Figure 1.

Engineering of the PPDA responsive orthogonal riboswitch for enhanced function. (A) Illustrating the mechanism of the orthogonal riboswitch showing small molecule mediated regulation of protein expression. Protein production is activated in response to pyrimido[4,5-d]pyrimidine-2,4-diamine (PPDA), which causes a change in the secondary structure of the eGFP 5′UTR, allowing translation to proceed. (B) An overview of the screening and optimization workflow employed in this study, highlighting the use of in vivo FACS selection and design of experiments to improve riboswitch function. This functional enhancement enabled development of four stress responsive riboswitch devices. (C) Schematic overview of a synthetic riboswitch device under regulation of the IPTG inducible P_tac promoter showing transcriptional and translational regulation of an N-terminally His tagged eGFP.