Journal
BIOTECHNOLOGY LETTERS
Volume 37, Issue 12, Pages 2445-2452Publisher
SPRINGER
DOI: 10.1007/s10529-015-1934-x
Keywords
Cell-wall weakening; Corynebacterium glutamicum; Cytoplasmic membrane fluidity; Electro-transformation; Single-strand DNA
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Funding
- Fundamental Research Funds for the Central Universities [JUSRP51306A, JUDCF13008, JUSRP51402A]
- Priority Academic Program Development of Jiangsu Higher Education Institutions
- Program of Introducing Talents of Discipline to Universities [111-2-06]
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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 x 10(7) cfu/mu g DNA of plasmid and 1.05 x 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.
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