Glutamine is an important amino acid that exists throughout the human body. Current measurement techniques for glutamine are unable to give spatio temporal information, and are not thermostable at biological temperatures, meaning they are not viable options for in vitro measurement of glutamine. This inability to measure glutamine within it’s natural environment means that we are unable to fully understand it’s functions. Glutamine binding protein (GlnBP) is a periplasmic protein that is found in e.coli cells, which when bound to glutamine undergoes a large conformational change, making it a perfect candidate as a FRET biosensor. Ancestral sequence reconstruction was performed to identify a statistically probable ancestor of this modern protein, which was more thermostable, but was also promiscuous for arginine. Point mutations were made in the binding region of the ancestral protein in an attempt to increase specificity to glutamine and decrease specificity to arginine. The mutant glutamine binding proteins were equipped with fluorophores, expressed, purified and assayed using spectrophotometry. Although still incomplete, we have been able to identify a number of mutations which can greatly increase specificity to glutamine. Once complete, we will have created a thermostable glutamine FRET biosensor. This sensor will be used to give spatio-temporal information about the functions of glutamine in in vitro experiments, furthering our understanding of the role of this important amino acid.