We have constructed a near-saturation level mutant library for Pseudomonas aeruginosa strain PAO1 using Tn5-derived transposons mapped to the PAO1 reference sequence. This chapter describes the high-throughput techniques used to generate and map the mutant strains. In addition, an analysis of the utility of this collection is presented based on changes to the annotation for the PAO1 genome in the past years, as well as the citation record for this collection. It is clear that many avenues of research have been accelerated by this collection and that additional large mutant strain collections will further aid in defining gene function and biological processes in pathogens.