Early Development of a Glyphosate Biosensor

Abstract

Glyphosate is a member of the class of molecules known as phosphonates and is the active ingredient in the herbicide “Roundup”. As the use of “Roundup Ready” crops increases across the globe, growing concerns regarding environmental contamination create demand for an effective method to detect glyphosate. The aim of our project was to convert a bacterial protein into a biosensor for glyphosate. We used phosphonate binding protein (PhnD), which naturally binds to phosphonates, but has a low affinity for glyphosate ((Kd ≈ 650μM). Our goal was to increase the affinity of PhnD for glyphosate. To accomplish this, we designed and then tested three mutants of PhnD for their ability to bind glyphosate. The mutant proteins were expressed recombinantly in bacteria, isolated, and tagged with the fluorescent reporter Alexa 488 for analysis of binding via fluorescence spectroscopy. We found that introducing asparagine or serine mutations at position 205 had no significant effect on affinity. However, an asparagine mutation at position 177 improved binding affinity for glyphosate by approximately 100-fold (Kd ≈ 8μM). While improvements need to be made in the future, this mutant in particular represents a significant step toward the creation of a useful biosensor and a reliable way to test glyphosate concentrations in soil.