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

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.