A vesicle bioreactor as a step toward an artificial cell assembly.
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 alpha-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 muM after 4 days. Oxygen diffusion and osmotic pressure are critical parameters to maintain expression and avoid vesicle burst.
Noireaux
Vincent
V
Center for Studies in Physics and Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
Libchaber
Albert
A
D000596:Amino Acids / Q000378:metabolism; D019149:Bioreactors; D002457:Cell Extracts / Q000235:genetics; D002463:Cell Membrane Permeability; D002474:Cell-Free System; D002477:Cells / Q000166:cytology* / Q000378:metabolism*; D004058:Diffusion; D004926:Escherichia coli / Q000166:cytology; D015870:Gene Expression; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism*; D009997:Osmotic Pressure; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D012265:Ribonucleotides / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus; D013997:Time Factors
D000596:Amino Acids; D002457:Cell Extracts; D006460:Hemolysin Proteins; D008081:Liposomes; D012265:Ribonucleotides; D010100:Oxygen