The authors have systematically constructed a set of precise, single-gene deletions of all nonessential genes in *Escherichia coli* K-12, creating the Keio collection. They used a one-step method involving PCR products with kanamycin resistance cassettes flanked by FRT sites to replace open-reading frames (ORFs) and generate in-frame deletions. Of the 4288 genes targeted, 3985 deletions were obtained, with two independent mutants saved for each gene to address potential issues in high-throughput studies. The Keio collection provides a valuable resource for systematic analyses of gene functions, regulatory networks, and mutational effects in a common strain background. The collection includes 303 genes, including 37 of unknown function, which are candidates for essential genes. The authors also discuss the distribution of essential genes and their conservation in other bacterial genomes, as well as the profiling of gene contributions to growth on rich and minimal media. The Keio collection is expected to be useful for functional genomics and systems biology studies in *E. coli* and other bacteria.The authors have systematically constructed a set of precise, single-gene deletions of all nonessential genes in *Escherichia coli* K-12, creating the Keio collection. They used a one-step method involving PCR products with kanamycin resistance cassettes flanked by FRT sites to replace open-reading frames (ORFs) and generate in-frame deletions. Of the 4288 genes targeted, 3985 deletions were obtained, with two independent mutants saved for each gene to address potential issues in high-throughput studies. The Keio collection provides a valuable resource for systematic analyses of gene functions, regulatory networks, and mutational effects in a common strain background. The collection includes 303 genes, including 37 of unknown function, which are candidates for essential genes. The authors also discuss the distribution of essential genes and their conservation in other bacterial genomes, as well as the profiling of gene contributions to growth on rich and minimal media. The Keio collection is expected to be useful for functional genomics and systems biology studies in *E. coli* and other bacteria.