Development of a Fast-Responding, Minimal Equipment Biosensor for Zinc Deficiency

McNerney, M., Georgia Institute of Technology
Styczynski, M. P., Georgia Institute of Technology

Zinc deficiency contributes to hundreds of thousands of childhood deaths annually, but current detection methods are cost-prohibitive for use in the regions most affected. Our group recently developed a low-cost bacterial biosensor that can produce different visible outputs based on the concentration of zinc in which the cells grow. However, these initial sensor cells always produce some pigment, which necessitates that they be grown from a very small inoculum so that initial coloration is minimal – thus requiring overnight assay times. To make an easy-to-use, field-deployable assay, we are engineering cells that completely repress pigmentation during growth and upon induction quickly produce different pigments based on zinc concentration. This would allow sample addition to concentrated pre-cultured cells, decreasing assay time from about 24 hours to less than 3 hours.

We first focused on reducing unwanted pigment expression and enabling fast production of the red pigment lycopene upon induction. A sensor that produces a pigment instead of fluorescent proteins is advantageous in resource-limited settings, since sensor readout can be interpreted without advanced equipment. However, complete repression of pigment production requires more precise control than what is necessary for most synthetic biology applications, since small amounts of enzyme can produce visible amounts of pigment, (especially over long times as may be necessary to pre-culture sensor cells). To combat unwanted pigment expression, three commonly used inducible promoter systems—the lac, ara, and T7 systems—were engineered to be more repressible. Despite orders of magnitude decreases in uninduced protein expression, even the most repressible systems still could not control lycopene production. Translational modifications proved much more effective at controlling lycopene production. When the original ribosomal binding sites for the crtE, crtB, and crtI genes were replaced with weak RBSs, all systems could fully repress lycopene production and produce visible lycopene within three hours of induction. Supplementation of metabolic precursor pathways further reduced the time required for visible pigmentation to 1.5 hours.

To engineer a zinc-sensitive response, we next incorporated zinc-responsive elements into the engineered promoter systems. Operator sites for the zinc-responsive transcription factor Zur were introduced into lac, ara, and T7 promoter systems to create systems that respond both to an exogenous inducer (either IPTG or arabinose) and to zinc. Promoters from all groups demonstrated a response to both exogenous inducer and zinc, and the best responding promoter was a T7-based promoter with binding sites for Zur and LacI. These promoter systems were used to control expression of violacein, and transcriptional switches and inducible protein degradation were explored as methods to increase the dynamic range of the sensor. Additional zinc-responsive promoters and pigment pathways can be added to create a multi-color litmus test for zinc concentration.

To engineer a system that produces specific pigments based on external state, translational control eliminated unwanted pigment expression, and transcriptional control enabled dual regulation. As the applications of synthetic biology expand to systems that require more precise control of metabolic state, multiple layers of regulation will likely be necessary to engineer the desired output.