Synthetic biology has advanced from the setup of basic genetic devices to the design of increasingly complex gene circuits to provide organisms with new functions. While many bacterial, fungal and mammalian unicellular chassis have been extensively engineered, this progress has been delayed in plants due to the lack of reliable DNA parts and devices that enable precise control over these new synthetic functions. In particular, memory switches based on DNA site-specific recombination have been the tool of choice to build long-term and stable synthetic memory in other organisms, because they enable a shift between two alternative states registering the information at the DNA level. Here we report a memory switch for whole plants based on the bacteriophage ϕC31 site-specific integrase. The switch was built as a modular device made of standard DNA parts, designed to control the transcriptional state (on or off) of two genes of interest by alternative inversion of a central DNA regulatory element. The state of the switch can be externally operated by action of the ϕC31 integrase (Int), and its recombination directionality factor (RDF). The kinetics, memory, and reversibility of the switch were extensively characterized in Nicotiana benthamiana plants.
Plant synthetic biology aims to contribute to global food security by engineering plants with new or improved functionalities. In recent years, synthetic biology has rapidly advanced from the setup of basic genetic devices to the design of increasingly complex gene circuits able to provide organisms with novel functions. While many bacterial, fungal and mammalian unicellular chassis have been extensively engineered, this progress has been delayed in plants due to their complex multicellular nature and the lack of reliable DNA devices that allow an accurate design of more sophisticated biological circuits. Among these basic devices, gene switches are crucial to deploying new layers of regulation into the engineered organisms. Of special interest are bistable genetic toggle switches, which allow a living organism to exist in two alternative states and switch between them with a minimal metabolic burden. Naturally occurring toggle switches control important decision-making processes such as cell fate and developmental events. We sought to engineer whole plants with an orthogonal genetic toggle switch to be able to regulate artificial functions with minimal interference with their natural pathways. Here we report a bistable toggle memory switch for whole plants based on the phage PhiC31 serine integrase and its cognate recombination directionality factor (RDF). This genetic device was designed to control the transcription of two genes of interest by inversion of a central DNA regulatory element. Each state of the device is defined by one transcriptionally active gene of interest, while the other one remains inactive. The state of the switch can be reversibly modified by the action of the recombination actuators, which were administered externally (e.g. via agroinfiltration), or produced internally in response to an inducible chemical stimulus. We extensively characterized the kinetics, memory, and reversibility of this genetic switch in Nicotiana benthamiana through transient and stable transformation experiments using transgenic plants and hairy roots. Finally, we coupled the integrase expression to an estradiol-inducible promoter as a proof of principle of inducible activation of the switch.
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