Establishment and application of a rapid and efficient method for gene replacement in Staphylococci
ZHU Tao1, DUAN Qin2, LI Chao-pin1, QU Di3
1.Department of Medical Parasitology, Wannan Medical College, Wuhu 241002, China; 2.Department of Stomatology, Wannan Medical College, Wuhu 241002, China; 3.Key Laboratory of Medical Molecular Virology, Ministry of Education and Health, Fudan University, Shanghai 200032, China
Abstract：We established a rapid and efficient method for creating knockout mutants in Staphylococci. The upstream and downstream homologous regions of the vraSR operon were combined by fusion PCR, ligated into temperature-sensitive shuttle plasmid pKOR1 by the Gateway cloning technology, and then transformed into a restriction-defective E. coli strain DC10B, yielding the recombinant plasmid pKOR1-vraSR. The plasmid was then electroporated into Staphylococcus epidermidis RP62A strain. Under the selective pressure of high temperature and chloramphenicol, the plasmid was firstly integrated into the chromosome of S. epidermidis through a single crossover event. The integrant strain was then subcultured, and the second crossover occurred subsequently in the absence of antibiotic selection, resulting in either wild-type strain or the mutant, which depended upon where the recombination event occurred. Finally, the culture was plated on the medium containing 1 μg/mL anhydrotetracycline for counter selection, several colonies were picked up and analyzed by PCR to identify the mutant. Result showed that a vraSR knockout mutant of the untransformable S. epidermidis RP62A was successfully generated in this study. In conclusion, the method for gene replacement in Staphylococci by using E. coli DC10B and shuttle plasmid pKOR1 is simple and efficient.
朱涛， 段芹， 李朝品， 瞿涤. 一种高效的葡萄球菌基因敲除方法的建立与应用[J]. 中国人兽共患病学报, 2015, 31(12): 1098-1102.
ZHU Tao, DUAN Qin, LI Chao-pin, QU Di. Establishment and application of a rapid and efficient method for gene replacement in Staphylococci. Chinese Journal of Zoonoses, 2015, 31(12): 1098-1102.
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2015, Vol. 31 
