Executive Summary : | The proposal proposes a genetically encoded fluorescence resonance energy transfer (FRET) nanosensor for measuring heparin levels in mammalian cells. This method offers advantages over traditional methods, such as high specificity, simplified detection processes, and wider dynamic range. The sensor is less invasive and can be optimized for different biological systems. Key questions include calculating heparin concentration, stability, specificity, and binding affinity. The study aims to design and construct the sensor, characterize it in vitro and in vivo, and develop affinity mutants. The proposed project aims to develop a heparin nanosensor protein in five years, focusing on key milestones such as design, optimization, specificity, in vitro and in vivo characterization. The project will involve searching for a suitable binding protein, amplifying sequences, and ligand titration curves. The nanosensor will be characterized in vitro using a microplate reader, and in vivo in eukaryotes using yeast and mammalian cells. The goal is to develop a genetically encoded FRET-based heparin sensor that can be non-invasively targeted to cellular and sub-cellular compartments of living cells, allowing for in vivo heparin concentration measurements with high spatial-temporal resolution. |