The reconstruction process has also been previously outlined (Feist et al, 2006 (link); Reed et al, 2006a (link)). Here, we provide certain details specific to this work. Starting from the metabolic network for iJR904 (Reed et al, 2003 (link)), additional reactions were added to the network based on E. coli-specific biochemical characterization studies (see Supplementary information for a complete list of references) and other reactions were removed (see Results). This process was aided by comparing the content of iJR904 with the EcoCyc database (see below). The E. coli genome annotation (Riley et al, 2006 (link)) was used as a citation source for biochemical characterization studies and a framework upon which translated metabolic proteins, and subsequently reactions, were assigned to form gene to protein to reaction (GPR) assignments. The SimPheny™ (Genomatica Inc., San Diego, CA) software platform was used to build the reconstruction. For each reaction entered into the reconstruction, the involved metabolites were characterized according to their chemical formula and charge determined using their pKa value for a pH of 7.2. Metabolite charge was determined using its pKa value(s). When the metabolite pKa was not available, charge was determined using the pKa of ionizable groups present in a metabolite (http://www.chemaxon.com/product/pka.html). All of the reactions entered into the network were designated as enzymatically catalyzed reactions or spontaneous reactions, were both elementally and charged balanced and are either reversible or irreversible. Reversibility was determined first from primary literature for each particular enzyme/reaction, if available (see Supplementary information for references). Additionally, general heuristic rules, like those applied by Kümmel et al (2006b) , were used to enter reversibility using knowledge about the physiological direction of a reaction in a pathway (sometimes including regulatory knowledge) and/or basic thermodynamic information (such as reactions hydrolyzing high-energy phosphate bonds are almost always irreversible). Furthermore, a thermodynamic analysis of reversibility was utilized to assign the directionality of some reactions (see above).