Riboswitch-Mediated Detection of Metabolite Fluctuations During Live Cell Imaging of Bacteria

Riboswitches are a class of noncoding RNAs that regulate gene expression in response to changes in intracellular metabolite concentrations. When riboswitches are placed upstream of genetic reporters, the degree of reporter activity reflects the relative abundance of the metabolite that is sensed by the riboswitch. This method describes how reporters for live cell imaging, such as yellow fluorescent protein (YFP), can be placed under genetic control by metabolite-sensing riboswitches in the bacterium Bacillus subtilis. Specifically, a protocol for generating a fluorescent YFP reporter, based on a c-di-GMP responsive riboswitch, is outlined below.