(265b) Development of a Fast-Responding, Minimal-Equipment Biosensor for Zinc Deficiency

Zinc deficiency causes hundreds of thousands of childhood deaths annually, and current diagnostic tools are too expensive for widespread use in affected areas. Our group is working to meet the need for a low-cost diagnostic tool by developing a bacterial biosensor that produces different visible outputs based on the concentration of zinc in which the cells grow. To make this sensor easy-to-use and field-friendly, we have now engineered cells that completely repress pigmentation during growth, and then upon induction produce different pigments based on zinc concentration. This allows sample addition to concentrated pre-cultured colorless cells, allowing assay results in under 3 hours.

To do this, we engineered hybrid promoters that respond to both an exogenous inducer (to enable pigmentation) and zinc (to control which pigment is produced) by adding operator sites for the zinc-responsive repressor Zur to standard inducible promoters. The best-performing hybrid promoter was incorporated into circuits with other zinc-responsive elements to create a multicolor zinc sensor that is colorless during preculture and, upon induction, produces one of three pigments to indicate relative zinc concentration.

To increase clinical relevance, we next moved Zur’s switching point from its natural threshold (0.1 μM zinc) to physiologically relevant serum zinc levels (5-10 μM). While analyte affinity of some sensors can be readily tuned, changing a transcription factor’s affinity for its analyte by orders of magnitude without altering DNA binding activity is challenging. The most effective strategy to move the switching point was controlling Zur expression with a zinc-activated promoter. Through modulation of transcription factor expression levels, we created a library of sensors with switching points at extracellular zinc concentrations between 1 and 20 μM.

Taken together, our efforts have enabled a biosensor with potential for future impact on millions of lives in the developing world, with design and tuning strategies that may be generalizable to other nutrients and sensors.