The resulting plasmid (pKX23) was verified by nucleotide sequencing and used as the template plasmid to make the linear recombineering substrate (Fig. 3b). The http://www.selleckchem.com/products/Roscovitine.html linear DNA was used to recombineer in RSW358 strains with pJAK12, pJAK14, or pJAK16. Selection of the recombinants was for Gmr. Transformants numbered > 4000 mL−1 for each,

and as expected, all were white on X-Gal-IPTG medium. Recombinants of pJAK12, pJAK14, and pJAK16 [pKX32 (Spr Gmr), pKX34 (Kmr Gmr), and pKX36 (Cmr Gmr), respectively] were verified by nucleotide sequencing. The aacC1-encoding SalI fragment was removed from each plasmid by digestion with SalI, religation, and transformation. Spr, Kmr, or Cmr transformants were selected, as appropriate for FG-4592 pJAK12, pJAK14, and pJAK16, respectively. As expected, Spr/Kmr/Cmr Gms transformants were blue on X-Gal-IPTG medium. Nucleotide sequencing confirmed the structures. The recombinants were named pJAK12 Blue, pJAK14 Blue, and pJAK16 Blue (Fig. 2b).

We also used the method to construct an oriTIncP-Gmr cassette and to provide the recombineering substrate for targeting it to the cat gene of pSIM9 (Fig. 2c). The template plasmid was constructed from pCR2.1 TOPO using HindIII, BamHI, NotI, XhoI, and XbaI (Fig. 3c). The oriT-encoding PCR product, made from pAA56 with flanking BamHI and NotI recognition sites (Table 2c), was inserted at the TA-cloning site, oriented by PCR with the appropriate primers, and verified by nucleotide sequencing to give pKR1. The aacC1 gene was cloned into NotI- and

XhoI-cleaved pKR1 to give the oriT-Gmr plasmid pKR2. HRI (260 bp) was cloned into HindIII- and BamHI-cleaved pKR2 to give pKR6, and HRII (275 bp) was ligated to XhoI- and XbaI-cleaved pKR6 to give pKR7. The MCS region of pKR7, which should have the elements for the recombineering substrate, was verified by nucleotide sequencing. Plasmid pKR7 was then used to make the recombineering substrate by PCR using primers Urease P1 and P8 (Table 2c). Gmr selection led to > 4000 colonies mL−1. One typical Gmr Cms oriTIncP pSIM9 derivative (pKR8) was shown by nucleotide sequencing to have the expected structure. In summary, we developed a method for making recombineering substrates with PCR primers that can be ≤ 35 nucleotides long (the ‘short-primer’ method). The method uses restriction endonuclease–based molecular cloning techniques to link GEs and regions of homology to make a recombineering substrate. A downside of the short-primer method is that it takes somewhat longer than the long-primer method to obtain the desired recombinant (about twice as long if the substrate is made from three cloned segments). The HRs of the short-primer method are easily changed to target the GEs to a different site. In addition, cloning of the segments requires that the PCR primers work to give the desired fragment, and each intermediate plasmid can be verified.