Cell-free expression (CFE) systems are a suite of methods that reconstitute complex cellular functions like transcription, translation, and metabolism outside the confines of a living cell. CFE systems have numerous biotechnological uses in sensing, biomanufacturing, medicine, basic research, and education. Most CFE systems are made from combining cellular lysates with a complex blend of excipients that improve activity. While the number of excipients makes exploring the combinatorial spaces challenging, high-throughput experimentation with acoustic liquid handling makes it feasible to optimize formulations if paired with an appropriate statistical framework.

Here we describe our use of design of experiments (DOE) to optimize excipient combinations for specific use cases of CFE. We pair our DOE with functional data analysis (FDA) to collapse activity over time measurements to metrics readily used for analysis. Initial formulation DOE examples range from five to 14 components. We further describe our efforts to push to higher scales, attempting mixture-process DOE designs with as many as 42 components using an experimental set-up that allows for 1,536 formulations to be tested at once.