A course-based undergraduate research experience (CURE) using transposon-directed insertion-site sequencing (TraDIS) to identify genes that are essential for bacteriophage P1 propagation in Escherichia coli

Understanding gene function through forward genetic screens is foundational to microbial genetics. Here, we describe the design and implementation of a multi-week course-based undergraduate research experience (CURE) embedded within a third-year Microbial Genetics course, developed over three iterations in trimester 3 of 2023–2025. In this CURE, students construct a high-density Tn 5 transposon insertion library in Escherichia coli , challenge the library with bacteriophage P1, and use transposon-directed insertion-site sequencing (TraDIS) coupled with statistical analysis (edgeR) to identify genes required for phage propagation. Students performed transposome electroporation, phage infection assays, genomic DNA extraction, analysis of high-throughput sequencing data on Galaxy, differential insertion analysis, data visualization, and ontology enrichment analysis. The project integrates wet-lab microbiology, molecular genetics, and bioinformatics while modeling an authentic forward genetic screen. This CURE emphasizes experimental design, genome-wide functional analysis, statistical reasoning, and interpretation of large-scale sequencing data. The curriculum is adaptable to other bacterial species, phage systems, or selective pressures, and provides a scalable framework for integrating authentic genomics research into upper-level microbiology courses.