Quantitative characterization of genetic parts and circuits for plant synthetic biology

Abstract

Plant synthetic biology promises immense technological benefits, including the potential development of a sustainable bio-based economy through the predictive design of synthetic gene circuits. Such circuits are built from quantitatively characterized genetic parts; however, this characterization is a significant obstacle in work with plants because of the time required for stable transformation. We describe a method for rapid quantitative characterization of genetic plant parts using transient expression in protoplasts and dual luciferase outputs. We observed experimental variability in transient-expression assays and developed a mathematical model to describe, as well as statistical normalization methods to account for, this variability, which allowed us to extract quantitative parameters. We characterized >120 synthetic parts in Arabidopsis and validated our method by comparing transient expression with expression in stably transformed plants. We also tested >100 synthetic parts in sorghum (Sorghum bicolor) protoplasts, and the results showed that our method works in diverse plant groups. Our approach enables the construction of tunable gene circuits in complex eukaryotic organisms.

Synthetic Botany

“…Protoplast transformation has been performed by electroporation (Fromm et al 1985;Ou-Lee et al 1986;Hauptmann et al 1987;Negrutiu et al 1987;Nishiguchi et al 1987;Jones et al 1989) or incubation in a PEG solution (Krens et al 1982;Potrykus et al 1985), and is established as one of the preferred methods for studying signaling pathways (reviewed in Sheen 2001). Furthermore, highthroughput protoplast transformation has been used to perform quantitative characterization of large libraries of genetic elements in Arabidopsis thaliana (Arabidopsis) and Sorghum bicolor (sorghum) (Schaumberg et al 2016).…”

Advancing Crop Transformation in the Era of Genome Editing

“…Most plant transformation tools have been developed on an ad hoc basis and not to rational standards that would facilitate design and assembly of larger synthetic biological circuits from individual parts or from quantitatively defined transfer functions (Schaumberg et al, 2016). Designing "mix-and-match" modular components (Liu et al, 2013;Liu and Stewart, 2015) for delivery of biological molecules might be a more useful strategy for plant biology researchers.…”

Plant synthetic biology for molecular engineering of signalling and development

“…Exogenous application may lead to a highly artificial spatial distribution of pathway activation, perturb the effect of endogenous hormones and provoke feedback regulation of endogenous hormone biosynthesis, signalling and transport pathways. Careful, multifaceted approaches that combine genetic tools, biosensors and mathematical modelling 57–59 could moderate the impact of these limitations.…”

“…1). New approaches for rapid prototyping of synthetic parts in plants 57 are key to developing the library of components needed to scale-up synthetic pathway engineering. Implementation of engineered networks in basal plant lineages may also be a means to accelerate the design–build–test engineering cycle, as these organisms often have smaller families of competing signalling components and more streamlined genome editing 58,60 .…”