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

Research output: Contribution to journalArticlepeer-review


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 languageEnglish (US)
Pages (from-to)405-418
Number of pages14
JournalACS synthetic biology
Issue number2
StatePublished - Feb 17 2023


  • 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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