Cell-free systems as complementary Test layers for protein engineering in biofoundry workflows.

Biofoundries have expanded the scale and design space of synthetic biology by combining laboratory automation with artificial intelligence-assisted protein engineering. As these workflows process larger numbers of variants, the Test phase becomes increasingly important: engineered proteins need to be characterized rapidly, quantitatively, and in formats that fit automated operation. Cell-free systems (CFSs) offer a practical way to meet these needs because they separate protein synthesis from cell growth and provide direct access to both the expressed proteins and the reaction environment. In this review, we discuss the complementary roles of lysate-based CFSs and PURE-type reconstituted CFSs as biochemical Test layers in biofoundry workflows. Their flexible use may broaden biofoundry applications and support more efficient Design-Build-Test-Learn cycles.