Optimization of the Electroporation Conditions for DNA Transformation of Staphylococcus carnosus
In this paper, the optimization of electroporation method for Staphylococcus carnosus is investigated. The various factors for electrotransformation are evaluated, including bacterial growth phase, electroporation parameters, pBT2 plasmid DNA concentratio
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Optimization of the Electroporation Conditions for DNA Transformation of Staphylococcus carnosus Qiang Gao, Mengxiao Wang, Changyan Yu, Lin Huang, Xiu Zheng and Yan Zhu
Abstract In this paper, the optimization of electroporation method for Staphylococcus carnosus is investigated. The various factors for electrotransformation are evaluated, including bacterial growth phase, electroporation parameters, pBT2 plasmid DNA concentration and structure, the effect of protoplast or other cell wall-weakening treatments, and the prepulse incubation temperatures, etc. The primary optimized electroporation method is consisted of 60 lL electrocompetent cells and 800–1,000 ng plasmid DNA using the electroporation parameters of 21 kV cm-1 field strength, 50 X resistance, and 25 lF capacitance by a Bio-Rad Gene Pulser XcellTM system. Using this optimized method, three different plasmids (pBT2, pCX19, and pTX15) are successfully transformed into S. carnosus, and the transformation efficiencies are stationary at around 1 9 103 transformants per lg plasmid DNA. Our results indicate that this electroporation method can be generally applied for foreign DNA transformation into S. carnosus host.
Keywords Electroporation Staphylococcus carnosus Plasmid Electrocompetent cells
Q. Gao (&) M. Wang C. Yu L. Huang Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic of China e-mail: [email protected] X. Zheng Y. Zhu The Logistic Service Group, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic of China
T.-C. Zhang et al. (eds.), Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012), Lecture Notes in Electrical Engineering 251, DOI: 10.1007/978-3-642-37925-3_182, Ó Springer-Verlag Berlin Heidelberg 2014
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182.1 Introduction As a non-pathogenic and Gram-positive bacterium, Staphylococcus carnosus is the only one bacterium fully recognized as food-grade GRAS (generally regarded as safe) in Staphylococci family [1, 2]. This microorganism plays an important role in the ripening of dry sausages and hams, and it has been used as a starter culture for dry meat manufacture in Europe since 1950s. Furthermore, due to the above significance, S. carnosus has been developed as a bacterial host system for foreign protein expression and cell surface display for decades [2]. The transformation step is of key importance for successful genetic manipulation. In general, electroporation is a mechanical method which transfers polar DNA molecules directly into the host cell with the aid of the high-voltage pulse lasted a few microseconds to a millisecond. Moreover, the advantages of this method are effective, rapid, and at low cost. Since the electroporation-mediated transformation of Staphylococcus aureus and S. epidermidis are quite successful [3], it would be preferable if
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