Engineering and screening of genetically encoded FRET Calcium indicators

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