Construction of a Sae-Deleted Mutant of Staphylococcus aureus by Homologous Recombination

Abstract:

In this study,two pairs of primers were designed according to the upstream and downstream sequence of sae gene and used to amplify the homologous arms by PCR.Then the upstream and downstream homologous arms were cloned into the shuttle vector pBT2 with the Em resistance gene fragment from the plasmid p646 inserted as a selection marker between the two sequences.The plasmid pBT2Δsae was constructed.The homologous recombination vector was subsequently transformed into S.aureus RN4220 by electroporation,S.aureus RNΔsae deletion mutant was successfully selected by homologous recombination at 40 ℃ and confirmed by PCR.Subsequently,the sae gene expression level was also valuated by RT-PCR.This study provided the useful tool for further exploring the regulation mechanism of sae gene in S.aureus.