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. Lanes 1, 2 + 3 were probed with antibody specific to the FLAG tag, lanes 4, 5 +6 were probed with antibody specific to ProteinA and lanes 7, 8 + 9 were probed with antibody specific to GFP. (C) SDS-PAGE analysis of the affinity-isolation of LacI::6 × His. Proteins were stained with Coomassie blue. Lane 1 shows protein standards, lane 2, whole cell extract, lane 3, LacI::6 × His affinity-isolate.