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 beta-galactosidase in the presence of caffeine, respectively. When tested with various concentrations of caffeine, the beta- galactosidase activity was proportional to the amount of caffeine, clearly indicating the caffeine dependent gene regulation by the caffeine riboswitch. (c) 2021 Published by Elsevier Inc.

Automated summary

The randomized aptamer domain of the theophylline riboswitch yielded a library with millions of variants, leading to the discovery of a caffeine-specific synthetic riboswitch through dual genetic selection. The riboswitch controlled the expression of target genes in the presence of caffeine, demonstrating caffeine-dependent gene regulation effectively.