Synthetic Riboswitches as Genetic and Analytical Tools

Abstract

The goal of this project was to identify a synthetic riboswitch for caffeine. The synthetic caffeine riboswitch will be useful to regulate gene expression in response to caffeine and detect caffeine in complex biological samples such as urine and blood. A riboswitch is a sequence of non-coding RNA that can specifically binds a ligand and thereby controls expression of genes associated with the biosynthesis, transport, or degradation of the specific ligand. In order to develop the caffeine synthetic riboswitch, we modified the theophylline-binding domain of a synthetic theophylline riboswitch. Caffeine is structurally similar to theophylline differing only by one N-methyl group. A total of 11 bases that showed significant interactions with theophylline in the theophylline riboswitch were randomized in the library design. The library was constructed by overlapping extension polymerase chain reaction and inserted into pTrp-TheoRS-CatUpp, a plasmid that encodes the upp-cat fusion gene under control of pTrp promoter. GH371 E. coli cells were transformed with the library and subject to a few rounds of the positive selection on chloramphenicol in the presence of caffeine and the negative election on 5-fluorouracil in the absence of caffeine. From the genetic screening, 192 individual single colonies were selected and tested for chloramphenicol resistance in the presence and absence of caffeine. Two clones clearly showed chloramphenicol resistance only in the presence of caffeine. These two caffeine riboswitch variants were sequence and will be further tested for calorimetric and fluorescence assays using beta-galatosidase (LacZ) and green fluorescence protein respectively as reporter genes.