DNA fragments, generated by PCR amplification, using pDOC-K as a template were cloned into pDOC-C, and the resulting donor plasmids used for gene doctoring. To PP2 date we have made deletions of the rpoS, fur, flhDC and
soxS genes in MG1655, O157:H7 Sakai, CFT073 and H10407 strains (data not shown). Functionality of the epitope tags To examine the functionality of the epitope tags we coupled each to the Lac repressor protein in MG1655. The experimental details and primer design for each recombination experiment are given in the methods section. For each epitope tag we identified more than 200 candidates that were kanamycin resistant, sucrose insensitive. After verification by PCR amplification and DNA sequencing of the chromosomal region (Figure 5; panel A), we tested the functionality of the epitope tags. The LacI::3 × FLAG, LacI::4 × ProteinA and LacI::GFP IACS-10759 mw fusion proteins were analyzed by Western blotting. Whole cell extracts were separated by SDS-PAGE and proteins transferred to nitrocellulose membranes, which were then probed with primary antibodies specific to the tag. The membranes were then washed and probed with secondary
antibodies conjugated to horse-radish peroxidase. Figure 5; panel B, shows an image of the membranes after exposure to X-ray film; the fusion proteins MK 8931 solubility dmso are indicated. In a recent study we validated the functionality of the LacI::3 × FLAG fusion protein by isolating
DNA fragments carrying LacI binding sites from cells . We also confirmed the fluorescence of the LacI::GFP fusion protein, in whole cells using fluorescent microscopy (data not shown). Finally, we tested the integrity of the 6 × His fusion proteins by isolating the protein fusion by affinity purification using nickel agarose affinity media (Qiagen). Purified proteins selleck compound were analysed by SDS-PAGE. Figure 5; panel C, shows a scanned image of the SDS-PAGE gel on which the fusion protein is highlighted. Figure 5 Verification and functionality of chromosomal lacI::tag fusions. (A) Ethidum bromide stained agarose gel showing DNA amplified by PCR from the lacI fusion strains. Lanes 1 and 6 are DNA markers, lanes 2, 3, 4 + 5 show DNA derived from lacI::6 × his, lacI::3 × FLAG, lacI::ProteinA and lacI::GFP respectively. (B) Western blot analysis of tagged strains. Lanes 1, 4 and 7 show protein standards. Lanes 2, 5 and 8 show wild-type MG1655. Lanes 3, 6 and 9 show the tagged strains.