Microdroplet assay development for metabolic engineering and synthetic biology applications

Abstract

Sustainable, efficient production of societally relevant chemicals in microorganisms is the epicenter of the field of metabolic engineering. Modern developments in DNA synthesis and sequencing have enabled high-information gene perturbation and protein engineering libraries that require reliable, reproducible, high throughput screening assays. Detailed herein are examples of utilizing growth-based assays and microfluidic screening to extract pertinent information from different gene perturbation libraries, microbial consortia, and engineered proteins. The first study details an approach to regulate gene expression in S. cerevisiae through utilization of a single-guide RNA library paired with dCas9 fused to either Mxi1 or VPR. Through these fusions and targets of guide RNAs, genes are either up or down regulated at increasing degrees. Using this library it was possible to identify target genes for moderate up and down regulation to improve growth on alternative carbon sources, namely glycerol and galactose. Final analysis of enriched guides via next generation sequencing identified moderate down regulation of essential genes, novel perturbations that would not have been isolated in traditional gene knock-out approaches. The studies detailed in chapters 3 and 4 delve into technical applications of microfluidics to screen for improved small molecule production. In chapter 2, the library detailed above is utilized to screen for gene targets that improve small molecule production in S. cerevisiae. This study also results in the development of a pico-injection microdroplet approach that utilizes cell-based biosensors to transduce small molecule production into a fluorescent signal. The study in chapter three characterizes this application further, ultimately leading identifying gene perturbations that improve early productivity or higher overall production, based on the time at which they were screened. In the fourth study, a previously developed CuAAC probe for extracellular electron transfer (EET) is ported into droplets to analyze an environmental microbial consortium. This study centers on the development of an oxygen-limited platform for anaerobic microbe cultivation in microdroplets, ultimately enriching environmental microorganisms previously uncharacterized for EET. This work expands the range of microorganisms compatible with this microdroplet system. In the final study, the microorganism array is expanded further through the development of a cytotoxicity assay for Sf9 insect cells is developed for use in microdroplets. This work lays a foundation for future applications to identify novel insecticidal toxins from engineered protein libraries. Collectively, these studies establish generalizable assays for high throughput screening of a wide array of organisms related to the field of synthetic biology.Biochemistr