Tuning Cell-Free Composition Controls the Time Delay, Dynamics, and Productivity of TX-TL Expression

The composition of cell-free expression systems (TX-TL) is adjusted by adding macromolecular crowding agents and salts. However, the effects of these cosolutes on the dynamics of individual gene expression processes have not been quantified. Here, we carry out kinetic mRNA and protein level measurements on libraries of genetic constructs using the common cosolutes PEG-8000, Ficoll-400, and magnesium glutamate. By combining these measurements with biophysical modeling, we show that cosolutes have differing effects on transcription initiation, translation initiation, and translation elongation rates with trade-offs between time delays, expression tunability, and maximum expression productivity. We also confirm that biophysical models can predict translation initiation rates in TX–TL using Escherichia coli lysate. We discuss how cosolute composition can be tuned to maximize performance across different cell-free applications, including biosensing, diagnostics, and biomanufacturing.