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

High-performance cell-free protein synthesis has transformative potential for synthetic biology, yet the prohibitive costs of Protein synthesis Using Recombinant Elements kits and the labor intensity of in-house preparation have restricted accessibility and scalability. We developed Purified components Optimized for Flexible protein expression using in vitro-produced translation factors (i-POPFLEX), a modular cell-free protein synthesis system in which 34 translation factors and a split T7 RNA polymerase are individually synthesized in vitro and assembled using automated liquid handling. This workflow minimizes manual input and supports parallelized production, generating complete, ready-to-use systems within 2 days. i-POPFLEX achieves up to 8.4-fold higher protein yields and a 96% cost reduction (27-fold lower cost) compared with commercial kits. Its flexible architecture also enables selective component inclusion for genetic code reprogramming and site-specific incorporation of noncanonical amino acids. By coupling modular design with automation, i-POPFLEX provides an accessible, customizable, and economically viable platform for next-generation biomanufacturing workflows.