TY - JOUR
T1 - Tuning Cell-Free Composition Controls the Time Delay, Dynamics, and Productivity of TX-TL Expression
AU - Vezeau, Grace E.
AU - Salis, Howard M.
N1 - Funding Information:
This project was supported in part by funds from the Air Force Office of Scientific Research (FA9550-14-1-0089), the Defense Advanced Research Projects Agency (FA8750-17-C-0254), and the Department of Energy (DE-SC0019090).
Publisher Copyright:
© 2021 American Chemical Society.
PY - 2021/10/15
Y1 - 2021/10/15
N2 - 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.
AB - 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.
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U2 - 10.1021/acssynbio.1c00136
DO - 10.1021/acssynbio.1c00136
M3 - Article
C2 - 34498860
AN - SCOPUS:85115621995
VL - 10
SP - 2508
EP - 2519
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
SN - 2161-5063
IS - 10
ER -