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. 2006 Dec 12;2:68. doi: 10.1038/msb4100099

Figure 3.

Figure 3

Characterization of a feedforward circuit with gel. The feedforward circuit of source So1 inhibiting switch Sw21 is characterized with both denaturing and non-denaturing gels. The total concentration of the source is 100 nM, and the total concentration of the switch template T21 is 75 nM. The total concentrations of activator A1 and A2 are 1 μM. (A) The nondenaturing gel data (red circles) and fluorometer data (blue circles) for the switch Sw21 states are compared. The gel data are quantitated by measuring the fluorescence of the OFF switch T21 band: [T21A1]=[T21tot]−[T21]. The fluorometer data and the model fit (black line) are the same as that of blue transfer curve in Figure 2E. (B) The total concentrations of inhibitors are measured from the denaturing gel and the concentrations of activator–inhibitor complexes are measured from the nondenaturing gel. Experimental data are plotted as circles and model fits are plotted as lines. (C) Denaturing gel stained with SYBR gold. Lane 1 contains a 10-base ladder, lanes 2 through 12 contain samples from the feedforward circuit with the concentration of ON source So1 template increasing from left to right. Lane 13 contains purified inhibitors, I1 and I2. Two DNA strands, So1-nt (either ON or OFF) and T12-t, form the source So1 template (either ON or OFF), while two DNA strands, T21-nt and T21-t, form the switch template, T21. (D) For the nondenaturing gel, three distinct excitation and emission scan results are overlayed (Texas Red-labeled T21: gray; Cy5-labeled A1: cyan; and FAM-labeled A2: magenta). Individual scan results are shown in Supplementary Figure S4. Lanes 1 through 11 contain samples from the feedforward circuit with the concentration of ON source So1 template increasing from left to right. Lane 12 contains the ON switch template T21A1 and the activator A1, lane 13 contains the OFF switch template T21 and the activator–inhibitor complex A1I1, and lane 14 contains the activator–inhibitor complex A2I2 and the activator A2. The ON switch template T21A1 has low fluorescence and is not clearly identified in lanes 1 through 3 and 12, presumably due to fluorescence resonance energy transfer (Marras et al, 2002) from Texas Red on T21 to Cy5 on A1.