Studying the Effectiveness of RNA Riboswitches

Riboswitches are regions of DNA found directly upstream of some genes. They act as genetic switches, turning gene expression on or off, depending on the presence of signaling molecules (ligands) in the cell. During transcription, the riboswitch RNA forms one of two mutually exclusive structures, one in the presence, the other in the absence of the ligand. One of these structures allows transcription, the other prohibits it by causing the RNA polymerase to detach prematurely. Riboswitches are naturally found in many bacteria. The behavior of riboswitches has been studied in vivo and in vitro, however the quantification of a riboswitch along with its variants has not been rigorously pursued. This project aims to create an in vivo assay in E. coli cells in order to test and quantify the effectiveness of the wild-type preQ 1 riboswitch controlling the Spinach2 aptamer. The fluorescent output of the Spinach2 aptamer (the gene under regulation) will be measured at different time intervals in E. coli cells grown in enriched media in the presence and in the absence of the preQ 1 ligand, and in the presence and in the absence of DFHBI-1T, the Spinach2 reporting molecule. The fluorescence measurements are consistent with expectations, in that there is greater fluorescence in media with DFHBI-1T and without preQ 1 compared to media with DFHBI-1T and with preQ 1 showing evidence for effective gene regulation in vivo. In the future this assay will be used to quantify and compare the effectiveness of variants of the preQ 1 riboswitch to optimize the system.