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 remain a minor player in the recent synthetic biology-driven transformation in bioproduction. Molecular tools and technologies for complex, multigene engineering in plants are as yet limited, with the challenge to enhance their 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, which contain a fusion origin of replication that enhances plasmid yield in both Escherichia coli and Rhizobium radiobacter. MAPS includes a new library of promoters and terminators with different activity levels; part sizes were minimized to improve construct stability and transformation efficiency. These promoters and terminators were characterized using a high-throughput protoplast expression assay. We observed a significant influence of terminators on gene expression, as the strength of a single promoter can change more than seven-folds in combination with different terminators. Changing the coding sequence changed the relative strength of promoter and terminator pairs, thus uncovering combinatorial gene regulation among all parts of a transcriptional unit. We further gained insights into the mechanisms of such interactions by analyzing RNA folding, with which we suggest a design principle for more predictive and context-independent genetic parts in synthetic biology of plant systems and beyond.