Analytical Sciences

Abstract − Analytical Sciences, 27(12), 1179 (2011).

Signal Enhancement by a Multi-layered Substrate for Mutagen Detection Using an SOS Response-induced Green Fluorescent Protein in Genetically Modified Escherichia coli
Hiroki ETOH, Mitsuru YASUDA, and Takuo AKIMOTO
School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura, Hachiouji, Tokyo 192-0982, Japan
In this paper, we describe a method to enhance the fluorescence signal of mutagen detection using SOS response-induced green fluorescence protein (GFP) in genetically modified Escherichia coli using a multi-layered substrate. To generate E. coli that express SOS response-induced GFP, we constructed a plasmid carrying the RecA promoter located upstream of the GFP gene and used it to transform E. coli BL21. The transformed strain was incubated with mitomycin C (MMC), a typical mutagen, and then immobilized on a multi-layered substrate with Ag and a thin Al2O3 layer on a glass slide. Since the multi-layered substrate technique is an optical technique with potential to enhance the fluorescence of fluorophore placed on top of the substrate, the multi-layered substrate was expected to improve the fluorescence signal of mutagen detection. We obtained an average 14-fold fluorescence enhancement of MMC-induced GFP in the concentration range 1 to 1000 ng/ml. In addition, the lower detection limit of MMC was improved using this technique, and was estimated to be 1 ng/ml because of an enlargement of the difference between the blank and the signal of 1 ng/ml of MMC.