Best practices for DNA template preparation towards improved reproducibility in cell-free protein production

Published

January 5, 2022

Author(s)

Eugenia Romantseva, Drew S. Tack, Nina Alperovich, David Ross, Elizabeth Strychalski

Abstract

Cell-free systems are increasingly used for in vitro protein production and offer a promising alternative to traditional, cell-based biomanufacturing[1-6]. At the state-of-the-art, cell-free systems can provide a high-throughput platform for on-demand biomanufacturing of therapeutics[3,7-9], antibodies[10-13], antimicrobials[1,14], and vaccines[15-18]. Additionally, cell-free systems can accelerate the design-build-test-learn cycle in engineering biology workflows through rapid prototyping of genetic circuits[19-26], expansion of the genetic toolbox[27-34], and development and optimization of biosensors[35-38]. Performance variability is a common challenge in cell-free protein production and hinders a wider adoption of these systems for both research and biomanufacturing. While the inherent stochasticity and complexity of biology likely contributes to variability, other systematic factors may also play a role, including the source and preparation of the cell extract, the composition of the supplemental reaction buffer, the facility at which experiments are conducted, and the human operator[39]. Variability in protein production could also arise from differences in the DNA template—specifically the amount of functional DNA added to a cell-free reaction and the quality of the DNA preparation in terms of contaminants and strand breakage. Here, we present protocols and suggest best practices optimized for DNA template preparation and quantitation for cell-free systems towards reducing variability in cell-free protein production.

Citation

Methods in Molecular Biology

Pub Type

Journals

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Keywords

Cell-free systems, cell-free protein production, TX-TL, DNA template preparation, automated DNA extraction, automated DNA purification

Citation

Romantseva, E. , Tack, D. , Alperovich, N. , Ross, D. and Strychalski, E. (2022), Best practices for DNA template preparation towards improved reproducibility in cell-free protein production, Methods in Molecular Biology, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=931675 (Accessed October 12, 2025)