FIG. 4.

Generation of pOmpX and pTA constructs for expression of OmpX in E. coli. (A) Schematic representation of the DNA fragment cloned into plasmid pPCR2.1-TOPO. The y1324 gene from Y. pestis KIM6⁺ with neighboring upstream and downstream regions (750 bp) was cloned into the pPCR2.1-TOPO plasmid, creating the pOmpX plasmid; the pTA plasmid (vector control) was made by deletion of the 766-bp fragment between EcoRI restriction enzyme-cut sites in the pOmpX plasmid and relegation of the backbone. (B) PCR analysis with M13 primers and confirmation of pOmpX and pTA constructs. Lane 1, DNA size marker (Fisher Scientific); lane 2, negative control (no DNA); lane 3, pOmpX; lane 4; pTA. (C) Analysis of OmpX-expressing E. coli TOP10 strains. E. coli Top10, E. coli Top10(pOmpX), and E. coli Top10(pTA) were grown to mid-exponential phase at 37°C and collected by centrifugation. Whole-cell lysate proteins were extracted, separated by SDS-PAGE, and stained with Coomassie blue. Molecular masses were estimated with a prestained Pageruler protein standard (Fermentas). The arrow indicates OmpX protein expressed by the pOmpX-carrying strain.