RA3 will utilize synthetic biology approaches to reconstruct plant signaling and metabolic pathways in orthogonal systems and obtain a quantitative understanding of pathway architectures within and between cells and their surroundings. These quantitative studies are technically challenging to perform on a cellular level in planta due to the molecular complexity of highly interconnected, often multicellular signaling and metabolic networks and pleiotropism. Reconstruction of plant pathways in heterologous systems enables rapid genetic permutation and testing of genetic diversity of cellular components and their directed evolution on a scale/throughput rate that is not possible in planta. RA3 will implement novel genetic, molecular, and imaging tools and approaches such as synthetic switches, promoters, regulatory modules, dynamic and programmable synthetic gene networks, quantitative biosensors, microfluidics, and 2D/3D tissue printing to accelerate scientific progress. These data will be integrated with mathematical modeling (RA4) to complement and inform the in planta studies proposed in RA1 and RA2.

RA3 will transition from the reconstruction of individual pathways (molecular modules) via multiple pathways within a cell (cellular modules) to interactions of a cell with its surroundings (cell-interface modules). In the future, this work will be extended with the development and deployment of printed cell clusters, tissues, and organoids for the study of functional and architectural features emerging from signaling and metabolic networks.