Figure 2

Optimization of induction signal. A mixture of 3 plasmids (pC₄N₂-R_HS/ZF3, pZBS-Tet-OFF, and pBI-EGFP-SEAP) was used for cell transfection; the total amount of plasmid DNA was constant at 0.5 μg per test point. As graphically represented, at each apex of the triangle shown, only one of the test plasmids is present (100% of mix), while opposite the matrix this plasmid is absent (0% of mix), with the two other plasmids present in equal proportions. White circles indicate ratios of plasmids evaluated for degree of induction of SEAP activity. Degree of induction reflects difference between growth without dimerizer and with tetracycline (OFF), versus with dimerizer and without tetracycline (ON). Based on the ON:OFF ratios obtained, the software program "Statistica" modeled the optimal response surface, shown as a contour plot on the triangle. Faded edges of the triangle represent limitations of the model, because it is meaningless whenever one of the components is absent. The optimal ratio for plasmid transfection with the DDC system was 1.5:1.5:1 (pC₄N₂-R_HS/ZF3:pZBS-Tet-OFF:pBI-EGFP-SEAP). This contrasted with a ratio of 3:1 (activator proteins:reporter) used for the original ARIAD system.