Building the Plant SynBio Toolbox through Combinatorial Analysis of DNA Regulatory Elements

While the installation of complex genetic circuits in microorganisms is relatively routine, the synthetic biology toolbox is severely limited in plants. Of particular concern is the absence of combinatorial analysis of regulatory elements, the long design-build-test cycles associated with transgenic plant analysis, and a lack of naming standardization for cloning parts. Here, we use previously described plant regulatory elements to design, build, and test 91 transgene cassettes for relative expression strength. Constructs were transiently transfected into Nicotiana benthamiana leaves and expression of a fluorescent reporter was measured from plant canopies, leaves, and protoplasts isolated from transfected plants. As anticipated, a dynamic level of expression was achieved from the library, ranging from near undetectable for the weakest cassette to a similar to 200-fold increase for the strongest. Analysis of expression levels in plant canopies, individual leaves, and protoplasts were correlated, indicating that any of the methods could be used to evaluate regulatory elements in plants. Through this effort, a well-curated 37-member part library of plant regulatory elements was characterized, providing the necessary data to standardize construct design for precision metabolic engineering in plants.

Automated summary

While installation of complex genetic circuits in microorganisms is routine, synthetic biology toolbox faces limitations in plants due to lack of combinatorial analysis, long design-build-test cycles, and naming standardization. This study designed, built, and tested 91 transgene cassettes using plant regulatory elements, showing different expression strengths in plant tissues with high correlation between canopies, individual leaves, and protoplasts. The efforts led to characterization of a 37-member plant regulatory element library, providing data for standardized construct design in plant metabolic engineering.