An mTurquoise2-Based Glucose Biosensor

Glucose is an important substrate for organisms to acquire energy needed for cellular growth. Despite the importance of this metabolite, single-cell information at a fast time-scale about the dynamics of intracellular glucose levels is difficult to obtain as the current available sensors have drawbacks in terms of pH sensitivity or unmatched glucose affinity. To address this, we developed a convenient method to create and screen biosensor libraries using yeast as a workhorse. This resulted in TINGL (Turquoise INdicator for GLucose), a robust and specific biosensor with an affinity that is compatible with intracellular glucose detection. We show that the sensor can be calibrated <i>in vivo</i> (i.e., intracellular) through equilibration of internal and external glucose in a yeast mutant unable to phosphorylate glucose. Using this method, we estimated dynamic glucose levels in budding yeast during transitions to glucose. We found that glucose concentrations reached levels up to approximately 1 mM as previously determined biochemically. Furthermore, the sensor showed that intracellular glucose dynamics differ based on whether cells are glucose-repressed or not. Finally, the human codon-optimized version (THINGL, Turquoise Human INdicator for GLucose) also showed a robust response after glucose addition to starved human cells, showing the versatility of the sensors. We believe that this sensor can aid researchers interested in cellular carbohydrate metabolism.