Figure 3.

Dose responsive analysis of the NO-sensing switch system. (A) NO release by different concentrations of DETA/NO in bacterial culture medium after incubation at 37 °C for 16 h. (B) Rluc8 intensity in SL. pNASR and SL. pSRluc8 cultured in the presence of indicated concentrations of DETA/NO for 16 h at 37 °C. Rluc8 intensity was measured with 0.2 μg of coelenterazine and normalized to the number of bacterial cells (OD₆₀₀). (C) Bioluminescence images of SL. pSRluc8 (upper panel) and SL. pNASR (bottom panel) from (B). w/o, without coelenterazine; w, with coelenterazine. (D) Western blot analysis of Rluc8 expression in bacterial samples from (B) using anti-Renilla luciferase antibody (upper panel). Rluc8 expression was normalized to DnaK expression, as detected with anti-DnaK antibody (bottom panel). (E) Switch activation verified by PCR amplification. Left, illustration of ON-state primer designation; right, PCR results using ON-state primer set to confirm switch activation in bacterial cells from (B). Bands appeared when fimS was switched and in the same direction as rluc8 (~1.1 Kbp, arrow).