Design and Implementation of Whole-Cell Biosensors for Micronutrient Detection in Low Resource Environments

Micronutrient deficiencies are a significant healthcare concern across the globe, affecting more than 2 billion people and causing billions of dollars in economic damage.  These conditions, though straightforward to treat, remain a problem because they are often difficult to recognize and diagnose, requiring lab tests that are prohibitively expensive in both material and human resources for those in developing or remote areas.  As obligate consumers of the same micronutrients, bacteria possess cellular machinery to control intracellular micronutrient levels and have corresponding regulatory mechanisms to respond to varying concentrations in their environment.  We are developing a novel diagnostic tool based on whole-cell bacterial sensors using designed genetic circuitry to direct existing or minimally engineered cellular machinery to trigger specific changes in color in response to defined micronutrient levels.  Such a test would be cheap, require minimal equipment and minimal training to administer and interpret, allow on-site diagnosis of micronutrient deficiencies in the populations most at risk, and facilitate epidemiological studies of such deficiencies and assessment of the efficacy of intervention strategies on a population scale. 

Towards this goal, we present work on a whole-cell biosensor for the detection of zinc, a key micronutrient; zinc deficiency is responsible for around 1.7% of global all-cause child mortality.  We have demonstrated a multi-output circuit using two native zinc-responsive E. coli transcription factors, ZntR and Zur, and we present a pigment-based reporter strategy based on the violacein and carotenoid pathways which obviates the need for additional detection equipment (which would be required for bioluminescent or fluorescent reporters) while maintaining unambiguous sensor output. We have demonstrated changes in cell color easily visible to the naked eye around medically relevant zinc concentrations and will discuss the strategies used to tune the sensor, including optimizing regulator production, vector selection, altering translational efficiency, and promoting protein degradation.  Using multiple mechanisms in tandem allowed the circuit to change output over a small fraction of the natural dynamic range of the transcription factors used and overcome the inherent challenges of using sequential pigmented metabolites in a single pathway as different circuit outputs. The reporting strategy is modular and is in principle adaptable to reporting other analytes for which differentially regulated transcription factors are available.  The resulting whole-cell biosensor is capable of distinguishing conditions that are high, intermediate, or low in zinc, and is currently being engineered for optimal response in human serum and reduction to practice in a format ready for use in the field.  This work represents the first E. coli whole-cell biosensor for zinc with human-readable output to minimize cost per assay and facilitate epidemiological study of zinc deficiency and evaluate the efficacy of nutritional intervention.