Mobius Assembly for Plant Systems highlights promoter-coding sequences-terminator interaction in gene regulation

Abstract 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.