Shamoo, Yousif2021-12-062022-12-012021-122021-12-03December 2Ganiga Prabhakar, Ramya. "Dynamics of a Synthetic Microbial Community in a Structured Droplet Environment." (2021) Diss., Rice University. <a href="https://hdl.handle.net/1911/111763">https://hdl.handle.net/1911/111763</a>.https://hdl.handle.net/1911/111763In nature, microbes live in multispecies communities where they compete for nutrients within a localized and sometimes structured environment. Outcomes of such microbial interactions are dynamic and can vary in response to environmental cues. To study community dynamics in laboratory conditions, the choice of culturing environments is therefore crucial. Studying microbial interactions using suspension cultures is challenging because it favors fast-growers over high-yielding cells. To this end, we developed emulsion droplets as a structured culturing method to investigate the population dynamics of a synthetic microbial community. We also developed a mathematical model to provide insights into the key parameters shaping the structure and function of such communities in droplets. In the first part of this project, to encapsulate competitive sub-communities we built microfluidic devices to generate water-in-oil droplets as discrete, picolitre-volume culture vessels. The ideal population sizes and culturing conditions for optimum bacterial growth in droplets were determined. We also engineered a microfluidic module for efficient droplet sorting based on fluorescence. Next, we test the dynamics of a synthetic microbial community within droplets. Here, we have rationally engineered competitive interactions among Producer, Non-producer, and the sensitive Receivers in Escherichia coli to study competitive outcomes in a well-mixed environment versus spatially structured conditions. The Producer strain kills a competing Receiver strain, mediated through secreted small molecules. The Non-producer strain is a phenotypic (and genotypic) variant of the Producer that cannot kill the Recipient strain as the gene for metabolite production is disrupted. We found that the secreted molecules diffuse between the droplets and therefore the droplets afford confinement of only the cells but not the secreted metabolite. Even in this condition, by encapsulating the competitive co-cultures in droplets, we successfully enriched the Producers from a large population of Non-producers and Receivers. We found that the metabolite concentration, the duration of bacterial incubation, the initial population sizes, and the droplet carrying capacity affect the Producer dynamics in the population. The final part of this thesis focuses on applying the insights from the population dynamics of the model community to enrich for novel antibiotic-producing Streptomyces roseosporus, by rationally competing against Enterococcus faecalis using the microfluidic platform. As daptomycin is ineffective against a resistant variant of E. faecalis, S roseosporus must trigger activation of a cryptic pathway or produce daptomycin-variant that is effective against E. faecalis for success in the population. Preliminary work in this project shows that the growth rates of the two populations must be further optimized for testing experimental evolution in droplets. Overall, in this thesis, we have established the microfluidic platform, validated the synthetic community dynamics, and set up initial work towards applying the platform for wild strains of bacteria. These insights can guide design principles to study the microbial community dynamics of natural and synthetic multispecies communities in the laboratory.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.Microfluidicsdropletsmicrobial communityThesis2021-12-06