摘要
旨在通过优化转化条件提高毕赤酵母电转化效率。采用不同的酵母细胞预处理液成分及浓度、不同的细胞体积、不同的质粒DNA用量,在不同的电压下进行转化,计数转化后在平板上产生的克隆数,通过计算每微克DNA获得的克隆数来评价转化效率。研究表明,用0.1 mol/L LiAc,0.01 mol/L DTT,0.6 mol/L sorbitol,0.01 mol/L Tris-HCl处理细胞可获得最佳转化效率;质粒DNA用量为10 ng时转化效率最高。使用0.1 cm电转杯,采用0.6 kV电压,对80μL细胞进行转化,转化效率最高,可达9.6×105/μg DNA;而使用0.2 cm电转杯,采用1.1 kV电压,对150μL细胞进行转化,转化效率最高,可达8.8×105/μg DNA。采用优化后条件,用10μg pPIC9K质粒DNA转化GS115,可获得1.1×106个转化子。此外通过对3种常用毕赤酵母载体转化效率的比较,发现pPIC9K的转化效率约是pPICZαA的160倍,是pGAPZαA的2 900倍。采用优化后的转化条件,毕赤酵母的电转化效率从103-4提升到了106。采用单个电转杯最多可获得1.1×106个转化子。
This paper aims to improve the transformation efficiency of Pichia pastoris by optimized electroporation. The transformation was carried on with different pretreatment buffer, cell volume, DNA amounts and electroporation voltage. The transformation efficiency was defined by counting the numbers after cultivating and calculating the clones per μg DNA. Research showed that a better transformation efficiency can be obtained when the pretreatment buffer is 0.1 mol/L LiAc, 0.01 mol/L DTT, 0.6 mol/L sorbitol, 0.01 mol/L Tris-HCl and the DNA amount is 10 ng. For the 0.1 cm cuvette, the highest transformation efficiency was 9.6×105 /μg DNA when the electroporation voltage was 0.6 kV and the cell volume was 80 μL. For the 0.2 cm cuvette, the highest transformation efficiency was 8.8×105/μg DNA when the electroporation voltage was 1.1 kV and the cell volume was 150 μL. At the same time, we compared the transformation efficiency of different type of plasmid and found that the transformation efficiency of pPIC9K was 160 times than that of pPICZαA and 2 900 times than that of pGAPZαA. By applying this developed protocol, it have improved the transformation efficiency of Pichia pastoris from 103-4 to 105 / μg DNA.
出处
《生物技术通报》
CAS
CSCD
北大核心
2013年第12期156-161,共6页
Biotechnology Bulletin
基金
国家自然科学基金项目(81101957)
十二五国家科技重大专项(2012ZX09301003-001-007)
关键词
毕赤酵母
电转化
转化效率
Pichia pastoris Electroporation Transformation efficiency
