An illustrative example of FOCAL. FOCAL is first used to couple cellular growth (vbio) with a chosen reaction flux (v2). (a) In the uncoupled system, v4 is coupled with v2 (that is, v4 ≥ 0 implies v2 ≥ 0) but vbio is not coupled with v2. (b) In the coupled case, vbio is coupled with v2 (vbio→ v2). Here, metabolite Aex is the only nutrient (no Gex), and a gene associated with v8 is deleted such that the upper pathway is required to synthesize metabolite F. Under these circumstances, flux through vbio requires flux through v2. Moreover, removal of v2 (along with v8) will result in a non-viable cellular mutant. (c) FOCAL can also be used to create substrate co-utilizing mutants where deletion of v3 and v8 requires the co-utilization of metabolites A and G in order to produce both biomass components, F and H.