The restriction endonuclease recognition sites are added to the primers (see Table 2). Ideally, the primers for the HRs are designed to anneal at a similar temperature because one primer of each pair (P1 and P6 in Fig. 1) will be used together to make the recombineering substrate. As proof of principal, our first project was to use the method to prepare the recombineering substrate to convert the Apr Kmr

plasmid pACYC177 to Spr Aps Kmr (Fig. 2a). Plasmid pCR2.1 TOPO was used to make the template plasmid. The four restriction endonucleases were KpnI, SpeI, XhoI, and XbaI (A, B, C, and D, respectively, in Fig. 1). PCR was used to create DNA fragments for the three segments: (1) the aadA gene flanked by SpeI and XhoI recognition sequences (primers P3 and P4, Table 2a); (2) the ‘left’ (as per Fig. 1 and www.selleckchem.com/products/VX-809.html the map of the MCS in pCR2.1 TOPO) homology region (HRI) flanked by KpnI and SpeI recognition sequences (primers P1 and P2 in Table 2a); and (3) the ‘right’ homology region learn more (HRII) flanked by XhoI and

XbaI recognition sequences (primers P5 and P6 in Table 2a). The nucleotide sequences of the PCR primers for the three DNA segments are given in Table 2a. The two HRs [HRI (282 bp) and HRII (256 bp)] directed the aadA segment to the bla gene of pACYC177. The aadA gene-containing segment was cloned into the TA-cloning site of pCR2.1 TOPO, oriented by PCR, and confirmed by nucleotide

sequencing. That plasmid (pJH020) was then used to clone HRI. The insert was confirmed by PCR, and the resulting plasmid (pJH022) was used to clone HRII to give the template plasmid (pJH023). The template region of pJH023 (Fig. 3a) was confirmed by nucleotide sequencing. pJH023 was used to generate the linear recombineering substrate by PCR with primers P1 the and P6. Recombineering in DH5α(pSIM9) cells with the linear PCR product gave > 4000 Spr colonies mL−1. The Spr colonies were Aps Kmr. One such colony yielded plasmid pJH027, which was verified by nucleotide sequencing to have the expected structure. In another project, the MCS-lacZα region that gives the blue/white screen for inserts of pBBR1MCS was added to the IncQ vectors pJAK12 (Spr), pJAK14 (Kmr), and pJAK16 (Cmr) (Fig. 2b). The MCS-lacZα region from pBBR1MCS was marked with Gmr by cloning into the MCS, a SalI fragment encoding aacC1 (Gmr). The template plasmid was constructed from pACYC177. The PsiI-lacZα-MCS::aacC1-ApaLI fragment was cloned into the PsiI–ApaLI region of pACYC177 to give pKX21. HRI and HRII targeted sequences adjacent to the identical MCS regions of the pJAK12/14/16 series.