Article | Published:

Quantitative characterization of genetic parts and circuits for plant synthetic biology

Nature Methods volume 13, pages 94100 (2016) | Download Citation

Abstract

Plant synthetic biology promises immense technological benefits, including the potential development of a sustainable bio-based economy through the predictive design of synthetic gene circuits. Such circuits are built from quantitatively characterized genetic parts; however, this characterization is a significant obstacle in work with plants because of the time required for stable transformation. We describe a method for rapid quantitative characterization of genetic plant parts using transient expression in protoplasts and dual luciferase outputs. We observed experimental variability in transient-expression assays and developed a mathematical model to describe, as well as statistical normalization methods to account for, this variability, which allowed us to extract quantitative parameters. We characterized >120 synthetic parts in Arabidopsis and validated our method by comparing transient expression with expression in stably transformed plants. We also tested >100 synthetic parts in sorghum (Sorghum bicolor) protoplasts, and the results showed that our method works in diverse plant groups. Our approach enables the construction of tunable gene circuits in complex eukaryotic organisms.

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Acknowledgements

We thank D. McCarthy for valuable help in preparing the manuscript. Financial support from the US Department of Energy (Advanced Research Projects Agency–Energy 2012 grant DE-AR0000311) and the US Department of Defense (Defense Threat Reduction Agency grant W911NF-09-10526) is gratefully acknowledged.

Author information

Author notes

    • Katherine A Schaumberg
    •  & Mauricio S Antunes

    These authors contributed equally to this work.

Affiliations

  1. School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado, USA.

    • Katherine A Schaumberg
    •  & Ashok Prasad
  2. Department of Biology, Colorado State University, Fort Collins, Colorado, USA.

    • Mauricio S Antunes
    • , Tessema K Kassaw
    • , Christopher S Zalewski
    •  & June I Medford
  3. Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, Colorado, USA.

    • Wenlong Xu
    •  & Ashok Prasad

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Contributions

K.A.S. designed and performed experiments, as well as a significant part of the data analysis, and contributed to manuscript writing. M.S.A. designed and performed experiments and contributed to manuscript writing. T.K.K. engineered many of the constructs, designed and performed experiments and contributed to manuscript writing. C.S.Z. designed and performed experiments and contributed to manuscript writing. W.X. performed data analysis, developed the camera correction method and contributed to manuscript writing. J.I.M. designed experiments, contributed to manuscript writing and supervised the overall project. A.P. designed data analysis, supervised the computational part of the project, and contributed to and supervised the writing of the paper. All authors contributed to editing of the paper and read the final version.

Competing interests

J.I.M. is founder and president of the small startup company Phytodetectors, Inc.

Corresponding authors

Correspondence to June I Medford or Ashok Prasad.

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DOI

https://doi.org/10.1038/nmeth.3659