In vivo screening for aptazyme-based bacterial riboswitches

In many synthetic biology applications, modular and easily accessible tools for controlling gene expression are required. In addition, in vivo biosensors and diagnostic devices will become more important in the future to allow for noninvasive determination of protein, ion, or small molecule metabolite levels. In recent years synthetic RNA-based switches have been developed to act as signal transducers to convert a binding event of a small molecule (input) into a detectable output. Their modular design allows the development of a variety of molecular switches to be used in biochemical assays or inside living cells. RNA switches developed by our group are based on the Schistosoma mansoni hammerhead ribozyme, a self-cleaving RNA sequence that can be inserted into any RNA of interest. Connection to an aptamer sensing a small molecule renders the cleavage reaction ligand-dependent. In the past we have successfully designed and applied such hammerhead aptazymes for the allosteric control of both bacterial and eukaryotic gene expression by affecting transcription elongation, translation initiation, or mRNA stability. In order to yield functional switches optimization of the connecting sequence between the aptamer and the HHR needs to be carried out. We have therefore developed an in vivo screening protocol detailed in this chapter that allows the identification of functional aptazymes in bacteria.