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 shaking incubator at 300 rotations per minute and 37 degrees Celsius. After 48 hours of incubation, clarify the bacterial culture medium by transferring the cell cultures to clean 500-milliliter polypropylene centrifuge bottles to centrifuge in a large-capacity, fixed-angle rotor at four degrees Celsius and 5,000 times G for 15 minutes.

Transfer the supernatant to clean centrifuge bottles by carefully pouring to centrifuge at 10,000 times G for 15 minutes. Next, transfer the supernatant to a 0.2-micron polyethersulphone vacuum-driven filter device of an appropriate size and connect the filtration device to a vacuum wall supply. If the filtration rate significantly drops, move the unfiltered material to a new device.

The filtered medium can be stored at four degrees Celsius overnight or can be further processed immediately. To check for the complete removal of the viable cells, spread an aliquot of the filtered supernatant on suitable agar plates and ensure the absence of any colonies after incubation at optimum conditions for the bacterial strain. For concentrating the filtered medium with a volume of 100 milliliters, load 90 milliliters of filtered culture medium onto the reservoir of centrifugal ultrafiltration device with a 100 kilodaltons molecular weight cutoff and centrifuge the medium in swinging bucket rotor at four degrees Celsius and 2,000 times G for 15 to 30 minute intervals until the volume of the medium in the top reservoir has been concentrated to less than 0.5 milliliters.

To top up the reservoir with any remaining filtered culture medium, remove the flow-through in the bottom of the device to rebalance. If the viscosity of the concentrated medium in the reservoir is visibly increased, dilute the medium with PBS and reconcentrate by centrifugation to reduce any non-extracellular vesicle, or EV proteins, smaller than the molecular weight cutoff of 100 kilodaltons. Then, transfer the concentrated medium to a low-protein binding tube to store at four degrees Celsius overnight.