Yili Ruan1, Linjiang Zhu2, Qi Li3,4. 1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, No 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China. summer.ruan19@gmail.com. 2. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, No 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China. zlj@jiangnan.edu.cn. 3. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, No 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China. liqi@jiangnan.edu.cn. 4. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214000, China. liqi@jiangnan.edu.cn.
Abstract
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.
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.
Authors: Yusuke Sasaki; Thomas Eng; Robin A Herbert; Jessica Trinh; Yan Chen; Alberto Rodriguez; John Gladden; Blake A Simmons; Christopher J Petzold; Aindrila Mukhopadhyay Journal: Biotechnol Biofuels Date: 2019-02-27 Impact factor: 6.040
Authors: Thomas Eng; Yusuke Sasaki; Robin A Herbert; Andrew Lau; Jessica Trinh; Yan Chen; Mona Mirsiaghi; Christopher J Petzold; Aindrila Mukhopadhyay Journal: Metab Eng Commun Date: 2019-12-02