FIG. 2.
Construction of a counterselectable integration plasmid, pCJK2, and derivatives. A constitutive promoter (P) was amplified by PCR from pFW11 and fused to the E. faecalis upp ORF to create the P-upp cassette, using overlap-extension PCR. This cassette was cloned into pORI280, generating pCJK2. Unique restriction sites that can be used to clone target DNA into the integration plasmid are indicated. To construct a plasmid for deletion of E. faecalis srtA, a chromosomal fragment encompassing a short segment of the end of srtA and sequences flanking srtA were amplified by PCR from either side of srtA from OG1RF chromosomal DNA, yielding the “srtA up” (upstream) and “srtA down” (downstream) amplicons. These amplicons were sequentially cloned into pCJK2 such that they are adjacent to each other, thereby creating the ΔsrtA allele. To construct a plasmid for exchange of wild-type prgB on pCF10 with the prgB1638 allele, prgB1638 was excised from pCWΩ1638 and subcloned into pCJK2, creating pCJK2-1. All pORI280-derived plasmids cannot replicate in E. faecalis.