Riboswitch and Transcription Factor-Based Cell-Free Biosensors for Human Performance Biomarkers

Cell-free systems offer the ability to develop portable, easy-to-use, robust, rapid and relatively inexpensive biosensors. One of the benefits of cell-free systems is the elimination of cell membrane transport barriers allowing for more accurate and sensitive detection of analytes of interest. This is especially useful for detection of human performance biomarkers with physiological concentrations in low picomolar – micromolar range. By leveraging the ability of regulatory RNAs (riboswitches) and regulatory proteins (transcription factors) to control gene expression we engineered E. coli cell-free expression systems to detect human performance biomarkers, specifically neurotransmitters dopamine and serotonin.

Synthetic riboswitches were designed by using a modular ‘mix-and-match’ approach. Dopamine and serotonin binding aptamers were combined with a ‘decoupled’ expression platform derived from a B. subtilis pbuE transcriptional riboswitch. The resultant riboswitch constructs were placed upstream of a green fluorescent protein, sfGFP, and their performance was tested in an E. coli cell-free expression system. We identified three functional riboswitches which exhibited >2-fold increase in fluorescence in the presence of micromolar concentrations of dopamine and serotonin.

We also demonstrated the functionality of a dual-signal dopamine sensor dependent on FeaR, an AraC family transcriptional regulator from E. coli, in a cell-free expression system. In the presence of dopamine, FeaR activates a tynA promoter inducing expression of red fluorescent protein and DOPA 4,5-dioxygenase from Mirabilis jalapa. DOPA 4,5-dioxygenase converts dopamine into a yellow betaxanthin pigment. As shown previously in E. coli cells, the combination of dual output signals reduced the interference from other catecholamine neurotransmitters in cell-free system. Furthermore, the cell-free biosensor exhibited much higher sensitivity (LOD ~ 100 nM) compared to a previously developed whole-cell dopamine biosensor (LOD ~ 1.43 µM).