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| 工业谷氨酸棒杆菌电转化整合效率的优化 |
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王慧梅1,2,黄勤勤1,2,梁玲1,2,黄钦耿1,2,吴松刚1,2,黄建忠1,2
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1.福建师范大学生命科学学院;2.工业微生物教育部工程研究中心
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| 摘要: |
| 研究不同因素对谷氨酸棒杆菌（Corynebacterium glutamicum）电转化效率的影响，探讨电转化的最适条件，提高电转化效率。以产L-异亮氨酸的谷氨酸棒杆菌工业菌株a11为受体菌，大肠杆菌（Escherichia coli） JM109及质粒pk18mobsacB为载体，通过电转化方法研究了菌体最佳感受态性能、复苏培养基高渗溶液浓度、最适电场强度及电击后的热激培养对电转化效率的影响。对于谷氨酸棒杆菌a11而言，使用无痕的自杀载体电击转化，在感受态细胞OD600 nm值为1.0~1.2，电场强度达到9.0 kV/cm，电转后46 ℃热激培养8 min，而且热激后继续保持37 ℃的适应性培养，电转化效率最高，达到1.8×103 cfu/μg DNA。实现了工业谷氨酸棒杆菌的电转化效率的提高，也为其它谷氨酸棒杆菌电转化效率的提高提供参考方法。 |
| 关键词: 谷氨酸棒杆菌 感受态细胞 电转化 整合 |
| DOI：10.14188/j.ajsh.2018.05.005 |
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| 基金项目:863国家高技术研究开发计划项目（2015AA021005） |
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| Optimization of electrotransformation integration efficiency of Corynebacterium glutamicum |
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WANG Huimei1,2,HUANG Qinqin1,2,LIANG Ling1,2,HUANG Qingeng1,2,WU Songgang1,2,HUANG Jianzhong1,2
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1.College of Life Science， Fujian Normal University， Fuzhou 350007， Fujian， China;2.Engineering;Research Center of Industrial Microbiology，Ministry of Education，Fujian Normal University，Fuzhou 350007，Fujian，China
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| Abstract: |
| To study the influence of different factors on the electrotransformation efficiency of Corynebacterium glutamicum, and to explore the optimal conditions for electrotransformation efficiency, the L-isoleucine-producing industrial strain C.glutamicum a11 was used as a recipiet, Escherichia coli JM109 and plasmid pk18mobsacB as vectors to study the effects of the best competencies of the bacteria, the concentration of the hypertonic solution in the resuscitation medium, the optimal electric field strength, and the heat shock culture after the electric shock on the electrotransformation efficiency. For industrial C. glutamicum a11, a non-invasive suicide vector was used for electroporation transformation. Under the conditions of OD600 nm value of the competent cells of 1.0~1.2, the electric field intensity of 9.0 kV/cm, the cell heat-culture at 46 °C for 8 min after electroporation and the adaptive culture maintained at 37 ℃, the electrotransformation efficiency reached up to 1.8×103 cfu/μg DNA. The improvement of the electrotransformation efficiency provides references for the further studies. |
| Key words: glutamicum competent cell electrotransformation integration |
