Fig. 1: Design of gene circuit and characterization of biosensor strains.

a Construction diagram of engineered bacteria. Programmable bacterial circuits can sense specific environmental signals and increase the transmission function of biosensors by amplifying gene switch. When lactate, H⁺ are present, or in anoxic condition, serine integrase TP901 driven by inductive promoter pLldR, pCadC, and pPepT can reverse the terminator orientation of the XOR gate, enabling the strong promoter pP7 transcript downstream genes. When φX174E is added to the circuit, engineered EcN would lyse itself with above environmental factors induction. b Relationship between environmental change and biosensor fluorescence or population intensity. Biosensor strains harboring mRFP were grown in specified environmental condition (0–10 mM lactate, pH 5.3, 5.8, 6.3 and 7.3, and 0% or 20% oxygen) for 48 h and their induction fluorescence signal were assayed (n = 3 biological replicates. Data are shown as mean ± s.e.m). c Biosensor strains responded to physiological cues. Cell culture medium supernatant from CT26 cancer cell line was collected every 12 h over 5 d and then cultured with biosensor strains. The hypoxic biosensor co-cultured with cell medium supernatant was grown under conditions with or without oxygen. After incubation at 37 °C for 12 h, mRFP fluorescence intensity of lactase (red), pH (green) and hypoxic (blue) biosensor strains was measured and recorded (n = 3 biological replicates. Data are shown as mean ± s.e.m). d Fluorescence microscopy observation. The fluorescence signals of lactate, pH and hypoxic biosensors under induced (10 mM, pH 5.3 and 0% O₂, respectively) and noninduced (0 mM, pH 7.3 and 20% O₂, respectively) conditions were observed using Olympus Bx53 (magnification: 40x). Bacteria were cultured in LB medium at 37 °C for 12 h. The scale bar marked in the lower right corner of the image represents 5 μm.