Systematic Evaluation of Genetic and EnvironmentalFactors Affecting Performance of Translational Riboswitches

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 integra...