A vesicle bioreactor as a step toward an artificial cell assembly

Area of Science:

• Biotechnology
• Synthetic Biology
• Biochemistry

Background:

• Cell-free expression systems offer a controllable environment for biological reactions.
• Encapsulating these systems in vesicles mimics cellular compartmentalization.
• Limitations in nutrient supply and waste removal hinder prolonged cell-free reactions.

Purpose of the Study:

• To develop a cell-like bioreactor using a cell-free expression system within phospholipid vesicles.
• To enhance the duration and yield of protein synthesis by controlling molecular transport.
• To overcome energy and material limitations inherent in traditional cell-free systems.

Main Methods:

• An Escherichia coli cell-free expression system was encapsulated in large unilamellar vesicles.
• Vesicles were formed in an oil-extract emulsion and transferred to a nutrient-rich feeding solution.
• Selective permeability was engineered by expressing alpha-hemolysin pore protein within the vesicles.

Main Results:

• Encapsulation in vesicles prolonged enhanced green fluorescent protein (eGFP) expression from 2 hours to 5 hours compared to bulk solution.
• Engineering selective permeability with alpha-hemolysin sustained protein expression for up to 4 days.
• Achieved a protein production of 30 muM after 4 days, demonstrating significantly increased reactor capacity.

Conclusions:

• Phospholipid vesicles serve as effective cell-like bioreactors for cell-free expression systems.
• Controlled nutrient permeability is crucial for overcoming limitations and extending reaction times.
• This approach offers a promising platform for sustained and high-yield bioproduction.