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

Abstract

Source: Watson, D. C., et.al. Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria. J. Vis. Exp. (2021)

This video demonstrates the isolation and concentration of extracellular vesicles (EVs) derived from the outer membrane of genetically engineered bacteria. The process involves culturing the bacteria to release EVs, followed by sequential centrifugation and filtration steps to remove cells and debris. Finally, ultrafiltration is used to concentrate the EVs for downstream analysis.

Protocol

1. Bacterial strains and culturing conditions

NOTE: Bacterial strains used in this study were Escherichia coli MP1, Akkermansia mucinophila, Bacteroides thetaiotaomicron, Bifidobacterium breve, and Bifidobacterium dentium.

1. For E. coli, use a sterile loop to inoculate single colonies into 250 to 1,000 mL of Luria-Bertani (LB) broth and incubate aerobically in a shaking incubator at 300 rpm and 37 °C for 48 h before processing the culture. For the recombinant E. coli MP1 strain harboring p114-mCherryClyluc, add chloramphenicol to the LB agar and broth at a final c....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5 mL flat cap, thin-walled PCR tubes	Thermo Scientific	3430	it is important to use thin-walled PCR tubes to obtain accurate readings with Qubit
16% Paraformaldehyde (formaldehyde) aqueous solution	Electron microscopy sciences	15700
250 mL Fiberlite polypropylene centrifuge bottles	ThermoFisher	010-1495
500 mL Fiberlite polypropylene centrifuge bottles	ThermoFisher	010-1493
65 mm Polypropylene Round-Bottom/Conical Bottle Adapter	Beckman Coulter	392077	Allows Vivacell to fit in rotor
Akkermansia mucinophila	ATCC	BAA-835
Amicon-15 (100 kDa MWCO)	MilliporeSigma	UFC910024
Avanti J-20 XPI centrifuge	Beckman Coulter		No longer sold by Beckman. Avanti J-26XP is closest contemporary model.
Bacteroides thetaiotaomicron VPI 5482	ATCC	29148
Bifidobacterium breve	NCIMB	B8807
Bifidobacterium dentium	ATCC	27678
Brain Heart infusion (BHI) broth	Himedia	M2101	After autoclaving, Both BHI broth and agar were introduced into the anaerobic chamber, supplemented with Menadione (1 µg/L), hematin (1.2 µg/L), and L-Cysteine Hydrochloride (0.05%). They were then incubated for at least 24 h under anaerobic conditions before inoculation with the anaerobic bacterial strains.
C-300 microfluidics cartridge	Spectradyne
Chloramphenicol	MP Biomedicals	ICN19032105
Escherichia coli HST08 (Steller competent cells)	Takara	636763
Escherichia coli MP1	Dr. Mark Goulian (gift)		commensal bacteria derived from mouse gut
Fiberlite 500 mL to 250 mL adapter	ThermoFisher	010-0151-05	used with Fiberlite rotor to enable 250 mL bottles to be used for smaller size of starting bacterial culture
Fiberlite fixed-angle centrifuge rotor	ThermoFisher	F12-6x500-LEX	fits 6 x 500 mL bottles
Lauria Bertani (LB) broth, Miller	Difco	244620