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      A vesicle bioreactor as a step toward an artificial cell assembly.

      Proceedings of the National Academy of Sciences of the United States of America

      Amino Acids, metabolism, Bioreactors, Cell Extracts, genetics, Cell Membrane Permeability, Cell-Free System, Cells, cytology, Diffusion, Escherichia coli, Gene Expression, Hemolysin Proteins, Kinetics, Liposomes, chemistry, Osmotic Pressure, Oxygen, Plasmids, Ribonucleotides, Staphylococcus aureus, Time Factors

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          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 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.

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          Author and article information

          Journal
          15591347
          539773
          10.1073/pnas.0408236101

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