The principles of our method illustrated using a simple metabolic network.

<p>Flux limits in this figure are represented by a thin black outline. Reaction fluxes are represented as shaded regions with flux magnitude proportional to <i>thickness</i>. Flux direction is not indicated. <b>Panel A</b>. Creation of the baseline flux limits (corresponding to in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036947#pone-0036947-g002" target="_blank">Figure 2</a>, Panel A)<b>.</b> Each reaction is given a flux limit corresponding to the maximum optimal flux solution over the two <i>in silico</i> nutrient uptake conditions. The shading is orange for <i>in silico</i> glucose growth, blue for <i>in silico</i> acetate and grey where the two solutions overlap. <b>Panel B.</b> Creation of the glucose expression-derived flux limits (corresponding to in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036947#pone-0036947-g002" target="_blank">Figure 2</a>, Panel B). Each flux limit shown in Panel A has been scaled by the level of gene expression for <i>in vivo</i> growth on glucose relative to the maximum gene expression for that reaction over both nutrient conditions. The arrows indicate two reactions for which gene expression was significantly lower on glucose than on acetate, resulting in significantly reduced flux limits. <b>Panel C</b>. Effect of the glucose expression-derived flux limits of Panel B on <i>in silico</i> glucose growth. The glucose optimal flux from Panel A (orange region) lies within the limits; biomass production is not changed. <b>Panel D.</b> Effect of the glucose expression-derived flux limits of Panel B on <i>in silico</i> acetate growth. The acetate optimal flux from Panel A (blue region) exceeds the flux limits for several reactions. (This is analogous to the optimal flux vector lying outside the flux cone in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036947#pone-0036947-g002" target="_blank">Figure 2</a>, Panel B.) Hence the flux limits will lead to smaller optimal fluxes for these reactions and reduced biomass production. Relative biomass production is therefore smaller for <i>in silico</i> acetate than for <i>in silico</i> glucose, and we conclude that glucose is the more likely carbon source for the expression data.