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Published October 15, 2021 | Supplemental Material
Journal Article Open

A Ratiometric Dual Color Luciferase Reporter for Fast Characterization of Transcriptional Regulatory Elements in Plants

Abstract

Plant synthetic biology requires precise characterization of genetic elements to construct complex genetic circuits that can improve plant traits or confer them with new characteristics. Transcriptional reporter assays are essential to quantify the effect of gene expression regulator elements. Additionally, transcriptional reporter systems are a key tool in understanding control of gene expression in biology. In this work, we construct and characterize a dual color luciferase ratiometric reporter system that possesses several advantages over currently used reporters. It is ratiometric, thus reducing variability and increasing consistency between experiments; it is fast, as both reporters can be measured at the same time in a single reaction, and it is less expensive to perform than current dual luciferase reporter assays. We have validated our system quantifying the transcriptional capability of a panel of promoters and terminators commonly used in synthetic biology with a broad range of expression magnitudes, and in a biologically relevant system, nitrate response.

Additional Information

© 2021 American Chemical Society. Received 18 June 2021. Published online 14 September 2021. Published in issue 15 October 2021. M.P.L. acknowledges support of a USDA BBT EAGER award (2019-67013-29104) and a USDA NIFA award (2018-67021-27964). G.S.D. is supported by the Resnick Sustainability Institute Postdoctoral Fellowship, and G.S.D. and S.M.B. acknowledge support of a USDA BBT-EAGER award (2019-67013-29012), and partial support by an HHMI Faculty Scholar fellowship. pRedF was a gift from Koen Venken (Addgene #118057). Author Contributions. E.G. and M.P.L. conceived the project and wrote the manuscript. E.G., G.S.D., S.M.B., and M.P.L. designed the experiments. E.G. performed the experiments and data analysis. E.G. and W.M. prepared the plasmids. G.S.D. performed the nitrate experiments. All authors have edited and commented on the manuscript and have given their approval of the final version. The authors declare no competing financial interest.

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Created:
August 22, 2023
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October 23, 2023