M. (Martijn) van Rosmalen MSc - Expertise
P.O. Box 513
5600 MB EINDHOVEN
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Yeast Display for Engineering new FRET-sensors Yeast display and other directed evolution approaches – randomly generating millions of mutants and selecting the best ones by thigh-throughput screening – have been very successful for engineering many different classes of proteins. However, to date the power that directed evolution offers has not yet been used for the engineering of genetically encoded FRET-sensors. FRET-sensors are valuable tools for noninvasively imaging cellular signaling molecules, metabolites, proteins, RNAs, post-translational modifications etc. inside living cells, but the toolbox of available FRET-sensors is still very limited owing to difficulties in their design and lack of an effective high-throughput approach to ‘evolve’ such sensors. The aim of my research is to apply yeast display and fluorescence activated cell sorting (FACS) to the development of new FRET sensors. Yeast display is a directed evolution approach in which a library of millions of yeast cells is constructed, each presenting a unique variant of a sensor fused to the Aga2p protein, which is tethered to the outside of the cell wall. Individual mutants are then selected by FACS based on their respective change in FRET in response to a specific ligand that the sensor is aimed to detect.