Quantitative characterization of genetic parts and circuits for plant synthetic biology

• Published in: Nature methods Vol. 13; no. 1; pp. 94 - 100
• Main Authors: Schaumberg, Katherine A, Antunes, Mauricio S, Kassaw, Tessema K, Xu, Wenlong, Zalewski, Christopher S, Medford, June I, Prasad, Ashok
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.01.2016; Nature Publishing Group
• Subjects: 38/77; 42; 42/44; 631/114/2114; 631/449/1659; 631/61; Arabidopsis; Arabidopsis thaliana; BASIC BIOLOGICAL SCIENCES; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical Engineering/Biotechnology; Chemical properties; Flowers & plants; Gene expression; Genetic aspects; genetic circuits; Genetic research; Life Sciences; Molecular biology; Plants - genetics; Proteomics; protoplasts; quantitative characterization; Sorghum; Sorghum bicolor; Stochastic Processes; Sustainable development; Synthetic biology; Synthetic Biology - methods; United States
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/nmeth.3659

Quantitative analysis of promoter-repressor pairs in plants will allow the design of predictable gene circuits in multicellular organisms. 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.