Developing a Fluorescence Biosensor for Glyphosate Using a Bacterial Protein

Oral Presentation

Prepared by S. Rizk, P. N'Guetta, M. Fink
Indiana University South Bend, 1700 Mishawaka Ave, 047 Northside Hall, South Bend, Indiana, 46634, United States


Contact Information: srizk@iusb.edu; 574-520-4653


ABSTRACT

Glyphosate is a phosphonate used to kill weeds by blocking pathways essential to plant growth. Currently, glyphosate is the most popular herbicide used around the globe. It has been recently classified as a probable carcinogen. It is also considered a potential environmental hazard. Several states are planning to restrict its use, while it has already been banned in the state of California. Hence, there is a need for the development of reliable detection methods for glyphosate in the soil, rivers or drinking water. This can help determine its concentration and effect on the environment. We took a protein engineering approach to develop a fluorescence-based biosensor for glyphosate. Phosphonate binding protein (PhnD) is a bacterial protein that binds to a variety of phosphonates with different affinities. This protein allows the bacteria to sequester phosphorus from the environment. Previous work showed that the protein binds to glyphosate but with very low affinity (dissociation constant 0.65 mM). Guided by the crystal structure of the PhnD, we constructed a set of mutations in the binding region in order to enhance binding affinity for glyphosate. One of the mutants showed nearly ~100-fold improvement in the binding affinity, allowing detection of glyphosate in the low micromolar range. The main advantage of using PhnD as a biosensor is that the protein undergoes a large conformational change upon binding to glyphosate. By attaching an environmentally sensitive fluorophore, binding can be detected by monitoring changes in fluorescence. Furthermore, the fluorescence change is ratiometric, allowing for a standardized way to correlate fluorescence ratios with glyphosate concentrations. We believe that this biosensor can be incorporated in a device that allows real-time detection of glyphosate levels in soil or water to help with environmental monitoring and decontamination efforts.