3-5-difluoro-4-hydroxybenzylidene-imidazolinone and bis(3--5-)-cyclic-diguanylic-acid

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 profluorescent, 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. In this updated chapter, we have added procedures on using biosensors in flow cytometry to detect exogenously added compounds. 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.

Topics: Aptamers, Nucleotide; Bacterial Proteins; Benzyl Compounds; Biosensing Techniques; Cloning, Molecular; Cyclic GMP; Escherichia coli Proteins; Flow Cytometry; Fluorescent Dyes; Imidazolines; Intravital Microscopy; Isopropyl Thiogalactoside; Microscopy, Fluorescence; Nucleic Acid Conformation; Phosphorus-Oxygen Lyases; Plasmids; Riboswitch; RNA; RNA, Transfer