DNA regulatory elements are fundamental parts for genetic circuit design. The generation and characterization of promoter libraries can greatly facilitate fine tuning of gene expression within a circuit. We have used DNase-Seq (Meyer and Liu 2014) to identify candidate regulatory elements (>50,000) controlling cell-preferential gene expression within maize leaves (Burgess and Reyna-Llorens; unpublished data). Current validation techniques involve fusing sequences to a reporter and analysing expression in planta, which requires testing each element in an individual plant, either through transient biolistic transformation or the generation of stable transgenics (Brown et al. 2011). Applying these techniques to whole regulatory element libraries is not feasible at a laboratory scale.
This project aims to develop a high-throughput screen for the analysis of promoter sequences driving expression of a reporter gene in plant protoplasts. Protoplasts will be transformed by plasmid reporter constructs consisting of a regulatory region fused to a minimal promoter and a fluorescent protein. The expression level of fluorescent reporter within each protoplast will be questioned by laser in situ excitation in the sorting microfluidic device which will be also used to separate out individual protoplasts. Protoplasts will be sorted into two pools according to a user-specified threshold of fluorescence intensity (Abalde-Cela et al. 2015). In analysing a library of elements, this procedure will be performed iteratively at ever decreasing thresholds of fluorescence intensity to sort regulatory elements by their promoter activity. To determine which regulatory regions are present in each fraction, DNA will be extracted and sequenced using Illumina technology for each pool. We envisage this device to be applicable to a range of plant species and cell types, and by coupling it to libraries of regulatory elements identified by DNase-Seq, it will rapidly increase the rate of identifying promoters for biotechnological applications.
Contract Research Staff, Department of Plant Sciences
Graduate Student, Department of Plant Sciences
PhD Candidate in Plant Synthetic Biology, Department of Plant Sciences
Postdoctoral Research Associate (PDRA) at Microdroplets Group, Department of Chemistry
Research Associate, Laboratory for Scientific Computing