Project Summary and Abstract Phase variation of gene expression enables bacteria to generate heterogenous populations and organize communities that collectively can withstand diverse environmental perturbations. This discrete ON/OFF pattern of gene expression occurs at multiple loci concurrently to create extensive phenotypic variation, but how expression from multiple phase variable loci is coordinated is unknown. We developed a breakthrough single- cell microfluidics technology to study phase variation at multiple loci directly, simultaneously, and over time to track specialized bacterial sub-populations and learn fundamental principles determining their relative abundances, rates of development, and interconnectedness. We learn these principles for Bacteroides fragilis, a crucial human gut symbiote and master of phase variation. B. fragilis directly inhibits pathogens such as Clostridium difficile and rapidly evolves a vast reservoir of mobile, phase variable antibiotic resistance genes. Studying phase variation mechanisms in B. fragilis will enhance engineering of human microbiota and rational design of symbiote-friendly antibiotics to limit evolution and subsequent mobilization of antibiotic-resistance genes. We combine single-cell microfluidics with genomics and biochemistry to specifically dissect a two-part regulatory system enabling coordinated phase variation: promoter inversion and termination control. To study these fundamental principles governing phase variable gene expression, I will be trained primarily in genomics and single-cell microfluidics by my co-mentors, Dr. Robert Landick and Dr. Ophelia Venturelli. Dr. Landick’s decades of experience studying fundamental mechanisms of prokaryotic gene regulation combined with Dr. Venturelli’s expertise in anaerobic bacteriology, engineering, and microfluidics provide me optimal training to achieve my career goal. The state-of-the-art facilities and resources provided by UW-Madison and the Departments of Biochemistry and Bacteriology provide me with the optimal environment in which I will carry out this project.