21ENGBIO Genomic switches for carbon dot activated combinatorial plant engineering

Plants have the potential to contribute to solving many of the global societal challenges, from food security to climate change. They are capable of producing most of the materials that society needs, from food, fibres, materials to an astounding diversity of secondary metabolites, and do this while sequestering carbon. Synthetic Biology has the potential to exploit these abilities of plants and make this list even longer, an excellent example being the recent successful production of COVID vaccine in tobacco plants. However, the development of plants as a system that is as straightforward to genetically 'program' in the way that for example bacteria can be is held back due to the lack of ease and flexibility in delivering the nucleotide 'apps' into the biological 'hardware'. Plant transformation can be slow, inefficient and frequently not work at all in genetically recalcitrant plants.
Our project aims to develop new tools to overcome this by developing new ways of using cutting-edge nanomaterial-based plant transformation. Due to the chemical functionalisation, this nanomaterial delivery is fast, very simple and flexible (the nanomaterial-nucleotide complex can be sprayed onto plants) and can be used on most species of plant. This means that we have to potential to develop 'genetic switches' or even 'genetic apps' for plants. We aim to see if the new nanomaterial delivery method can be used to deliver 'spray-on' switches by using them in combination with 'pre-loaded software' (synthetic sequences stably integrated into the plant genome) that, when switched on changes the colour of the plant by producing a scarlet pigment that the switches will turn on or off. We also aim to see if both the switches and the genes they control can be delivered in tandem by nanomaterials. We hope that this combined approach will start to develop the technology needed to make programable plants that would than have an astounding array of possibilities for the application of engineering biology.

A recently emerged field is Plant Bionanotechnology, or Plant Bionics: the application of functional nanomaterials to plant biology, including the use of nanomaterials to deliver nucleotides (NTs) into plants. Several groups have shown that carbon nanomaterials are effective in delivering both RNA and DNA into plant cells. We have pioneered the development of a plant transformation system based around amine-terminated, PEG-functionalised fluorescent carbon dots (CDs) as carriers of genetic material (RNA, plasmid DNA or linear DNA). When functionalised with PEG chains, a positively charged CD is produced that interacts electrostatically with negatively charged nucleotides, resulting in the formation of a nanoplex. These nanoplexes are highly water soluble, and when applied in water to plants (via watering or foliar spray) have been startlingly effective at the efficient delivery of even sizable plasmids into mature, whole plants. CDs are therefore capable of forming the basis of a plant transient transformation system that is fast, flexible in its route of delivery and the range of nucleotides delivered, and can be utilised for wide ranging molecular methodologies including novel gene expression, gene knock out and gene editing. This flexibility makes it ideal to combine with recent advances in plant synthetic biology. We aim to take synthetic multi-gene pathways driven by an inverted 'switch' promoter that, when triggered result in clear phenotypes (such as the biosynthesis of scarlet betalains). 'Spray-on' CD nanoplexes will deliver plasmids expressing the integrase targeting the switch promoter and 'flip' the promoter to the correct orientation, resulting in the expression of the multi-gene pathway. This would be an initial, simple proof of concept, but would demonstrate the possibility of the range of interchangeable components that could be easily added into this flexible system.