Mechanistic Insights into Cell-Free Gene Expression through an Integrated -Omics Analysis of Extract Processing Methods

Blake J. Rasor; Payal Chirania; Grant A. Rybnicky; Richard J. Giannone; Nancy L. Engle; Timothy J. Tschaplinski; Ashty S. Karim; Robert L. Hettich; Michael C. Jewett

doi:10.1021/acssynbio.2c00339

Mechanistic Insights into Cell-Free Gene Expression through an Integrated -Omics Analysis of Extract Processing Methods

Blake J. Rasor, Payal Chirania, Grant A. Rybnicky, Richard J. Giannone, Nancy L. Engle, Timothy J. Tschaplinski, Ashty S. Karim, Robert L. Hettich^*, Michael C. Jewett^*

^*Corresponding author for this work

Chemical and Biological Engineering

Research output: Contribution to journal › Article › peer-review

6 Scopus citations

Abstract

Cell-free systems derived from crude cell extracts have developed into tools for gene expression, with applications in prototyping, biosensing, and protein production. Key to the development of these systems is optimization of cell extract preparation methods. However, the applied nature of these optimizations often limits investigation into the complex nature of the extracts themselves, which contain thousands of proteins and reaction networks with hundreds of metabolites. Here, we sought to uncover the black box of proteins and metabolites in Escherichia coli cell-free reactions based on different extract preparation methods. We assess changes in transcription and translation activity from σ⁷⁰ promoters in extracts prepared with acetate or glutamate buffer and the common post-lysis processing steps of a runoff incubation and dialysis. We then utilize proteomic and metabolomic analyses to uncover potential mechanisms behind these changes in gene expression, highlighting the impact of cold shock-like proteins and the role of buffer composition.

Original language	English (US)
Pages (from-to)	405-418
Number of pages	14
Journal	ACS synthetic biology
Volume	12
Issue number	2
DOIs	https://doi.org/10.1021/acssynbio.2c00339
State	Published - Feb 17 2023

Funding

The authors thank Adam Silverman for helpful conversations about experiments and the literature. This work was funded by the U.S. Department of Energy Office of Science, Biological and Environmental Research Division (BER) Genomic Science Program (GSP) under Contract no. DE-SC0018249. B.J.R. was supported by a National Defense Science and Engineering Graduate Fellowship (Award ND-CEN-017-095). G.A.R. was supported by the National Science Graduate Research Fellowship Program under Grant no. DGE-1842165. M.C.J. gratefully acknowledges the David and Lucile Packard Foundation and the Camille Dreyfus Teacher-Scholar Program. This manuscript has been co-authored by UT-Battelle, LLC under Contract no. DE-AC05-00OR22725 with the U.S. Department of Energy. The authors thank Adam Silverman for helpful conversations about experiments and the literature. This work was funded by the U.S. Department of Energy Office of Science, Biological and Environmental Research Division (BER) Genomic Science Program (GSP) under Contract no. DE-SC0018249. B.J.R. was supported by a National Defense Science and Engineering Graduate Fellowship (Award ND-CEN-017-095). G.A.R. was supported by the National Science Graduate Research Fellowship Program under Grant no. DGE-1842165. M.C.J. gratefully acknowledges the David and Lucile Packard Foundation and the Camille Dreyfus Teacher–Scholar Program. This manuscript has been co-authored by UT-Battelle, LLC under Contract no. DE-AC05-00OR22725 with the U.S. Department of Energy.

Keywords

TX-TL
cell-free protein synthesis
in vitro
metabolomics
proteomics

ASJC Scopus subject areas

Biomedical Engineering
Biochemistry, Genetics and Molecular Biology (miscellaneous)

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10.1021/acssynbio.2c00339

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title = "Mechanistic Insights into Cell-Free Gene Expression through an Integrated -Omics Analysis of Extract Processing Methods",

abstract = "Cell-free systems derived from crude cell extracts have developed into tools for gene expression, with applications in prototyping, biosensing, and protein production. Key to the development of these systems is optimization of cell extract preparation methods. However, the applied nature of these optimizations often limits investigation into the complex nature of the extracts themselves, which contain thousands of proteins and reaction networks with hundreds of metabolites. Here, we sought to uncover the black box of proteins and metabolites in Escherichia coli cell-free reactions based on different extract preparation methods. We assess changes in transcription and translation activity from σ70 promoters in extracts prepared with acetate or glutamate buffer and the common post-lysis processing steps of a runoff incubation and dialysis. We then utilize proteomic and metabolomic analyses to uncover potential mechanisms behind these changes in gene expression, highlighting the impact of cold shock-like proteins and the role of buffer composition.",

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AU - Engle, Nancy L.

AU - Tschaplinski, Timothy J.

AU - Karim, Ashty S.

AU - Hettich, Robert L.

AU - Jewett, Michael C.

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AB - Cell-free systems derived from crude cell extracts have developed into tools for gene expression, with applications in prototyping, biosensing, and protein production. Key to the development of these systems is optimization of cell extract preparation methods. However, the applied nature of these optimizations often limits investigation into the complex nature of the extracts themselves, which contain thousands of proteins and reaction networks with hundreds of metabolites. Here, we sought to uncover the black box of proteins and metabolites in Escherichia coli cell-free reactions based on different extract preparation methods. We assess changes in transcription and translation activity from σ70 promoters in extracts prepared with acetate or glutamate buffer and the common post-lysis processing steps of a runoff incubation and dialysis. We then utilize proteomic and metabolomic analyses to uncover potential mechanisms behind these changes in gene expression, highlighting the impact of cold shock-like proteins and the role of buffer composition.

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Mechanistic Insights into Cell-Free Gene Expression through an Integrated -Omics Analysis of Extract Processing Methods

Abstract

Funding

Keywords

ASJC Scopus subject areas

Access to Document

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