Methods of constructing Staphylococcus epidermidis gene mutants via different plasmids
LOU Qiang; ZHU Tao; Wang Jia-xue; WU Yang; Zhu Yu-li; QU Di
Key Laboratory of Medical Molecular Virology of Ministry of Education and Public Health of China; Institutes of Biomedical Sciences and Institute of Medical Microbiology, Shanghai Medical School, Fudan University, Shanghai 200032, China
Abstract：Objective To construct Staphylococcus epidermidis gene knock-out mutants with different plasmids. Methods Recombinant plasmids were constructed for homologous recombination of the two component signal transduction system genes (arlS、saeRS and lytS) of S. epidermidis with two different shuttle plasmids, pMAD and pBT2, transformed to S. aureus RN4220 by electroporation and then transformed into S. epidermidis 1457. S. epidermidis mutants were selected using various antibiotics and biomarkers and identified by PCR and biochemistry experiments. Results With pMAD or pBT2 plasmid, we separately constructed S. epidermids genes knock-out homologous recombinant plasmids, pMAD-ΔsaeRS, pBT2-ΔarlS and pBT2-ΔlytS. S. epidermidis 1457 was transformed with the recombinant plasmids and the gene knocking out mutant was screened out, respectively. Only one cycle was needed for screening out SE1457-ΔsaeRS mutant and 5-10 cycles were required for screening out SE1457-ΔarlS and SE1457-ΔlytS mutant. The biofilm forming of SE1457-ΔarlS mutant decreased by 90.96% and no significant difference was observed between SE1457-ΔlytS、SE1457-ΔsaeRS mutants and SE1457 parent strain. Conclusion pMAD method can be used to construct S. epidermidis mutant strain simply and effectively.
