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张穗生,郭媛,韦廷宗,裴建新,黎贞崇,陈东,黄志民,黄日波.Red重组系统介导下大肠杆菌改造体系的优化[J].广西科学,2010,17(2):160-163. [点击复制]
- ZHANG Sui-sheng,GUO Yuan,WEI Ting-zong,PEI Jian-xin,LI Zhen-chong,CHEN Dong,HUANG Zhi-min,HUANG Ri-bo.Optimizing the Conditions for Red-mediated Recombinogenic Engineering of E.coli[J].Guangxi Sciences,2010,17(2):160-163. [点击复制]
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| Red重组系统介导下大肠杆菌改造体系的优化 |
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张穗生1, 郭媛2, 韦廷宗1, 裴建新1, 黎贞崇1, 陈东1, 黄志民1, 黄日波1
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| (1.广西科学院国家非粮生物质能源工程技术研究中心, 广西南宁 530007;2.广西大学生命科学与技术学院, 广西南宁 530004) |
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| 摘要: |
| 对Red重组系统介导下大肠杆菌进行遗传改造的转化条件、诱导条件、复苏条件三方面实验参数进行优化研究。结果表明:电转化加入的DNA为20ng,Red重组系统感受态细胞培养浓度为OD600=0.60,所需的电击感受态细胞数为3×108;对大肠杆菌基因做双敲除时,突变第2个基因的阿拉伯糖诱导最适时间比突变首个基因延长,诱导浓度为40μm,复苏时间变化对体系影响不显著。 |
| 关键词: Red重组系统 转化条件 诱导条件 复苏条件 |
| DOI： |
| 投稿时间：2009-07-09修订日期：2009-09-03 |
| 基金项目:广西科学院基本科研业务费资助项目"甘蔗渣生产丁醇技术和工艺研究"(08YJ16SW05),广西科学研究与技术开发计划项目"利用禾本纤维生产丁醇菌株的选育及其中试"(桂科攻0992022-1)资助。 |
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| Optimizing the Conditions for Red-mediated Recombinogenic Engineering of E.coli |
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ZHANG Sui-sheng1, GUO Yuan2, WEI Ting-zong1, PEI Jian-xin1, LI Zhen-chong1, CHEN Dong1, HUANG Zhi-min1, HUANG Ri-bo1
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| (1.National Engineering Research Center for Non-food Biorefinery, Guangxi Academy of Sciences, Nanning, Guangxi, 530007, China;2.College of Life Science and Technology, Guangxi University, Nanning, Guangxi, 530004, China) |
| Abstract: |
| Red recombination system, one of important tools for gene replacement, has been applied in Escherichia coli metabolism engineering.Optimization of experimental parameters of this system is necessary for improving the efficiency of mutation. The goal of our research is to optimize the manipulation parameters by comparing the effect of transformation conditions, induction condition and cell recovery conditions on the gene knock-out results of E.coli.The optimized transformation condition was that 20ng DNA is added into 3×108 cells per transformation, culture used for preparation of competent cell is at concentration of 0.60 OD600 value.The induction time required for knocking-out the second gene was relative longer comparing with that for first gene, the ideal concentration for arabinose induction was 40μm. No obvious influence was found when the recovery time was adjusted. |
| Key words: lambda red recombination transformation condition induction condition cell recovery condition |
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