The Twinkle Factory labeling technology enables the specific fluorescent labeling of any protein of interest (POI). It is based on the instantaneous formation of a fluorescent molecular assembly between the small (14 kDa), genetically encoded, protein tag FAST and various fluorogenic ligands (TFFluorogens). TFFluorogens strongly fluoresce only when bound to FAST, enabling to detect and image FAST-tagged proteins with high contrast without the need of washing the excess of fluorogenic ligands. The labeling of FAST-tagged proteins with a TFFluorogen is non-covalent and can be reversed if necessary by washing. Furthermore, FAST does not require molecular oxygen for being fluorescent, unlike fluorescent proteins, enabling to fluorescently label proteins in weakly oxygenated or anaerobic environments, helpful for biofilm imaging, or for any anaerobe imaging, e.g., Clostridium.

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