BME PhD Prospectus Presentation: Eric Bueno

Title: "Single-Cell Analyses of Horizontal Gene Transfer in Escherichia coli"

Advisory Committee: Mary J. Dunlop, PhD - BME (Research Advisor) Samagya Banskota, PhD - BME (Chair) Alexander Green, PhD - BME Miguel Jimenez, PhD - BME Joseph Larkin, PhD - Biology, Physics

Abstract: Bacterial conjugation is the form of horizontal gene transfer (HGT) most commonly used to transfer antibiotic resistance genes between cells, facilitating transfer of genetic material both within and between species. The F-plasmid, the first conjugative plasmid discovered in Escherichia coli, encodes machinery enabling gene transfer across gram-negative Enterobacteriaceae. F and F-like plasmids are highly prevalent in E. coli clinical isolates, motivating the need to understand the dissemination of these conjugative vehicles in pursuit of finding strategies to limit their spread. There exist numerous studies which elucidate the broad patterns of conjugation events across microbial consortia in bulk culture. However, conjugation is inherently a cell-to-cell interaction requiring physical contact; population-level analyses are unable to determine which phenotypes are predisposed toward successful gene transfer. Recent single-cell resolution studies have suggested significant heterogeneity in conjugation efficiency across isogenic populations. Herein, I propose three aims to leverage single-cell resolution microscopy, microfluidics, and computational tools to provide critical insight into conjugative transfer: (1) characterize the conjugation dynamics of single cells undergoing F-plasmid-driven HGT under varying conditions; (2) determine whether single-cell characteristics persist through cell lineages or arise stochastically; (3) investigate how chromosomal genes modulate conjugation efficiency within microbial communities. I will present preliminary data demonstrating the burden imposed on recipient cells post-conjugation and propose an experimental framework to resolve the genetic and cellular determinants of conjugation heterogeneity within isogenic populations.