fig2

Figure 2. Increased expression of PhoE results in increased production of PhoE-containing OMVs. (A) CBB-stained SDS-PAGE gel showing WCL and OMVs obtained from BL21(DE3) omp8 cultures expressing PhoE from pEH3-PhoE. The gel displays samples from cultures that were not induced (0) or induced with 10, 50, or 100 µM IPTG. The amount of culture loaded, indicated by OD₆₀₀ equivalents, is shown. The position of processed PhoE, based on its expected molecular weight, is marked on the left. The open asterisk () indicates pre-processed PhoE (with signal peptide), while the dot (●) indicates chloramphenicol acetyltransferase encoded by the pEH3-PhoE plasmid; (B) Results of NTA performed on OMVs isolated from BL21(DE3) omp8 cultures expressing different levels of PhoE. The left panel shows the number of vesicles measured per OD₆₀₀ of culture, while the right panel illustrates the fold increase relative to cultures with no IPTG added. Bar graphs were generated using GraphPad. PhoE: Outer membrane porin PhoE; OMV: outer membrane vesicle; CBB: Coomassie brilliant blue; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; WCL: whole cell lysate; IPTG: isopropyl β-D-1-thiogalactopyranoside; OD600: optical density at 600 nm; NTA: nanoparticle tracking analysis.