Optical tracking of a stress-responsive gene amplifier applied to cell-based biosensing and the study of synthetic architectures

A synthetic regulatory construct based on a two-stage amplifying promoter cascade is applied to whole-cell biosensing. Green fluorescent protein (GFP) and red fluorescent protein (RFP) enable two-component tracking of the response event, enabling the system to exhibit increased sensitivity, a lower limit of detection, and a unique optical density-free assessment mode. Specifically, the recA and tac promoters are linked by the LacI repressor in Escherichia coli, where DNA-damage activates the recA promoter and the up-regulation of GFP and LacI proteins. LacI represses the tac promoter, down-regulating the otherwise constitutive mCherry transcription. The response of the construct was compared with two singly tagged, conventional recA promoter-reporter constructs: recA::gfpmut3.1 and recA::mCherry. Using a miniature LED-based flow-through optical detector developed for remote sensing applications, limits of detection for the dual reporter construct reached as low as 0.1 nM MMC. By comparison, single-ended reporters recA::mCherry and recA::gfpmut3.1 achieved best limits of detection of 0.25 nM and 2.0 nM, respectively. An approach to three-component optical analysis, based on a system of detectors with coupled calibration equations enables accurate assessments of the red fluorescence, green fluorescence, and biomass of sensor cell suspensions. The system approach is effective at overcoming interferences caused by optically dense samples and overlapping fluorescence spectra. Such a technique may be useful in studying the biological mechanisms which underlie the synthetic regulatory device of this work and others.