Systematic Evaluation of Genetic and Environmental Factors Affecting Performance of Translational Riboswitches

[Image: see text] Since their discovery, riboswitches have been attractive tools for the user-controlled regulation of gene expression in bacterial systems. Riboswitches facilitate small molecule mediated fine-tuning of protein expression, making these tools of great use to the synthetic biology community. However, the use of riboswitches is often restricted due to context dependent performance and limited dynamic range. Here, we report the drastic improvement of a previously developed orthogonal riboswitch achieved through in vivo functional selection and optimization of flanking coding and noncoding sequences. The behavior of the derived riboswitches was mapped under a wide array of growth and induction conditions, using a structured Design of Experiments approach. This approach successfully improved the maximal protein expression levels 8.2-fold relative to the original riboswitches, and the dynamic range was improved to afford riboswitch dependent control of 80-fold. The optimized orthogonal riboswitch was then integrated downstream of four endogenous stress promoters, responsive to phosphate starvation, hyperosmotic stress, redox stress, and carbon starvation. These responsive stress promoter–riboswitch devices were demonstrated to allow for tuning of protein expression up to ∼650-fold in response to both environmental and cellular stress responses and riboswitch dependent attenuation. We envisage that these riboswitch stress responsive devices will be useful tools for the construction of advanced genetic circuits, bioprocessing, and protein expression.