目(11PJ1400100)
中央高校基本科研业务费专项资金项目(2011D10512)
关键词
酿酒酵母
转化效率
电转化
DTT
LiAc
saccharomy cescerevisiae
transformation efficiency
electroporation
DTT
LiAc
