Round Table Seminar: Cindy Ast, Frommer Lab - Carnegie Institution - Plant Department, Designing the next Generation of Sensors – The Matroshkas.

We recently developed a set of novel activity state sensors called AmTracs. AmTracs are genetically encoded single fluorophore sensors based on circularly permuted (cp) variants of the Enhanced Green Fluorescent Protein (EGFP) inserted into the Ammonium Transporter AMT1;3. All sensors are specific for ammonium, dose-dependent and reversible in their response, which is strictly linked to the transport activity of the AMT. Thorough photophysical analysis revealed that modifying the linker amino acids as well as folding and maturation properties of the fused EGFP variant modulates the sensor’s fluorescence intensity and response. Currently we are exploring new strategies of rendering AmTrac ratiometric. One approach is based on the insertion of an orange or red fluorescent protein into a loop of the cpEGFP in AmTrac. These sensor constructs, named Matroshkas, are at a very early stage of development but provide a promising perspective of ratiometric sensor design.