PhD Student North Carolina State University Durham, North Carolina
Body of Abstract: Understanding gene expression regulation is central to the development of biotechnological solutions for several pressing agricultural problems. The advent of synthetic biology methods has opened the door to programmable genetic constructs in plants that confer tunability and spatiotemporal regulation to gene expression. These tools can be applied in a variety of agricultural research projects to create customizable promoters that drive the expression of genes of interest. However, the relationship between promoter elements within the promoter of a gene and that gene’s expression levels is not well defined. To explore this relationship, we are building synthetic promoters harboring up to ten transcription factor binding sites using GoldenBraid technology and cloning these promoters upstream of a reporter gene. This is done by inserting one to ten copies of a protospacer, a 23bp recognition sequence for a dCas9-based synthetic transcription factor, into a 1kb neutral promoter sequence that has no known transcription factor binding sites in plants. We assemble the reporter gene to express an mCherry-Luciferase fusion protein as a readout and transiently co-express it along with a dCas9 activation system in Nicotiana benthamiana to test the effects of protospacer position, orientation, angular phase shift, copy number, and spacing on reporter expression. Preliminary results show the following patterns: constructs with protospacers in either the sense or antisense orientation confer comparable levels of gene expression, increasing protospacer copy number boosts reporter activity, and gene expression peaks when the protospacer is a particular distance away from the core promoter. This work is expected to shed light on the rules of nature dictating promoter architecture that, in turn, determine gene expression levels, ultimately paving the way to the creation of tunable expression systems. We will apply this knowledge to build stimulus-inducible constructs to confer hormone-regulated expression to genes of interest in Arabidopsis thaliana.