Development of RNA genetic tools

Understanding context performance of riboswitches

Figure 1b

Riboswitches are attractive tools for the regulation of gene expression in bacterial systems, making these tools of great use to the synthetic biology community. However, the use of riboswitches is often restricted due to codon context dependent performance, and limited dynamic range. We recently explored the cause and effect of sequence context sensitivity for translational ON riboswitches located in the 5’ UTR, by constructing and screening a library of N-terminal synonymous codon variants. By altering the N-terminal codon usage we were able to obtain RNA devices with a broad range of functional performance properties (ON, OFF, fold-change). The study provides a novel multi-variant analysis framework by which to rationalize the codon context performance of allosteric RNA-devices, that serves as a platform for future riboswitch context engineering endeavours (ACS SynBio 2018).

Graphical Abstract

The behaviour of the derived riboswitches was then 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 optimised 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 (ACS SynBio Bio 2019).


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