Isolation and Enrichment of Outer Membrane-Derived Extracellular Vesicles from Bacteria

Start by inoculating a colony of genetically engineered bacteria into a liquid medium.

These bacteria express a fluorescent fusion protein in the outer membrane, an external protective layer.

Incubate for the bacteria to release extracellular vesicles (EVs), membrane-bound nanostructures involved in intercellular communication, which incorporate the fluorescent protein.

Transfer the culture into a bottle and centrifuge at low speed to pellet the cells.

Transfer the supernatant containing EVs, cellular proteins, and cell debris. Centrifuge at high speed, and remove the supernatant containing debris.

Filter the supernatant through a membrane to eliminate any remaining bacteria and debris.

Inoculate a sample on agar and incubate it. The lack of bacterial colony formation confirms the absence of bacteria.

Transfer the solution to an ultrafiltration device with a semipermeable membrane and centrifuge to trap the EVs while eliminating smaller proteins.

Collect the concentrated EVs in a tube for downstream analysis.

Begin by inoculating single colonies of Escherichia coli into 250 to 1000 milliliters of Luria-Bertani, or LB broth, using a sterile loop. Then, incubate the culture aerobically in a s