Figure 2. Overall strategy of PCR ligation mutagenesis.

The mutant strain was made as follows: 1. Construction of recombinant plasmids. Two pairs of gene-specific primers were designed. P1 and P2 are used to amplify the 5′ upper stream homologous arm of LuxS gene, while P3 and P4 amplify the 3′ region flanking the target gene. Each DNA fragment was ligated to the VECTOR according to the manufacturer′s instruction. The recombinant plasmid was then transformed into E. coli DH5a competent cells. Positive clones of transformed cells were selected and sequenced, named as pEASYLuxS-up-amp-kana-LuxS-down. Under these conditions, P2 has an expanded HindIII site, whereas P3 has an expanded XhoI site, both attached to their 5′ ends. 2. Transformation of E. faecalis . E. faecalis was transformed by electroporation according to the protocol. The E. faecalis LuxS gene knockout was identified by PCR with primers P1 to P4