A FRET-based color-changing whole-cell biosensor for visual detection of 2,4-dinitrotoluene

A whole-cell biosensor for detection of 2,4-dinitrotoluene (2,4-DNT), a landmine-related compound, that changes the fluorescence color of cells through Förster resonance energy transfer (FRET) was developed. Two genetic constructs were designed to induce a color change in response to 2,4-DNT. The first encodes Tobacco Vein Mottling Virus (TVMV) protease under the control of the yqjF promoter, a promoter known to respond to 2,4-DNT. The second encodes TagRFP and mAmetrine connected by a linker peptide containing a TVMV protease recognition sequence. These genes were introduced into separate plasmids and co-transformed into Escherichia coli BL21(DE3) to construct the biosensor. In the absence of 2,4-DNT, the cells exhibited yellow fluorescence due to FRET between mAmetrine and TagRFP. In the presence of 2,4-DNT, expression of TVMV protease cleaved the linker peptide, disrupted FRET, and resulted in green fluorescence. A clear fluorescence color change from yellow to green was observed in response to 0-1 mM 2,4-DNT. Cleavage of the linker peptide by TVMV protease was confirmed by SDS-PAGE. These results demonstrate the feasibility of a color-changing microbial biosensor for visual detection of 2,4-DNT. This strategy provides a visual output independent of signal intensity.