Here we describe the systematic construction of well-defined, in-frame, single-gene deletions of all nonessential genes in Escherichia coli K-12. The principal strategy is based on the method for one-step inactivation of chromosomal genes in E. coli K-12 established by Datsenko and Wanner (1), namely, the replacement of a target gene with a selectable antibiotic-resistant marker generated by polymerase chain reaction (PCR) using oligonucleotide DNA primers homologous to the gene flanking regions. The advantages of this method include complete deletion of an entire open reading frame and precise design eliminating polar effects for the downstream genes on E. coli chromosome.