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Simple Extract Preparation Methods for E. coli-Based Cell-Free Expression

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Cell-Free Gene Expression

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

Abstract

Cell-free protein synthesis (CFPS) is a powerful platform for synthetic biology, allowing for the controlled expression of proteins without reliance on living cells. However, the process of producing the cell extract, a key component of cell-free reactions, can be a bottleneck for new users to adopt CFPS as it requires technical knowledge and significant researcher oversight. Here, we provide a detailed method for implementing a simplified cell extract preparation workflow using CFAI media. We also provide a detailed protocol for the alternative, 2x YPTG media-based preparation process, as it represents a useful benchmark within the cell-free community.

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Acknowledgments

Special thanks to our lab mates, Philip Smith and Byungcheol So for their support and feedback. All figures were created with BioRender.com. This work was supported by the Bill and Linda Frost Fund, Center for Applications in Biotechnology’s Chevron Biotechnology Applied Research Endowment Grant, and the National Science Foundation (NSF-1708919).

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, CA, USA

    Alissa C. Mullin, Taylor Slouka & Javin P. Oza

Authors
  1. Alissa C. Mullin
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  2. Taylor Slouka
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  3. Javin P. Oza
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Corresponding author

Correspondence to Javin P. Oza .

Editor information

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

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Mullin, A.C., Slouka, T., Oza, J.P. (2022). Simple Extract Preparation Methods for E. coli-Based Cell-Free Expression. 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_2

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  • DOI: https://doi.org/10.1007/978-1-0716-1998-8_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1997-1

  • Online ISBN: 978-1-0716-1998-8

  • eBook Packages: Springer Protocols

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