Fig. 3: Increasing sensor activation with addition of an integrase-based switch.
From: Probiotic acoustic biosensors for noninvasive imaging of gut inflammation
a Plasmid diagram of the optimized thiosulfate sensor thsS(t3)R-bARGSer with an integrase-based switch to create thsS(t3)R-Bxb1_P7-bARGSer. Addition of thiosulfate induces expression of the Bxb1 integrase which causes irreversible site-specific recombination between the attP and attB sites that reverses the orientation of the strong constitutive P7 promoter to activate bARGSer expression. BURST signal-to-background ratio (SBR) (b) and representative images (c) of the optimized thiosulfate sensor with and without the Bxb1 integrase-based switch at varying thiosulfate concentrations. See Fig. S6a, b for the corresponding xAM data. d Plasmid diagram of the optimized thiosulfate sensor thsS(t3)R-GFP_mCherry with an integrase-based switch to create thsS(t3)R-Bxb1_P7-GFP_mCherry. Mean GFP fluorescence measured via flow cytometry (e) and representative histograms (f) of the optimized thiosulfate sensor with and without the Bxb1 integrase-based switch at varying thiosulfate concentrations. In b and e, points represent biological replicates (N = 4), curves represent fits to the Hill equation (see Table S2 for fitted parameters), and numbers next to dashed arrows indicate maximal fold changes. All strains were grown on plates with varying concentrations of thiosulfate at 37 °C for 20–24 hours (see Fig. S5 for images of the plates) and suspended in PBS for ultrasound imaging and flow cytometry. For ultrasound imaging, cells were cast in agarose phantoms at a concentration of 5 × 108 cells/mL. See Fig. S6c–j for the corresponding data for the tetrathionate sensor.
