Extended Data Fig. 2: Alternative circuit design for maximizing total GFP dynamic range.
From: Population-level amplification of gene regulation by programmable gene transfer
(A) Schematic of the inducible ADEPT circuit. In this design, GFP is no longer ssrA-tagged, allowing a higher accumulation, and is regulated under the lactose-inducible promoter pLac, adding a new layer for gene expression control. All other control mechanisms (ATc, Kan, and Lin) remain the same. (B-D) The alternative design was tested under various inducer combinations by measuring the pTarget-carrying fraction (Fp) (B), normalized GFP intensity (C), and total GFP intensity (D). (B) For Fp measurement, cells were grown overnight in LB media supplemented with 25 μg/mL Cm + 50 μg/mL Kan in a 2-mL deep well plate. Cultures were diluted 1/1000-fold into 20 media conditions with combinations of ATc (0, 0.01, 0.1, 1, 10, and 100 ng/mL), Lin (0 and 3.2 mM), and Kan (0 and 50 μg/mL). After 4 hours, samples were plated on LB agar plates containing 25 μg/mL Cm ± 50 μg/mL Kan for CFU counting. Fp was calculated as the ratio of CFU from Cm + Kan plates from Cm-only plates. Dashed and solid lines represent the mean of four technical replicates. (C-D) For GFP measurement, overnight cultures were diluted 10-4-fold into the same 20 media conditions. No inducer was added for GFP expression since leaky expression under pLac was sufficient to observe the effect of ATc, Kan, and Lin. Samples were placed in a black-walled 96-well plate with 50 µl of mineral oil to prevent evaporation. After 15 hours at 37 °C, cell density (OD600) and GFP intensity (Ex: 488 nm; Em: 510 nm) were measured. The normalized GFP signal (C) showed a trend similar to total GFP intensity (D), demonstrating the robustness and modularity of the ADEPT design.
