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Cell Extracts from Bacteria and Yeast Retain Metabolic Activity after Extended Storage and Repeated Thawing
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    Cell Extracts from Bacteria and Yeast Retain Metabolic Activity after Extended Storage and Repeated Thawing
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    • Blake J. Rasor
      Blake J. Rasor
      Department of Chemical and Biological Engineering,  Chemistry of Life Processes Institute  and  Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States
    • Ashty S. Karim
      Ashty S. Karim
      Department of Chemical and Biological Engineering,  Chemistry of Life Processes Institute  and  Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States
    • Hal S. Alper
      Hal S. Alper
      Institute for Cellular and Molecular Biology  and  McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas 78712, United States
      More by Hal S. Alper
    • Michael C. Jewett*
      Michael C. Jewett
      Department of Chemical and Biological Engineering,  Chemistry of Life Processes Institute  and  Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States
      Robert H. Lurie Comprehensive Cancer Center  and  Simpson Querrey Institute, Northwestern University, Chicago, Illinois 60611, United States
      *E-mail: [email protected]
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    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2023, 12, 3, 904–908
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    https://doi.org/10.1021/acssynbio.2c00685
    Published February 27, 2023
    Copyright © 2023 American Chemical Society

    Abstract

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    Cell-free synthetic biology enables rapid prototyping of biological parts and synthesis of proteins or metabolites in the absence of cell growth constraints. Cell-free systems are frequently made from crude cell extracts, where composition and activity can vary significantly based on source strain, preparation and processing, reagents, and other considerations. This variability can cause extracts to be treated as black boxes for which empirical observations guide practical laboratory practices, including a hesitance to use dated or previously thawed extracts. To better understand the robustness of cell extracts over time, we assessed the activity of cell-free metabolism during storage. As a model, we studied conversion of glucose to 2,3-butanediol. We found that cell extracts from Escherichia coli and Saccharomyces cerevisiae subjected to an 18-month storage period and repeated freeze–thaw cycles retain consistent metabolic activity. This work gives users of cell-free systems a better understanding of the impacts of storage on extract behavior.

    Copyright © 2023 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.2c00685.

    • Materials and Methods, Supp. Figure S1 (representative time courses from reactions with E. coli extract that was stored for 3 to 18 months), Supp. Figure S2 (representative time courses from reactions with S. cerevisiae extract that was stored for 3 to 18 months), Supp. Figure S3 (reactions require higher concentration of E. coli extract for complete glucose consumption), Supp. Figure S4 (SDS-PAGE comparing wildtype and enriched cell extracts), Supp. Figure S5 (lyophilized reactions retain metabolic activity), Supp. Table S1 (relative metrics for E. coli extract performance over 18 months), and Supp. Table S2 (relative metrics for S. cerevisiae extract performance over 18 months) (PDF)

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    This article is cited by 2 publications.

    1. Hyeongwoo Park, Haneul Jin, Dayeong Kim, Joongoo Lee. Cell-Free Systems: Ideal Platforms for Accelerating the Discovery and Production of Peptide-Based Antibiotics. International Journal of Molecular Sciences 2024, 25 (16) , 9109. https://doi.org/10.3390/ijms25169109
    2. Fang Ba, Yufei Zhang, Xiangyang Ji, Wan‐Qiu Liu, Shengjie Ling, Jian Li. Expanding the toolbox of probiotic Escherichia coli Nissle 1917 for synthetic biology. Biotechnology Journal 2024, 19 (1) https://doi.org/10.1002/biot.202300327
    3. Fang Ba, Yufei Zhang, Xiangyang Ji, Wan-Qiu Liu, Shengjie Ling, Jian Li. Expanding the toolbox of probiotic Escherichia coli Nissle 1917 for synthetic biology. 2023https://doi.org/10.1101/2023.06.05.543671

    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2023, 12, 3, 904–908
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acssynbio.2c00685
    Published February 27, 2023
    Copyright © 2023 American Chemical Society

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