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.