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Figure 1. OMV production is increased upon overexpression of the BAM complex and PhoE. (A) CBB-stained SDS-PAGE gel (Left panel) and W-blot incubated with anti-BAM complex antibodies (Right) of WCL and OMVs obtained from cultures of BL21(DE3) WT or BL21(DE3) omp8 (omp8) harboring the empty vector pTRC-His9a (EV), pJH114 expressing the BAM complex (BAM), or pEH3-PhoE expressing PhoE (PhoE). Shown are the relevant parts of the CBB-stained gel, containing samples induced with 100 µM IPTG, and the corresponding regions of the Western blot, containing samples either induced (+) or not induced (-) with 100 µM IPTG. The OD₆₀₀ equivalents of the culture loaded in each sample are indicated. Molecular weight markers are shown on the right, and the positions of BAM subunits are marked on the left based on their expected molecular mass. The black asterisk () indicates mature PhoE protein; the open asterisk () denotes pre-processed PhoE (with signal peptide) that accumulates upon overexpression; and the dot (●) indicates chloramphenicol acetyltransferase encoded by the pEH3-PhoE plasmid; (B) Results of NTA of OMVs isolated from BL21(DE3) WT and omp8 cultures containing EV, BAM, or PhoE. The left panel shows the number of vesicles per OD₆₀₀ of culture, the middle panel depicts the increase in vesicle number relative to WT with EV, and the right panel displays the average vesicle diameter. Bar graphs were generated using GraphPad; (C) Transmission electron microscopy images of BL21(DE3) omp8 cells induced for BAM expression (Top) and OMVs from BL21(DE3) omp8 harboring EV (bottom left) or BAM (bottom right). The scale bar in the top right corner represents 100 nm. Black arrowheads () indicate vesicles in the top image, and open arrowheads () indicate multilamellar OMVs in the bottom left image. OMV: Outer membrane vesicle; BAM: β-barrel assembly machinery; PhoE: outer membrane porin PhoE; CBB: Coomassie brilliant blue; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; WCL: whole cell lysate; EV: empty vector; IPTG: isopropyl β-D-1-thiogalactopyranoside; OD600: optical density at 600 nm; NTA: nanoparticle tracking analysis; WT: wild type; TEM: transmission electron microscopy.