室温电感受态细菌细胞可提高DNA转化和重组工程效率。
Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.
作者信息
Tu Qiang, Yin Jia, Fu Jun, Herrmann Jennifer, Li Yuezhong, Yin Yulong, Stewart A Francis, Müller Rolf, Zhang Youming
机构信息
Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China.
Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, Campus E8.1, 66123 Saarbrücken, Germany.
出版信息
Sci Rep. 2016 Apr 20;6:24648. doi: 10.1038/srep24648.
Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and recombineering efficiency in E. coli and several other gram-negative bacteria thereby economizing time and cost. Increased transformation efficiency of large DNA molecules is a significant advantage that might facilitate the cloning of large fragments from genomic DNA preparations and metagenomics samples.
细菌感受态细胞对于每个分子生物学实验室进行克隆、构建DNA文库以及诱变而言都是必不可少的。在各种转化方法中，电穿孔法被发现具有最佳的转化效率。以往的电穿孔方法是基于在冰冷条件下洗涤并对细菌细胞进行电穿孔，这会使细胞变得脆弱且容易死亡。在此，我们提出了基于简单温度转换的方法，该方法提高了大肠杆菌及其他几种革兰氏阴性菌中的DNA转化和重组工程效率，从而节省了时间和成本。大型DNA分子转化效率的提高是一个显著优势，这可能有助于从基因组DNA制备物和宏基因组学样品中克隆大片段。
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