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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By using different TFFluorogens, the spectral properties of the FAST-tagged protein can be changed without the need to switch protein tags, providing an experimental versatility not encountered with fluorescent proteins.

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The labeling of FAST-tagged proteins can be furthermore restricted to the cell-surface by using TFFluorogens unable to cross efficiently the cell membrane, enabling the study of protein trafficking at the membrane with various fluorimetric techniques, even including non-imaging flow cytometry.

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The use of the Twinkle Factory labeling technology implies cloning and expression of the FAST-tagged protein, and labeling of the resulting fusion with the TFFluorogen of choice. Note that proteins of interest can be expressed with FAST as either an N- or a C-terminal fusion.

References

Princeps paper and history of FAST

A small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo. Proceedings of the National Academy of Sciences (PNAS) 113(3), 497–502 (2016). M.-A. Plamont, E. Billon-Denis, S. Maurin, C. Gauron, F. M. Pimenta, C. G. Specht, J. Shi, J. Querard, B. Pan, J. Rossignol, K. Moncoq, N. Morellet, M. Volovitch, E. Lescop, Y. Chen, A. Triller, S. Vriz, T. Le Saux, L. Jullien & A. Gautier – archives ouvertes HALrequest

Dynamic multi-color protein labeling in living cells. Chemical Science 8, 5598–5605 (2017). C. Li, M.-A. Plamont, H. Sladitschek, V. Rodrigues, I. Aujard, P. Neveu, T. Le Saux, L. Jullien & A. Gautier – request

High-throughput, image-based screening of pooled genetic-variant libraries. Nature methods14(12), 1159 (2017). G. Emanuel, J. R. Moffitt & X. Zhuang

Fluorogenic probing of membrane protein trafficking. Bioconjugate Chemistry 29, 1823–1828 (2018). C. Li, A. Mourton, M.-A. Plamont, V. Rodrigues, I. Aujard, M. Volovitch, T. Le Saux, F. Perez, S. Vriz, L. Jullien, A. Joliot & A. Gautier – archives ouvertes HALrequest

Phenolic Pyrogallol Fluorogen for Red Fluorescence Development in a PAS Domain Protein. Chemistry of Materials30(5), 1467-1471 (2018). Kim, J. K., Lee, H. A., Lee, H., & Chung, H. J.

Circularly permuted fluorogenic proteins for the design of modular biosensors. ACS Chemical Biology 13, 2392–2397 (2018). A. G. Tebo, F. M. Pimenta, M. Zoumpoulaki, C. Kikuti, H. Sirkia, M.-A. Plamont, A. Houdusse & A. Gautier – archives ouvertes HALrequest

Improved chemical-genetic fluorescent markers for protein imaging in living cells, Biochemistry 57, 5648–5653 (2018). A. G. Tebo, F.M. Pimenta, Y. Zhang & A. Gautier – request

Spying on cells with chemical-genetic hybrids (Espionner les cellules avec des hybrides chémogénétiques). L’Actualité Chimique 435, 31–35 (2018)A. Gautier – request

Red‐shifted substrates for FAST fluorogen‐activating protein based on the GFP‐like chromophores. Chemistry–A European Journal (2019). N. V. Povarova, S. O. Zaitseva, N. S. Baleeva, A. Y. Smirnov, I. N. Myasnyanko, M. B. Zagudaylova, … & A. S. Mishin

A split fluorescent reporter with rapid and reversible complementation. Nat. Commun. 10, 2822 (2019). A. G. Tebo & A. Gautier

About low-oxygen and anaerobic conditions

A strongly fluorescing anaerobic reporter and protein-tagging system for Clostridium organisms based on the Fluorescence-Activating and Absorption-Shifting Tag (FAST) protein. Appl. Environ. Microbiol., AEM-00622 (2019). H. E. Streett, K. M. Kalis & E. T. Papoutsakis

Engineering Clostridium organisms as microbial cell-factories: challenges & opportunities. Metabolic engineering 50, 173-191 (2018). K. Charubin, R. K. Bennett, A. G. Fast & E. T. Papoutsakis

The inducible chemical-genetic fluorescent marker FAST outperforms classical fluorescent proteins in the quantitative reporting of bacterial biofilm dynamics. Scientific reports 8(1), 10336 (2018). A. Monmeyran, P. Thomen, H. Jonquière, F. Sureau, C. Li, M.-A. Plamont, … & N. Henry

About super resolution and single-molecule imaging

Single-molecule localization microscopy with the Fluorescence-Activating and absorption-Shifting Tag (FAST) system. ACS Chemical Biology (2019). E. M. Smith, A. Gautier & E. M. Puchner

Live cell super resolution imaging by radial fluctuations using fluorogen binding tags. Nanoscale 11(8), 3626-3632 (2019). M. Venkatachalapathy, V. Belapurkar, M. Jose, A. Gautier & D. Nair

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