(157az) Regulation of Gene Expression in E. coli Using Theophylline Sensing Riboswitches

Fine-tuned regulation of gene expression is highly sought after for biological engineering applications. One manner in which this ability has been achieved is through the isolation, creation, and implementation of RNA riboswitches. Riboswitches are composed of two functional domains: the aptamer and the functional domain. A functional domain alters into a different secondary conformational state as a result of the aptamer binding to its respective metabolite, which can either block or enable transcription or translation. The theophylline aptamer, one of the most studied RNA aptamers, has been attached to a switch in order to achieve gene regulation with in Escherichia coli. In this work, we have studied principles guiding riboswitch function in E. coli through use of trans-acting switches or cis-acting theophylline hammerhead ribozymes placed in the 5ʹ untranslated region to control expression of the gfp gene. These riboswitch constructs have then been analyzed for fold activation of the target gene when in the presence and absence of theophylline. Addition of spacers or anti-ribosomal binding site stems to the riboswitches greatly affected the expression of gfp when cells were cultured in the presence of theophylline.