Automated, modular assembly of reconstituted cell-free systems from in vitro-produced components

Area of Science:

• Synthetic Biology
• Biochemistry
• Molecular Biology

Background:

• Cell-free protein synthesis (CFPS) is crucial for synthetic biology but limited by high costs and labor-intensive preparation of commercial kits.
• Existing CFPS methods face challenges in accessibility and scalability due to expensive reagents and complex protocols.

Purpose of the Study:

• To develop an accessible, scalable, and cost-effective modular cell-free protein synthesis system.
• To overcome the limitations of current commercial CFPS kits through automation and in-vitro production of components.

Main Methods:

• Developed Purified components Optimized for Flexible protein expression using in vitro-produced translation factors (i-POPFLEX), a modular CFPS system.
• Synthesized 34 translation factors and a split T7 RNA polymerase individually in vitro.
• Assembled components using automated liquid handling for parallelized production within 2 days.

Main Results:

• Achieved up to 8.4-fold higher protein yields compared to commercial kits.
• Reduced costs by 96% (27-fold lower cost) compared to commercial kits.
• Demonstrated flexibility for genetic code reprogramming and noncanonical amino acid incorporation.

Conclusions:

• i-POPFLEX offers an accessible, customizable, and economically viable platform for next-generation biomanufacturing.
• The modular design and automation of i-POPFLEX significantly improve protein synthesis efficiency and reduce costs.
• This system enhances the scalability and accessibility of high-performance cell-free protein synthesis for diverse applications.