On the Friday before Thanksgiving, IUPUI hosted the 24th annual IU Undergraduate Research Conference. Biology major Pierre-Emmanuel N'Guetta travelled downstate to present his work over the past several years under his mentor and biochemistry professor Shahir Rizk. N'Guetta represented IU South Bend amazingly well and came in 3rd place for best poster from the entire IU system. He was first selected as one of the finalists and then was required to give an impromptu presentation in front of a panel of judges. The title and abstract of his presentation are given below. Congratulations on a wonder performance!
Engineering a Fluorescent Biosensor for the Detection of Herbicide Glyphosate
Glyphosate is the active ingredient in the herbicide Roundup and is used to kill weeds. Nowadays, glyphosate is the most popular herbicide used around the globe. It has been classified as a probable carcinogen. Several states are planning to restrict its use. Hence, there is a need for the development of detection methods for GP. The E. coli phosphonate binding protein naturally binds to GP, making it a good candidate for the development of a glyphosate fluorescent biosensor. This happens with a very low affinity limiting PhnD ability to detect low concentrations of GP. Our goal is to increase the affinity of PhnD for glyphosate. PhnD undergoes a conformational change upon binding to its ligand. We can take advantage of this conformational hange to develop PhnD into a fluorescent sensor for lyphosate, where a fluorescent reporter group is attached o PhnD. In this work, we introduced a mutation in the inding pocket of PhnD (E177N) that improved affinity by 100-fold. We also sought to take advantage of the equilibrium property by engineering antibody fragments that bind specifically to the closed form of PhnD. We utilized engineered antibody fragments (Fabs) to improve the affinity for GP. The Fabs were designed to stabilize the bound form of PhnD. Five Fabs were tested, one was found to increase affinity of GP to PND by an additional 10-fold. The combined effect of the mutation and the Fab result in a 1000-fold affinity enhancement, allowing the development of sensitive biosensors for GP pollution.
