Development of Novel Sentinel Species-Based Bacterial Biosensors for Estrogenic Compounds

Estrogenic compounds are an important class of chemicals that have the ability to modulate the function of the nuclear hormone receptor, estrogen receptor beta (ERβ). These compounds can be found naturally in the environment or chemically synthesized, and have been linked to health risks ranging from altered sexual development to breast cancer. Our previous work resulted in an Escherichia coli cellular biosensor that is comprised of an engineered protein scaffold with an inserted human ERβ ligand-binding domain (LBD) fused to a thymidylate synthase (TS) reporter enzyme. Through growth in thymineless media, the biosensor can distinguish between estrogen agonists and antagonists based on the resulting E. coli growth phenotype. This research seeks to extend the ERβ biosensor method to include sentinel animal species, such as the zebra finch and wood frog, which serve as early indicators of the presence of environmental toxins. Preliminary findings using ERβ biosensors from several mammals demonstrated species-specific responses to ligands, suggesting species-specific susceptibility to certain endocrine disruptors. Using protein sequence alignments and PCR cloning, novel zebra finch and wood frog-based ERβ biosensors were created and each biosensor was validated against five known estrogens using the bacterial assay method. Mammalian and fish biosensors were also tested and a general trend of unique responses to weak and strong estrogens based on animal class was observed. The animal biosensors will be moved into higher sensitivity constructs in an attempt to improve ligand response. Adaptation of the ERβ biosensor to sentinel species, in particular, will enable the prevention and treatment of diseases through early detection of estrogenic compounds in the environment and expand the bacterial biosensor library. The creation of additional animal ERβ bacterial biosensors will also provide a broader understanding of how drug response deviates across species.