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

Published

August 10, 2025

Author(s)

Fernanda Piorino Macruz de Oliveira, Chad Alan Sundberg, Elizabeth Strychalski, Eugenia Romantseva

Abstract

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 over the course of a CFE reaction and enable the development of predictable and reproducible CFE technologies. To date, comprehensive characterization of these systems has posed a formidable metrology challenge, as it requires time-course measurements of extraneous reactions involving endogenous components in addition to transcription and translation of a target gene added exogenously to the CFE reaction. Seeking to provide more informative characterization that is still easy to conduct and complements current quality control, we present a measurement framework for transcription and translation dynamics. We use different nucleic acid templates to characterize a suite of extracts prepared in house, as well as extracts and reconstituted systems available commercially. Notably, we include measurements of low-performing systems to probe 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 practices, provide informative data for developing CFE technologies, and be adopted for routine characterization.

Citation

ACS Synthetic Biology

Pub Type

Journals

Keywords

cell-free expression systems, transcription, translation, characterization, metrology

Citation

Piorino Macruz de Oliveira, F. , Sundberg, C. , Strychalski, E. and Romantseva, E. (2025), Characterizing Cell-Free Transcription and Translation Dynamics with Nucleic Acid-Based Assays, ACS Synthetic Biology (Accessed September 24, 2025)