Engineering and screening of genetically encoded FRET Calcium indicators

摘要: Fluorescent protein sensors have gained great importance in research as they exhibit a number of advantages over synthetic dyes. They can be targeted precisely, large populations cells imaged simultaneously, and allow for chronic imaging approaches. Many them however still suffer from comparably low signal changes. Improving fluorescent tedious time-consuming. For this reason, efforts been made not only to improve existing sensors, but also develop better strategies them. In work, novel large-scale bacterial based screening assay was established complement rational design. Sensor expression, stimulation, bacteria, well the handling amounts data created by such were optimized. While new adapted other applications, it is especially suited genetically encoded Ca2+ indicators basis FRET (Forster Resonance Energy Transfer). We used optimize utilizing binding Troponin C fused between proteins ECFP cpCitrine. The resulting ‘Twitch’ sensor series exhibited dynamic range up 1000% ratio change, sensitivity fast kinetics. second approach, we attempted similar deploying red-shifted proteins. To end, further conducted orange mKOκ FRET, mKOκ. developed utilized troponin Dreiklang (photoswitchable) addition It bright change approximately 170%. In summary, procedures presented thesis, will facilitate development biosensors, already employed highly effective indicators.