Biology subjects

Sundberg, C. A.

Publications and source records attributed to Sundberg, C. A..

2 recordsLinked to original sources

bioRxiv2025

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.

synthetic biology↗

bioRxiv2025

Design-driven optimization of low-cost reagent formulations for reproducible and high-yielding cell-free gene expression

Access to recombinant proteins is vital in basic science and biotechnology research. Cell-free gene expression systems provide one approach to address this need, but widespread utilization remains limited by the cost, complexity, and inconsistency of current platforms. To address these limitations, we carry out a multi-dimensional definitive screening design to reduce the number of reagent components and remove costly secondary energy substrates. From more than 1,200 reagent formulations, we discover a simple and reproducible system based on 12 components. The optimized reagent formulation can produce 2.4 {+/-} 0.3 g/L of protein product at the 15-L scale ([~]$55/gprotein) and 3.7 {+/-} 0.2 g/L ([~]$36/gprotein) at the 4-mL scale with oxygen supplementation. This provides an 84 to 99% reduction in cost over previous cell-free reagent formulations. We further show that the optimized reagent formulation can produce nucleoside triphosphates from nucleotides and ribose and that it is robust to failure across batches of cell lysates, users/locations, and in the synthesis of different proteins. Specifically, we demonstrate the production of fifteen therapeutically relevant products, including full-length aglycosylated monoclonal antibodies. We anticipate that our optimized reagent formulation will further democratize the use of cell-free systems for protein manufacturing and synthetic biology applications.

synthetic biology↗