Figure 1.

Titration effects of decoy sites on response output in engineered non-autoregulatory strains. (A) Schematic diagram of the experimental design of TF titration by decoys. The active TF, phosphorylated PhoB (PhoBp), can bind to both endogenous chromosomal sites and the plasmid-borne decoy sites. (B and C) Reduction of phoA reporter fluorescence by decoys. Reporter outputs (B) were tracked in strain RU1988 (LAC, P_phoA-yfp) with plasmids harboring different numbers of decoy sites. Time 0 refers to the time when bacteria were resupended in Pi-limited media. Lines with corresponding shaded ranges indicate mean ± SD from 8 individual wells from one representative experiment. Relative promoter activities (C) were derived using the reference promoter activity at 8.2 μM PhoB with no decoy sites. Diamond symbols illustrate data from individual cultures and horizontal bars indicate the averages. (D) Dependence of decoy effects on DNA affinity and total PhoB concentrations. Three reporter strains with decreasing promoter affinities for PhoBp, RU1989 (P_phoB*), RU1988 (P_phoA) and RU1990 (P_phnC), were assayed. Data are shown as mean ± SD. All data were from at least two independent experiments with eight individual cultures per experiment. Solid and dashed lines represent modeled data at corresponding total PhoB concentrations.