(175a) Fine-Tuning Expression of Genes in E. coli Using a Theophylline-Sensing Hammerhead Riboswitch

Riboswitches are regulatory elements composed of RNA that can be used to control gene expression inside of cells. They have been used in bacteria, yeast, and mammalian cells, and have been shown to work through a variety of mechanisms. Each riboswitch contains an aptamer domain, which selectively binds a specific target molecule- usually a metabolite- and a functional domain that controls whether the gene is turned ON or OFF through allosteric means.

We have constructed various theophylline-sensing hammer head riboswitches, which we placed upstream of the lacZ gene for expression in E. coli. Multiple plasmids were created with varied spacers to test the optimization of the gene spacing to the proximity to the riboswitch. The switch contains an anti-Ribosomal Binding Site (RBS) fragment, hindering binding when in the absence of theophylline. In the presence of the metabolite, the switch undergoes a catalytic self-cleavage, which exposes the RBS, allowing for ribosomal binding and translation. Plasmid pEcSWAP115 yielded the highest lacZ expression as measured via the Miller Assay; however, the OFF conformation does have leaky expression in the absence of theophylline.