A comprehensive genome-scale reconstruction of Escherichia coli metabolism—2011 Jeffrey D Orth, Tom M Conrad, Jessica Na, Joshua A Lerman, Hojung Nam, Adam M Feist, and Bernhard Ø Palsson present a new genome-scale metabolic reconstruction of Escherichia coli K-12 MG1655, named iJO1366. This model includes 1366 genes, 2251 metabolic reactions, and 1136 unique metabolites. It was updated using experimental data from 1075 gene knockout strains, compared with previous models and experimental data to ensure accurate predictions, and mapped to the genomes of all sequenced E. coli strains, identifying hundreds of unannotated genes. The model is expected to be widely used for studying E. coli systems biology and metabolic engineering. The model was built by updating the iAF1260 model, which had 2077 reactions, 1039 metabolites, and 1260 genes. The new model includes 107 new genes and one removed gene. It was validated by comparing predicted growth phenotypes with experimental data and by identifying gaps in the model. The model was also compared with other reconstructions, including the Model SEED and EchoLocation databases. The iJO1366 model was used to predict the growth of E. coli on various carbon, nitrogen, phosphorus, and sulfur sources, as well as the essentiality of genes in glucose and glycerol minimal media. The model was also used to map to other E. coli and Shigella strains, identifying hundreds of unannotated genes in these organisms. The model includes a wide range of metabolic functions and is the most complete E. coli metabolic reconstruction to date. It is expected to continue to serve as a basis for the metabolic reconstruction of other bacteria and to be an important tool in microbial systems biology for years to come.A comprehensive genome-scale reconstruction of Escherichia coli metabolism—2011 Jeffrey D Orth, Tom M Conrad, Jessica Na, Joshua A Lerman, Hojung Nam, Adam M Feist, and Bernhard Ø Palsson present a new genome-scale metabolic reconstruction of Escherichia coli K-12 MG1655, named iJO1366. This model includes 1366 genes, 2251 metabolic reactions, and 1136 unique metabolites. It was updated using experimental data from 1075 gene knockout strains, compared with previous models and experimental data to ensure accurate predictions, and mapped to the genomes of all sequenced E. coli strains, identifying hundreds of unannotated genes. The model is expected to be widely used for studying E. coli systems biology and metabolic engineering. The model was built by updating the iAF1260 model, which had 2077 reactions, 1039 metabolites, and 1260 genes. The new model includes 107 new genes and one removed gene. It was validated by comparing predicted growth phenotypes with experimental data and by identifying gaps in the model. The model was also compared with other reconstructions, including the Model SEED and EchoLocation databases. The iJO1366 model was used to predict the growth of E. coli on various carbon, nitrogen, phosphorus, and sulfur sources, as well as the essentiality of genes in glucose and glycerol minimal media. The model was also used to map to other E. coli and Shigella strains, identifying hundreds of unannotated genes in these organisms. The model includes a wide range of metabolic functions and is the most complete E. coli metabolic reconstruction to date. It is expected to continue to serve as a basis for the metabolic reconstruction of other bacteria and to be an important tool in microbial systems biology for years to come.