Glycolysis is a critical metabolic pathway that is dysregulated in many diseases, including cancer, which is often characterized by a metabolic shift that increases the glycolytic flux, leading to excessive lactate generation, which can serve as an energy source – the Warburg effect. Many aspects of the mechanisms and role of dysregulated glycolysis in disease remain unclear, as current methods for both lactate and glycolytic flux measurements require cell lysis, therefore making compartment-specific dynamic measurements impossible. The aim of this study is to develop biosensors for lactate and upper glycolytic intermediates such as glucose-6-phosphate, as representative of the glycolytic flux. These will allow to image glycolysis dynamically in living cells, specifically, with a suitable dynamic range, and subcellular resolution. By inserting a fluorescent protein (FP) in the transcription factors LldR for lactate, and CggR for upper glycolytic intermediates, several promising biosensor candidates were generated for CggR, but not LldR. To find alternative candidates for a functional lactate biosensor, a phylogenetic analysis was performed on LldR. As a next step, functional constructs will be optimized by generating a library by random mutagenesis. In preparation, expression conditions were optimized, and two scripts were written in Python that would facilitate the analysis and selection of promising candidates. Once optimized, these biosensors will become an invaluable tool for glycolytic flux measurements and will allow us to address several biological questions on the role of glycolysis in health and disease, including the regulation of the Warburg effect in cancer cells.
Generating a Lactate Biosensor: a new tool to analyse cancer cell metabolism in vivo
Pérez Chávez, I. ((PhD) Student), Ezeriņa, D. (Promotor), Messens, J. (Promotor). 2020
Student thesis: Master's Thesis