Fig. 4: Colorimetric assay for bactosensor-mediated bile salts detection.

a Schematic diagram for the design and operation procedure of the TcpP18–LacZ system for bile salt detection. The TcpP18–LacZ uses the TcpP loop variant V18 as sensing module for lower LOD and higher sensitivity and LacZ as a colorimetric reporter, with CPRG as a substrate which is converted to chlorophenol red turning the reaction from yellow to purple. b Bile salt specificity profile of the TcpP variant V18 characterized using TcpP18–LacZ sensor. Response of TcpP18–LacZ was quantified as ΔA580 (the difference in absorbance at 580 nm (A₅₈₀) with or without ligand bile salts). The bar graph corresponds to the mean value of three replicates performed in triplicate on three different days (n = 3 biologically independent samples). Error bars: ±SD. Cells growing in the exponential phase were incubated with bile salts for 4 h before flow cytometry analysis. c Optimization and quantification of TcpP18–LacZ response to the bile salt glycodeoxycholic acid (GCDCA). The LOD and response dynamic range of TcpP18–LacZ were fine-tuned by varying the cell concentration and incubation time. Data points correspond to the mean value of three replicates performed in triplicate on three different days (n = 3 biologically independent samples). Error bars: ±SD. See “Methods”, main text, and SI for details.