Stages of data addition (+) and method runs ([>]) in the workflow.

<p>Letters and numbers in parentheses refer, respectively, to input data and to the stages of the workflow. (1) Geometric FBA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079195#pone.0079195-Smallbone4" target="_blank">[32]</a> is run, yielding a thermodynamically feasible, stationary flux distribution matching the flux data; (2) a sub-network of flux-carrying reactions is exported, (3) consistent values of all metabolite concentrations and equilibrium constants are determined, (4) kinetic constants appearing in the sub-network are balanced, (5) kinetic rate laws and associated kinetic constants are inserted into the model, and (6) the maximal reaction rates are adjusted to reproduce the steady-state fluxes calculated before. The laboratory data feeding into “Flux” can include quantitative metabolomic measurements of metabolites, and also dynamically calculated flux values for the network. The lower half of the diagram shows a cycle of experimentation and modelling: here the result of the MCA can be used to target which rate laws should be measured <i>in vitro</i> after measurement, this rate law can be substituted into the model, with a view to better fit the observed perturbation behaviour. All grey arrows refer to aspects of the workflow where additional data can be added in as knowledge increases. The pathway shown is a truncated version of the trehalose pathway. Abbreviations: T6P = trehalose 6-phosphate; aaT = - trehalase; Glu = Glucose; G6P = glucose 6-phosphate.