A vesicle bioreactor as a step toward an artificial cell assembly

TLDR

Oxygen diffusion and osmotic pressure are critical parameters to maintain expression and avoid vesicle burst. The authors encapsulated an E. coli cell‑free expression system in large unilamellar phospholipid vesicles produced by an oil–extract emulsion, transferred them into a feeding solution of ribonucleotides and amino acids, and expressed the α‑hemolysin pore protein to create selective permeability that prolongs transcription–translation.

Abstract

An Escherichia coli cell-free expression system is encapsulated in a phospholipid vesicle to build a cell-like bioreactor. Large unilamellar vesicles containing extracts are produced in an oil–extract emulsion. To form a bilayer the vesicles are transferred into a feeding solution that contains ribonucleotides and amino acids. Transcription–translation of plasmid genes is isolated in the vesicles. Whereas in bulk solution expression of enhanced GFP stops after 2 h, inside the vesicle permeability of the membrane to the feeding solution prolongs the expression for up to 5 h. To solve the energy and material limitations and increase the capacity of the reactor, the α-hemolysin pore protein from Staphylococcus aureus is expressed inside the vesicle to create a selective permeability for nutrients. The reactor can then sustain expression for up to 4 days with a protein production of 30 μM after 4 days. Oxygen diffusion and osmotic pressure are critical parameters to maintain expression and avoid vesicle burst.