Characterizing Cell-Free Transcription and Translation Dynamics with Nucleic Acid–Based Assays

Characterization of cell-free expression (CFE) systems
must expand
beyond single spectrophotometric measurements of a green fluorescent
protein to provide meaningful metrics of system performance during
a CFE reaction and enable the development of predictable and reproducible
CFE technologies. To date, comprehensive characterization of these
systems has posed a formidable measurement challenge, as it requires
time-course measurements of reactions involving endogenous components
in addition to transcription and translation of a target genetic circuit
added exogenously to the CFE reaction. To provide more informative
characterization that is still easy to conduct and complements current
practices, we demonstrate a measurement framework for transcription
and translation dynamics. We use different nucleic acid templates
to characterize a suite of Escherichia coli extracts prepared in-house, as well as extracts and reconstituted
systems available commercially. Notably, we include measurements of
low-performing systems to assess the sensitivity of our measurement
framework and elucidate metrics indicative of system performance.
For all these CFE systems, we compute reaction metrics to enable quantitative
comparison. We believe this is an accessible measurement framework
that can complement existing characterization, provide informative
data for developing CFE technologies, and be adopted for routine characterization.