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

Grace E. Vezeau, Howard M. Salis

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2508-2519
Number of pages12
JournalACS Synthetic Biology
Volume10
Issue number10
DOIs
StatePublished - Oct 15 2021

All Science Journal Classification (ASJC) codes

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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