Plant biotechnology predominantly relies on a restricted set of genetic parts with limited capability to customize spatiotemporal and conditional expression patterns. Synthetic gene circuits have the potential to integrate multiple customizable input signals through a processing unit constructed from biological parts to produce a predictable and programmable output. Here we present a suite of functional recombinase-based gene circuits for use in plants. We first established a range of key gene circuit components compatible with plant cell functionality. We then used these to develop a range of operational logic gates using the identify function (activation) and negation function (repression) in Arabidopsis protoplasts and in vivo, demonstrating their utility for programmable manipulation of transcriptional activity in a complex multicellular organism. Specifically, using recombinases and plant control elements, we activated transgenes in YES, OR and AND gates and repressed them in NOT, NOR and NAND gates; we also implemented the A NIMPLY B gate that combines activation and repression. Through use of genetic recombination, these circuits create stable long-term changes in expression and recording of past stimuli. This highly compact programmable gene circuit platform provides new capabilities for engineering sophisticated transcriptional programs and previously unrealized traits into plants.
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All raw values of data presented in this study, output of statistical tests and summaries (including central tendency and variation), list of primer sequences and the sequences of the plasmids used in this study are available in a Zenodo repository64 with the identifier 10.5281/zenodo.6381286.
R code used to perform statistical tests and generate plots is available in a Zenodo repository64 with the identifier 10.5281/zenodo.6381286.
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We would like to thank B. Johnston for advice on R code; M. Oliva and D. Collings for help with confocal image analysis; I. Roux for suggestion of the in vitro recombinase assay; Y.-H. Chooi for the kind donation of purified recombinant Cre enzyme; B. Crawford for the kind donation of a pOp6 promoter-containing plasmid; C. Helliwell for graciously providing the LhGR seeds used in this study; and W. Wong for generous donation of mammalian BLADE plasmids. The plasmid sequencing data were generated on instrumentation supported by the Australian Cancer Research Foundation Centre for Advanced Cancer Genomics and Genomics WA. This work was supported by the following grants to R.L.: Australian Research Council (ARC) Centre of Excellence in Plant Energy Biology (CE140100008), ARC DP210103954, NHMRC Investigator Grant GNT1178460, Silvia and Charles Viertel Senior Medical Research Fellowship and Howard Hughes Medical Institute International Research Scholarship. T.S. was supported by the Hackett Postgraduate Research Scholarship. M.A.K. was supported by an International Postgraduate Research Scholarship. B.K. was supported by the CSIRO Synthetic Biology Future Science Platform.
The authors declare no competing interests.
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Lloyd, J.P.B., Ly, F., Gong, P. et al. Synthetic memory circuits for stable cell reprogramming in plants. Nat Biotechnol (2022). https://doi.org/10.1038/s41587-022-01383-2