通过削弱谷氨酸棒杆菌的细胞壁并增加细胞质膜流动性来提高其电转化效率。
Improving the electro-transformation efficiency of Corynebacterium glutamicum by weakening its cell wall and increasing the cytoplasmic membrane fluidity.
作者信息
Ruan Yili, Zhu Linjiang, Li Qi
机构信息
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, No 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China.
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214000, China.
出版信息
Biotechnol Lett. 2015 Dec;37(12):2445-52. doi: 10.1007/s10529-015-1934-x. Epub 2015 Sep 9.
OBJECTIVE
To improve the transformation efficiency of Corynebacterium glutamicum cells with heterogenous plasmid DNA and single-strand DNA (ssDNA) using a methodology based on electro-transformation.
RESULTS
A semicomplex hypertonic medium was selected with addition of glycine and DL-threonine to weaken cell walls and addition of Tween 80 and isonicotinic acid hydrazide to increase cytoplasmic membrane fluidity. Their contents were optimized by response surface methodology. Cell growth, electro-transformation buffer, and transformation protocol were also optimized. Temporary heating inactivation of the host restriction enzyme showed a significant effect. Finally, a high transformation efficiency of 3.57 ± 0.13 × 10(7) cfu/μg DNA of plasmid and 1.05 × 10(6) Str (R) cfu per 10(9) viable cells with a ssDNA was achieved.
CONCLUSION
The results shed light on the application in functional genomics and genome editing of C. glutamicum.
目的
采用基于电转化的方法提高谷氨酸棒杆菌细胞对异源质粒DNA和单链DNA（ssDNA）的转化效率。
结果
选择一种半复合高渗培养基，添加甘氨酸和DL-苏氨酸以削弱细胞壁，添加吐温80和异烟肼以增加细胞质膜流动性。通过响应面法对其含量进行了优化。还对细胞生长、电转化缓冲液和转化方案进行了优化。宿主限制酶的临时加热失活显示出显著效果。最终，实现了质粒DNA的高转化效率，为3.57±0.13×10⁷cfu/μg DNA，单链DNA每10⁹个活细胞有1.05×10⁶Str（R）cfu。
结论
这些结果为谷氨酸棒杆菌在功能基因组学和基因组编辑中的应用提供了启示。
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