一种快速脱盐制备大肠杆菌电转受态细胞的方法

doi:10.14088/j.cnki.issn0439-8114.2020.17.039

湖北农业科学 ›› 2020, Vol. 59 ›› Issue (17): 170-174.doi: 10.14088/j.cnki.issn0439-8114.2020.17.039

一种快速脱盐制备大肠杆菌电转受态细胞的方法

蔡松¹, 杨东成¹, 唐梅¹, 熊圆圆², 王金华¹, 王永泽¹

1.湖北工业大学生物工程与食品学院,武汉 430068;
2.武汉科诺生物科技股份有限公司,武汉 430068

收稿日期:2019-12-18 发布日期:2020-10-23
通讯作者: 王永泽（1976-）,男,副教授,博士,主要从事生物能源与生物材料方向研究,（电子信箱）wyzzju@126.com。
作者简介:蔡松（1994-）,男,贵州赫章人,在读硕士研究生,研究方向为蛋白药物与疫苗工程,（电话）13477082264（电子信箱）csaiong@126.com。
基金资助:
国家“十二五”支撑计划项目（2012BAD27B03）; 山东技术创新计划项目（201720311004）

Method for preparing Escherichia coli electrotransformation competent cells by rapid desalting

CAI Song¹, YANG Dong-cheng¹, TANG Mei¹, XIONG Yuan-yuan², WANG Jin-hua¹, WANG Yong-ze¹

1. School of Food and Biological Engineering,Hubei University of Technology,Wuhan 430068, China;
2. Wuhan Ke-nuo Biotechnology Co., Ltd,Wuhan 430068, China

Received:2019-12-18 Published:2020-10-23

摘要/Abstract

摘要： 为了建立一种高效快捷的大肠杆菌（Escherichia coli）电转化方案,采用类似密度梯度离心的方法,使用角转子离心机达到快速除盐的目的。研究发现,以20%甘油（V/V）/1.5%甘露醇（m/V）溶液作为梯度介质,采用菌悬液与梯度介质比例为1∶5 （V/V）时形成的密度梯度可有效地进行离心除盐,离心后的细胞以20%甘油（V/V）/1.5%甘露醇（m/V）溶液重悬,随后在电场强度为15 kV/cm的条件下进行电转化能得到较高的转化效率,而在室温条件下进行电转化试验不能提高转化效率。本研究仅通过2次离心,电转化效率可达到4.2×10⁹ CFU/μg DNA,大大简化了试验流程。

关键词: 电转化, 感受态细胞, 脱盐, 大肠杆菌（Escherichia coli）

Abstract: A similar density gradient centrifuge method was adopted to establish an efficient and fast protocol for Escherichia coli electrotransformation. Angle rotor centrifuge was used to achieve the goal of rapid desalination of cells using 20% glycerol （V/V） /1.5% mannitol （m/V） solution as gradient medium. The ratio of bacteria suspension to gradient medium was 1∶5 （V/V） to form a density gradient, which led to effectively centrifugal desalination. After centrifugation the cells were resuspended in a mixture of 20% glycerol （V/V）/1.5% mannitol （m/V）, sequentially electroporated at 15 kV/cm. This procedure was suitable for effective desalination. Electroporation at room temperature did not improve transformation efficiency. In this study, the electransformation efficiency can reach 4.2×10⁹ CFU/μg DNA only by two centrifugation, which greatly simplifies the experimental procedure.

Key words: electrotransformation, competent cells, desalting, Escherichia coli

中图分类号:

Q812

蔡松, 杨东成, 唐梅, 熊圆圆, 王金华, 王永泽. 一种快速脱盐制备大肠杆菌电转受态细胞的方法[J]. 湖北农业科学, 2020, 59(17): 170-174.

CAI Song, YANG Dong-cheng, TANG Mei, XIONG Yuan-yuan, WANG Jin-hua, WANG Yong-ze. Method for preparing Escherichia coli electrotransformation competent cells by rapid desalting[J]. HUBEI AGRICULTURAL SCIENCES, 2020, 59(17): 170-174.

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一种快速脱盐制备大肠杆菌电转受态细胞的方法

Method for preparing Escherichia coli electrotransformation competent cells by rapid desalting

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