Title: Synthetic Biology and the Development of Synthetic Switches in Plants

Speaker: Prof. Ashok Prasad, Dept. of Chemical & Biological Engineering School of Biomedical Engineering Cell and Molecular Biology Program Molecular, Cellular and Integrative Neuroscience Colorado State University, Fort Collins, USA

Abstract: Synthetic biology is the engineering of biologically based or inspired systems that display functions that do not exist in nature. Though the genetic engineering techniques it is based on have a longer history, synthetic biology is often said to have taken off when the first synthetic gene regulatory systems were developed. These were a synthetic genetic switch and a genetic oscillator, that were built in E. coli in the year 2000. In the last two decades there has been a significant advance in the complexity of synthetic genetic systems that we can design for model microorganisms, as well as mammalian cells. However, 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. Plant synthetic biology is of great interest since it can potentially lead to sustainable green technologies for human needs. In this talk I will give a brief account of the achievements and challenges of synthetic biology and discuss our own work for the development of a synthetic genetic toggle switch in a plant, in collaboration with a plant biologist group at CSU. 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 (plant cells without the cell wall) 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.