Isolation and Characterization of OMVs from E. coli

OMVs_Δ60 were prepared growing the E. coli BL21(DE3)Δ60 strain in an EZ control bioreactor (Applikon Biotechnology, Schiedam, The Netherlands) until the end of the exponential phase at 30 °C, pH 6.8 (±0.2), dO₂ > 30%, 280–500 rpm. OMVs were then purified and quantified as previously described [16 (link)]. Briefly, the culture supernatant was separated from biomass by centrifugation at 4000× g for 20 min. After filtration through a 0.22 μm pore size filter (Millipore, Burlington, MA, USA), OMVs were isolated, concentrated and diafiltrated from the supernatant using Tangential Flow Filtration (TFF) with a Cytiva Äkta Flux system (Marlborough, MA, USA). OMVs were quantified using DC protein assay (Bio-Rad, Hercules, CA, USA). OMV proteins were separated using a 4–12% gradient polyacrylamide gel (Invitrogen, Waltham, MA, USA) and finally stained with Coomassie Blue (Giotto, Sesto Fiorentino, Italy).