Mobius Assembly for Plant Systems uncovers combinatorial interactions among promoters, coding sequences, and terminators in gene regulation

Plants are the primary biological platforms for producing food, energy, and materials in agriculture; however, they are largely untouched by synthetic biology-driven transformation in bioproduction technologies. Molecular tools for complex, multigene engineering are as yet limited, with development underway to enhance stability and predictivity. Here, we present a new standardized and streamlined toolkit for plant synthetic biology, Mobius Assembly for Plant Systems (MAPS). It is based on small plant binary vectors (pMAPs) that contain a fusion origin of replication that enhances plasmid yield in both E. coli and Agrobacterium . MAPS includes a new library of promoters and terminators with different activity levels; the parts were made small in size to improve construct stability and transformation efficiency. These promoters and terminators were characterized using a high-throughput protoplast expression assay. Our findings show a significant influence of terminators on gene expression, as the strength of a promoter can change more than 7 folds with the different terminators. Additionally, we have observed that changing the coding sequence changes the relative strength of promoter and terminator pairs, uncovering combinatorial interactions among all parts of a transcriptional unit. We further gained insights into the mechanisms of such interactions by analyzing RNA folding. These results contribute to improving stability, predictability, and orthogonality in synthetic biology of plant systems and beyond.