Fig. 2: The synthetic EcN outfitted with modified T0SS can secrete protein-loaded OMVs with high encapsulation ratio and package multiple distinct cargos in individual OMVs.

a Western blot analysis of GFP abundance in OMV samples from EcNΔnlpI-OG (abbreviated as “OG”) and EcNΔnlpI-GR (abbreviated as “OGR”) and EcNΔnlpI (abbreviated as “NC”) using an anti-GFP antibody. b The synthetic EcN outfitted with modified T0SS and endogenous loading system was able to secrete protein-loaded OMVs with an encapsulation ratio of 97.9%. c The OMV encapsulation ratio of GFP was tested by Nano-flow cytometry, which was calculated as the ratio of GFP-loaded OMVs to total OMVs derived from EcNΔnlpI-OG, using EVMembrane Red to stain the membrane of OMVs. d Western blot analysis of RFP abundance in OMV samples from EcNΔnlpI-OR (abbreviated as “OR”), EcNΔnlpI-GR (abbreviated as “OGR”) and EcNΔnlpI (abbreviated as “NC”) using an anti-RFP antibody. e The synthetic OMVs of engineered EcN could encapsulate multiple protein payloads simultaneously in individual OMV vehicles, making the synthetic OMV a promising cascade biocatalyst. f Engineered OMVs were able to simultaneously encapsulate GFP and RFP in individual OMVs, as visualized via Polar-SIM super-resolution microscope. Scale bar=2 μm. a, d, f Each experiment was repeated three times independently with similar results. b, e Created in BioRender. Gong, X. (2025) https://BioRender.com/i12g295.