Analytical biophotonics techniques such as steady-state fluorescence spectroscopy and fluorescence microscopy have been proven to be important tools in the study of genetically engineered bacterial sensors for plant antigenotoxicity. The assay involves the use of <i>E. coli</i> RS4U tagged with the red fluorescent protein (RFP). The cells emit red fluoresce in direct proportion to the genotoxicant present. Antigenotoxicity is seen as an act of preventing the DNA-damage induced expression of RFP. Thus, co-treatment of the cells with the genotoxicant and antigenotoxicant plant samples resulted to the reduction of the RFP fluorescence.
A new antioxidant activity assay based on the reactive oxygen species (ROS)-inducible bacterial strain (<i>E. coli</i> DPD2511) is described. The strain harbors the plasmid <i>pKatG::luxCDABE</i> and responds to hydrogen peroxide treatment by increasing light emission at 490 nm. Antioxidant capacity is evaluated through the ability of an agent to inhibit the hydrogen peroxide-induced bioluminescence of <i>E. coli</i> DPD2511. Applicability of the developed assay in detecting levels of antioxidants in various aqueous plant extracts is demonstrated. The assay was validated against 2,2-diphenylpicrylhydrazyl (DPPH) assay, a known antioxidant assay.
The glucose-mediated conformational changes in the glucose binding protein (GBP) have been exploited in the development of fluorescence based glucose sensors. The fluorescence response is generated by a polarity sensitive dye attached to a specific site. Such fluorescent sensors respond to submicromolar glucose at diffusion-controlled rates mimicking the wild type. However, such sensors have been limited to <i>in vitro</i> glucose sensing because of the preliminary dye-labeling step. In the study described here, the dye-labeling step is omitted by genetically encoding the GBP with two green fluorescent mutants namely, the green fluorescent protein (GFP) and the yellow fluorescent protein (YFP) in the N- and C-terminal ends, respectively. These two GFP mutants comprise a fluorescence resonance energy transfer (FRET) donor and acceptor pair. Thus, when glucose binds with GBP, the conformational changes affect the FRET efficiency yielding a dose-dependent response. A potential application for this FRET-based glucose biosensor is online glucose sensing in bioprocessing and cell culture. This was demonstrated by the measurement of glucose consumption in yeast fermentation. Further development of this system should yield <i>in vivo</i> measurement of glucose in bioprocesses.