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    A Temperature-Controlled Cell-Free Expression System by Dynamic Repressor
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    • Junzhu Yang
      Junzhu Yang
      Key Laboratory of Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
      More by Junzhu Yang
    • Chen Wang
      Chen Wang
      Key Laboratory of Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
      More by Chen Wang
    • Yuan Lu*
      Yuan Lu
      Key Laboratory of Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
      *Email: [email protected]
      More by Yuan Lu
      Orcidhttps://orcid.org/0000-0003-4500-6230
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    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2022, 11, 4, 1408–1416
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    https://doi.org/10.1021/acssynbio.1c00641
    Published March 23, 2022
    Copyright © 2022 American Chemical Society
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    Abstract

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    The cell-free protein synthesis (CFPS) system is a typical protein production platform in the field of synthetic biology. However, there are limitations in controlling protein synthesis in the CFPS system. Compared with the traditional method of adding chemicals, temperature is an ideal control switch to achieve precise spatiotemporal control with few side effects. Hence, the design of a temperature-controlled cell-free protein synthesis (tcCFPS) system based on E. coli was carried out with the repressor cI protein in this study. The corresponding tcCFPS achieved a 143-fold dynamic protein expression level at 37 °C than that at 30 °C. Besides, the artificial cell controlled by temperature was constructed to expand the applications of tcCFPS. This study provides a new effective method for active protein synthesis in a cell-free system and a potential means of drug synthesis and delivery.

    Copyright © 2022 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.1c00641.

    • Preparation of cell extract; Inner and outer solution preparation for artificial cell system; DNA part sequence; The feature of cell extracts in the research; Information of promoter, RBS and part gene sequence; Introduction of all plasmids used in the research; The expression of two temperature-controlled plasmids in cell system; The fluorescent background value of tcCFPS; The expression of tcCFPS using the Origami (DE3) cell extract; The expression of tcCFPS with the addition of purified cI protein; Western blotting analysis for cI-BS cell extract; Determining the concentration of cI proteins; Mean fluorescence change curve of pcI at different temperatures; The standard curve of cI DNA and GFP DNA concentration and the CT value; Confocal imaging of liposome without the pcI in different glucose concentration; The correlation between FSC and SSC (PDF)

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    Cited By

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    This article is cited by 7 publications.

    1. Pao-Wan Lee, Sebastian J. Maerkl. Regulatory Components for Bacterial Cell-Free Systems Engineering. ACS Synthetic Biology 2024, 13 (12) , 3827-3841. https://doi.org/10.1021/acssynbio.4c00574
    2. Carolina Monck, Yuval Elani, Francesca Ceroni. Genetically programmed synthetic cells for thermo-responsive protein synthesis and cargo release. Nature Chemical Biology 2024, 20 (10) , 1380-1386. https://doi.org/10.1038/s41589-024-01673-7
    3. Junzhu Yang, Chi-Kit Sou, Yuan Lu. Cell-free biocatalysis coupled with photo-catalysis and electro-catalysis: Efficient CO2-to-chemical conversion. Green Energy & Environment 2024, 9 (9) , 1366-1383. https://doi.org/10.1016/j.gee.2023.10.002
    4. Sónia Siquenique, Shanny Ackerman, Avi Schroeder, Bruno Sarmento. Bioengineering lipid-based synthetic cells for therapeutic protein delivery. Trends in Biotechnology 2024, 14 https://doi.org/10.1016/j.tibtech.2024.08.004
    5. Xiaowen Cheng, Chen Wang, Junzhu Yang, Dong Liu, Yuting Liao, Bin Wang, Sanyang Han, Xue Zhang, Hao Zheng, Yuan Lu. Nanotransducer‐Enabled Wireless Spatiotemporal Tuning of Engineered Bacteria in Bumblebee. Small 2023, 19 (36) https://doi.org/10.1002/smll.202301064
    6. Fanqi Ma, Haiyan Liu, Feng Shi, Youhe Xiang, Zhengyu Fan. Quorum sensing-mediated dynamic regulation of 4-hydroxyisoleucine biosynthesis in Corynebacterium glutamicum. World Journal of Microbiology and Biotechnology 2023, 39 (7) https://doi.org/10.1007/s11274-023-03633-0
    7. Ying-Chen Yi, Shih-I Tan, Ruei-En Hu, Chuan-Chieh Hsiang, Jia-Yi Lin, Sefli Sri Wahyu Effendi, I-Son Ng. Exploring temperature-mediated plasmid replication as a reversible and switchable protein expression system in genetic Escherichia coli. Journal of the Taiwan Institute of Chemical Engineers 2023, 144 , 104751. https://doi.org/10.1016/j.jtice.2023.104751
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    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2022, 11, 4, 1408–1416
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acssynbio.1c00641
    Published March 23, 2022
    Copyright © 2022 American Chemical Society
    Request reuse permissions

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