Dual genetic selection of the theophylline riboswitch with altered aptamer specificity for caffeine.

The aptamer domain of the theophylline riboswitch was randomized to generate a library containing millions of different variants. Dual genetic selection utilizing the cat-upp fusion gene was performed for the library, which successfully led to the identification of a caffeine-specific synthetic riboswitch. When a chloramphenicol-resistance gene was expressed under control of this riboswitch, E. coli cells showed chloramphenicol resistance only in the presence of caffeine. When inserted upstream of the gfpuv or lacZ gene, the caffeine riboswitch induced the expression of green fluorescent protein or β-galactosidase in the presence of caffeine, respectively. When tested with various concentrations of caffeine, the β-galactosidase activity was proportional to the amount of caffeine, clearly indicating the caffeine-dependent gene regulation by the caffeine riboswitch.