Kellenberger Colleen A, Hallberg Zachary F, Hammond Ming C
Department of Chemistry, University of California, Berkeley, Berkeley, CA, 94720, USA.
Methods Mol Biol. 2015;1316:87-103. doi: 10.1007/978-1-4939-2730-2_8.
The development of fluorescent biosensors is motivated by the desire to monitor cellular metabolite levels in real time. Most genetically encodable fluorescent biosensors are based on receptor proteins fused to fluorescent protein domains. More recently, small molecule-binding riboswitches have been adapted for use as fluorescent biosensors through fusion to the in vitro selected Spinach aptamer, which binds a pro-fluorescent, cell-permeable small molecule mimic of the GFP chromophore, DFHBI. Here we describe methods to prepare and analyze riboswitch-Spinach tRNA fusions for ligand-dependent activation of fluorescence in vivo. Example procedures describe the use of the Vc2-Spinach tRNA biosensor to monitor perturbations in cellular levels of cyclic di-GMP using either fluorescence microscopy or flow cytometry. The relative ease of cloning and imaging of these biosensors, as well as their modular nature, should make this method appealing to other researchers interested in utilizing riboswitch-based biosensors for metabolite sensing.
荧光生物传感器的发展源于实时监测细胞代谢物水平的需求。大多数可基因编码的荧光生物传感器基于与荧光蛋白结构域融合的受体蛋白。最近，小分子结合核糖开关通过与体外筛选的菠菜适体融合而被用作荧光生物传感器，该适体可结合绿色荧光蛋白发色团的促荧光、细胞可渗透小分子模拟物DFHBI。在此，我们描述了制备和分析核糖开关 - 菠菜tRNA融合体以在体内实现配体依赖性荧光激活的方法。示例程序描述了使用Vc2 - 菠菜tRNA生物传感器通过荧光显微镜或流式细胞术监测细胞内环二鸟苷酸水平的变化。这些生物传感器相对容易克隆和成像，以及它们的模块化性质，应该会使这种方法对其他有兴趣利用基于核糖开关的生物传感器进行代谢物传感的研究人员具有吸引力。
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