Fig. 1

Light-switchable synthetic circuit with tunable activation threshold and spatial resolution. (a) Schematic diagram of the circuit. The CAG promoter is constitutively expressing the photoactive transactivator GAVPO. Upon blue light illumination GAVPO forms a homodimer, which then initiates the transcription of TetR::mCherry::NLS from the pU5 promoter. GFP is under the control of TetR::mCherry::NLS-repressible promoter CMV(tetO2). Dox can release the repression. (b) Cells were illuminated with blue light (1.25 W m⁻²) for different durations (dark, 10 min, 30 min, and 3 h) in the absence of Dox, followed by 24 h incubation in dark. Data are presented as mean ± SEM (n = 3). (c) A square area, which is indicated by white arrows in the upper panel, was illuminated by blue light (1.25 W m⁻²) for 24 h. In the middle and low panel, the boundary between illuminated and dark area was indicated by the blue line. The right part of each picture is the illuminated area, while the left part is the dark area. Cells shown in the low panel were treated with 1 μg/ml of Dox. Scale bar is 2 mm in the upper panel, and 100 μm in the middle and low panel. (d) Cells with different levels of TetR::mCherry::NLS differentially responded to Dox. The upper panel shows the mCherry intensity of the cells illuminated with blue light (1.25 W m⁻²) for 1 h (weak, red line), 5 h (moderate, green line), or 20 h (strong, blue line), respectively. The lower panel shows GFP intensity of cells treated with different concentration of Dox after illumination. The data are presented as mean ± SEM (n = 3). Data are fitted to a modified Hill equation (dashed lines). The EC50s for the three curves are 2.70 ± 0.15 ng/ml (red), 4.74 ± 0.13 ng/ml (green), and 35.81 ± 1.03 ng/ml (blue). The Hill coefficients for the three curves are 1.81 ± 0.15 (red), 1.77 ± 0.11 (green), and 1.67 ± 0.06 (blue)