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.

Whole cell biosensing via recA::mCherry and LED-based flow-through fluorometry.

A miniature flow-through optical cell has been developed with the potential for integration into a stand-alone, potentially disposable whole-cell biosensor platform. The compact and inexpensive optical system is comprised of closely coupled light-emitting diodes (LEDs), light-to-frequency (LTF) photodiodes, and celluloid filters. The system has been optimized to measure fluorescent reporters produced by cultures of biosensor cells in liquid suspension. As demonstration subjects, Escherichia coli cells carrying medium-copy plasmids with fluorescent reporter fusions to the rec promoter were exposed to the DNA-damaging agent mitomycin C (MMC). As reporter proteins, green fluorescent protein (GFP) and red fluorescent protein (RFP) were compared for suitability in the compact instrument. The RFP mCherry outperformed GFP (GFPmut3.1) as a reporter protein in the developed system on two counts. First, measurement distortions due to high optical density suspensions are minimal using RFP compared to GFP. Second, the limit of detection for MMC is estimated at 0.25nM for recA::mCherry and 2.0nM for recA::gfpmut3.1. Finally, a measurement method is presented whereby multiple channels of optical data are calibrated in an integrated fashion to allow simultaneous measurement of fluorescence and biomass concentration. The method substantially eliminates optical distortions due to dense samples and thus obviates the conventional need for sample dilution prior to measurement.