Etoh Hiroki, Yasuda Mitsuru, Akimoto Takuo
School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura, Hachiouji, Tokyo 192–0982, Japan.
Anal Sci. 2011;27(12):1179-83. doi: 10.2116/analsci.27.1179.
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 Al(2)O(3) 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.
在本文中，我们描述了一种方法，该方法使用多层底物，通过在基因工程改造的大肠杆菌中利用SOS反应诱导的绿色荧光蛋白（GFP）来增强诱变剂检测的荧光信号。为了生成表达SOS反应诱导的GFP的大肠杆菌，我们构建了一个携带位于GFP基因上游的RecA启动子的质粒，并使用它来转化大肠杆菌BL21。将转化后的菌株与典型诱变剂丝裂霉素C（MMC）一起孵育，然后固定在载玻片上带有银和薄氧化铝（Al₂O₃）层的多层底物上。由于多层底物技术是一种有潜力增强置于底物顶部的荧光团荧光的光学技术，因此预计多层底物可改善诱变剂检测的荧光信号。在1至1000 ng/ml的浓度范围内，我们获得了MMC诱导的GFP平均14倍的荧光增强。此外，使用该技术提高了MMC的检测下限，由于空白与1 ng/ml MMC信号之间的差异增大，估计检测下限为1 ng/ml。
