Biology

Prof. Ashok Prasad
Colorado State University

Synthetic biology can be said to have taken off when the first synthetic switch and oscillators were built in E. coli in 2000. Despite significant advances in model microorganisms, synthetic biology has not really advanced in more complicated multicellular organisms. In particular, despite a long history of plant genetic engineering, the complexity of plant biology appears to have prevented the development of synthetic gene circuits in plants. However plant synthetic biology is of great interest since it can potentially lead to sustainable green technologies for human needs. In this talk I will discuss the development of a synthetic genetic toggle switch in a plant. This project is a collaborative project between two research groups: a plant biologist group and my research group. A key step in the rational design of synthetic networks is the quantitative characterization of components to enable predictive modeling. However this poses special difficulties for plants, since stably transforming plants is time consuming and can take months. An alternative strategy is the use of transient protoplast assays for quantitative testing of components. We developed an experimental test bed for characterizing externally inducible repressors using protoplasts, but found that transient protoplast assays show significant experimental variability that makes direct quantitative comparisons yield incorrect results. We studied some of the sources of variability and developed a mathematical model to normalize the data and make quantitative comparisons between different inducible repressors. Despite some remaining uncertainties we showed that protoplast assays could approximately predict quantitative properties of synthetic circuits in stably transformed plants. We tested hundreds of repressible promoters, and carried out a statistical analysis of the quantitative data to uncover design principles for building synthetic inducible repressors in plants (Nature Methods, v13, pp94–100, (2016)). We used the quantitative characterization to select promoter-repressor pairs that could work together as a genetic toggle switch. Two different switches were constructed; plants stably transformed and then tested using a luciferase reporter. Quantitative analysis of the results confirms that one of them is a bistable genetic toggle switch, the first ever in a plant. Though challenges of achieving desirable design parameters and predictability remain, our work demonstrates that synthetic circuits that function in a predictable manner can be developed even for complex organisms that undergo sexual reproduction and go through several developmental stages.