Integrating and amplifying signals from riboswitch biosensors

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Michael Goodson, Svetlana Harbaugh, Nancy Kelley-Loughnane

UES, United States

Biosensors offer a built-in energy supply and inherent sensing machinery that when exploited correctly may surpass traditional sensors. However, biosensor systems are still limited when compared to traditional sensors in signal production and in their inability to integrate information from multiple sensors. Signal integration in biosensors has the potential to reduce false positive responses and increase specificity, for example if two different sensors that detect the same ligand produce an output only when both are activated. Similarly, signal integration will enable sensing of combinations of ligands that indicate nefarious activities when detected together. Amplifying signal output will increase the detection range of the biosensor, and increase sensitivity since activation of a few cells will induce reporter protein expression over the entire sensor. To address these issues we are making use of recent advances in synthetic biology to create biological ‘circuits’, by utilizing plasmids to act as logic gates in E. coli and connecting them by quorum sensing molecule ‘wires’. RNA-based translational control switches, or riboswitches, leverage conformational changes in the structure of RNA molecules when bound to a specific ligand to regulate the accessibility of a ribosome-binding site of a transcribed gene. Using the Registry of Standard Biological Parts and previously identified riboswitches, we have designed plasmids that confer AND gate logic when transformed into E. coli. The output of the AND gate is the production of a quorum signaling molecule. This molecule serves as the input to a signal amplification circuit that initiates the overproduction of a fluorescent protein. In this presentation we describe the production of these circuits, and quantify the changes in fluorescent output and sensitivity that they confer.