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A Suite of Constitutive Promoters for Tuning Gene Expression in Plants

  • Andy Zhou
    Andy Zhou
    Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California 94720, United States
    Feedstocks Division, Joint BioEnergy Institute, Emeryville, California 94608, United States
    Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California 94705, United States
    More by Andy Zhou
  • Liam D. Kirkpatrick
    Liam D. Kirkpatrick
    Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California 94720, United States
    Feedstocks Division, Joint BioEnergy Institute, Emeryville, California 94608, United States
    Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California 94705, United States
  • Izaiah J. Ornelas
    Izaiah J. Ornelas
    Feedstocks Division, Joint BioEnergy Institute, Emeryville, California 94608, United States
    Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California 94705, United States
    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, United States
  • Lorenzo J. Washington
    Lorenzo J. Washington
    Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California 94720, United States
    Feedstocks Division, Joint BioEnergy Institute, Emeryville, California 94608, United States
    Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California 94705, United States
  • Niklas F. C. Hummel
    Niklas F. C. Hummel
    Feedstocks Division, Joint BioEnergy Institute, Emeryville, California 94608, United States
    Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California 94705, United States
  • Christopher W. Gee
    Christopher W. Gee
    Feedstocks Division, Joint BioEnergy Institute, Emeryville, California 94608, United States
    Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California 94705, United States
  • Sophia N. Tang
    Sophia N. Tang
    Feedstocks Division, Joint BioEnergy Institute, Emeryville, California 94608, United States
    Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California 94705, United States
    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, United States
  • Collin R. Barnum
    Collin R. Barnum
    Biochemistry, Molecular, Cellular and Developmental Biology Graduate Group, University of California, Davis, California 95616, United States
  • Henrik V. Scheller
    Henrik V. Scheller
    Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California 94720, United States
    Feedstocks Division, Joint BioEnergy Institute, Emeryville, California 94608, United States
    Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California 94705, United States
  • , and 
  • Patrick M. Shih*
    Patrick M. Shih
    Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California 94720, United States
    Feedstocks Division, Joint BioEnergy Institute, Emeryville, California 94608, United States
    Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California 94705, United States
    Innovative Genomics Institute, University of California, Berkeley, California 94720, United States
    *Email: [email protected]
Cite this: ACS Synth. Biol. 2023, 12, 5, 1533–1545
Publication Date (Web):April 21, 2023
https://doi.org/10.1021/acssynbio.3c00075
Copyright © 2023 American Chemical Society

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    Abstract

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    The need for convenient tools to express transgenes over a large dynamic range is pervasive throughout plant synthetic biology; however, current efforts are largely limited by the heavy reliance on a small set of strong promoters, precluding more nuanced and refined engineering endeavors in planta. To address this technical gap, we characterize a suite of constitutive promoters that span a wide range of transcriptional levels and develop a GoldenGate-based plasmid toolkit named PCONS, optimized for versatile cloning and rapid testing of transgene expression at varying strengths. We demonstrate how easy access to a stepwise gradient of expression levels can be used for optimizing synthetic transcriptional systems and the production of small molecules in planta. We also systematically investigate the potential of using PCONS as an internal standard in plant biology experimental design, establishing the best practices for signal normalization in experiments. Although our library has primarily been developed for optimizing expression in N. benthamiana, we demonstrate the translatability of our promoters across distantly related species using a multiplexed reporter assay with barcoded transcripts. Our findings showcase the advantages of the PCONS library as an invaluable toolkit for plant synthetic biology.

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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.3c00075.

    • Pairwise Pearson correlation of mRNA abundance between chosen samples to enrich for PCONS promoters (PDF)

    • Table S1: PCONS sequences; Table S2: primer sequences; Table S3: samples used for PCONS; Table S4: plasmid information (XLSX)

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    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

    Cited By

    This article is cited by 1 publications.

    1. Tasmia Islam, Swapna Kalkar, Rachel Tinker-Kulberg, Tetyana Ignatova, Eric A. Josephs. The “Duckweed Dip”: Aquatic Spirodela polyrhiza Plants Can Efficiently Uptake Dissolved, DNA-Wrapped Carbon Nanotubes from Their Environment for Transient Gene Expression. ACS Synthetic Biology 2024, 13 (2) , 687-691. https://doi.org/10.1021/acssynbio.3c00620

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