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Best Practices for DNA Template Preparation Toward Improved Reproducibility in Cell-Free Protein Production

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Cell-Free Gene Expression

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2433))

Abstract

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 (Cole et al. ACS Synth Biol 8:2080–2091, 2019). 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 toward reducing variability in cell-free protein production.

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Authors and Affiliations

  1. National Institute of Standards and Technology, Gaithersburg, MD, USA

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

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  1. Eugenia F. Romantseva
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  2. Drew S. Tack
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  3. Nina Alperovich
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  4. David Ross
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  5. Elizabeth A. Strychalski
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Correspondence to Eugenia F. Romantseva .

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Editors and Affiliations

  1. Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, and Center for Synthetic Biology, Northwestern University, Evanston, IL, USA

    Ashty S. Karim

  2. Department of Chemical and Biological Engineering, Chemistry of Life Processes Institute, Center for Synthetic Biology, Robert H. Lurie Comprehensive Cancer Center, and Simpson Querrey Institute, Northwestern University, Evanston, IL, USA

    Michael C. Jewett

1 Electronic Supplementary Materials

Supplementary File 1

Excel template for building a 6-point eGFP calibration curve. The volumes of eGFP and 1× PBS are determined from the measured concentration of the stock recombinant eGFP for triplicate measurements of the calibration curve in three locations in a 384-well plate. (XLSX 12 kb)

Supplementary File 2

Excel template for assembling cell-free reactions. The volumes of DNA and nuclease-free water are determined based on the measured concentration of the prepared solution of DNA and the minimal recommended DNA concentration of 5 nmol/L per sample, for triplicate measurements of each cell-free reaction in a 384-well plate. (XLSX 13 kb)

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Romantseva, E.F., Tack, D.S., Alperovich, N., Ross, D., Strychalski, E.A. (2022). Best Practices for DNA Template Preparation Toward Improved Reproducibility in Cell-Free Protein Production. In: Karim, A.S., Jewett, M.C. (eds) Cell-Free Gene Expression. Methods in Molecular Biology, vol 2433. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1998-8_1

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  • DOI: https://doi.org/10.1007/978-1-0716-1998-8_1

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1997-1

  • Online ISBN: 978-1-0716-1998-8

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