Improving the photostability of bright monomeric orange and red fluorescent proteins

Shaner, Nathan C.; Lin, Michael Z.; McKeown, M.; Steinbach, Paul; Hazelwood, Kristin L.; Davidson, Michael W.; Tsien, Roger Y.

doi:10.1038/nmeth.1209

Cited by 923 publications

(958 citation statements)

References 32 publications

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“…Control experiments with IPTG and preirradiated PA‐IPTG solutions at equivalent concentrations were also performed. Whereas red fluorescence was observed 1.5 h after induction with soluble IPTG, in agreement with RFPs maturation half time of 100 min,31 the irradiated PA‐IPTG samples took ≈1.5 h longer to display visible levels of red fluorescence ( Figure 3 a). The time between induction with PA‐IPTG and protein expression is intrinsic to this system and has been associated to the formation of photolysis intermediates that get hydrolyzed to active IPTG by machinery within the E. coli (Figure 1; Figure S2, Supporting Information) 24, 25…”

supporting

confidence: 73%

Toward Light‐Regulated Living Biomaterials

et al. 2018

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Living materials are an emergent material class, infused with the productive, adaptive, and regenerative properties of living organisms. Property regulation in living materials requires encoding responsive units in the living components to allow external manipulation of their function. Here, an optoregulated Escherichia coli (E. coli)‐based living biomaterial that can be externally addressed using light to interact with mammalian cells is demonstrated. This is achieved by using a photoactivatable inducer of gene expression and bacterial surface display technology to present an integrin‐specific miniprotein on the outer membrane of an endotoxin‐free E. coli strain. Hydrogel surfaces functionalized with the bacteria can expose cell adhesive molecules upon in situ light‐activation, and trigger cell adhesion. Surface immobilized bacteria are able to deliver a fluorescent protein to the mammalian cells with which they are interacting, indicating the potential of such a bacterial material to deliver molecules to cells in a targeted manner.

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supporting

confidence: 73%

Toward Light‐Regulated Living Biomaterials

et al. 2018

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“…Numerous GFP variants exhibit some degree of (generally undesirable) reversible photoswitching 4,23,24 . We found that the fluorescence of the yellow fluorescent protein Citrine 25,26 , a derivative of GFP, can be reversibly modulated to a small extent by alternate irradiation with light of 365 nm (on switching) and 405 nm (off switching), whereas fluorescence is excited at 515 nm.…”

Section: Generation Of the Rsfp Dreiklangmentioning

confidence: 99%

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

et al. 2011

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VOLUME 29 NUMBER 10 OCTOBER 2011 nature biotechnology A r t i c l e sFluorescent proteins (FPs) 1 whose fluorescence can be reversibly or irreversibly switched by optical irradiation have opened new opportunities for the imaging of cells. They have facilitated in vivo protein-tracking schemes 2,3 , applications based on singlemolecule observations 4,5 and fluorescence microscopy with subdiffraction resolution [6][7][8][9][10] .Still, photoswitchable proteins have not displayed their full potential, because proteins that are just photoactivatable 11-13 can be switched only once, which implies that repeated measurements with the same molecule are impossible. On the other hand, photochromic or reversibly switchable fluorescent proteins (RSFPs) can be repeatedly photoswitched between a fluorescent and a nonfluorescent state by irradiation with light of two different wavelengths. However, in all previously characterized RSFPs, the wavelength used for generating the fluorescence emission is identical to one of the wavelengths used for switching the fluorescence on or off. The result is a complex interlocking of switching and fluorescence readout [14][15][16][17][18][19][20][21][22] , impeding or even precluding many applications, including fluorescence nanoscopy (super-resolution microscopy). Hence, the identification of an RSFP in which the generation of fluorescence is disentangled from switching has long been pursued. RESULTS Generation of the RSFP DreiklangNumerous GFP variants exhibit some degree of (generally undesirable) reversible photoswitching 4,23,24 . We found that the fluorescence of the yellow fluorescent protein Citrine 25,26 , a derivative of GFP, can be reversibly modulated to a small extent by alternate irradiation with light of 365 nm (on switching) and 405 nm (off switching), whereas fluorescence is excited at 515 nm. However, the achievable contrast was low, especially at pH values >6, rendering the reversible switching of Citrine unusable (Supplementary Fig. 1).To further develop this unusual switching behavior, we performed extensive random mutagenesis as well as directed PCR-mediated mutagenesis on a plasmid encoding Citrine. We transformed Escherichia coli with the plasmid, and screened with an automated home-built fluorescence microscope for bacterial colonies expressing fluorescent proteins whose fluorescence was excited with green light (515 nm) and which could be reversibly photoswitched from a fluorescent state to a long-lived nonfluorescent state by irradiation with near-UV (405 nm) light and back to a fluorescent state by UV (365 nm) light (Fig. 1a). In several consecutive screening rounds ~70,000 individual clones were analyzed. Finally, we identified a mutant differing from Citrine at four positions (Citrine-V61L, F64I, Y145H, N146D) ( Supplementary Fig. 2), which can be effectively switched and excited to fluoresce. We named this switchable fluorescent protein Dreiklang, the German word for a three-note chord in music.At thermal equilibrium, Dreiklang adopts the brightly fluorescent ...

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“…The vectors used express a fluorescent protein under the control of the spleen focus-forming virus (SFFV) promoter. To complement the colors of the vectors LeGO-G2 (expressing EGFP, green) and LeGO-V2 (expressing Venus, yellow), 17 four other fluorescent proteins were cloned: namely, EBFP2 (blue; a gift from Robert Campbell) (plasmid 14891; Addgene), 36 T-Sapphire (violet excitable green; a gift from Oliver Griesbeck), 37 mOrange2 (orange; a gift from Lalita Ramakrishnan) (plasmid 30175; Addgene), 38 and dKatushka2 (red; a gift from Lalita Ramakrishnan) (plasmid 30181; Addgene). 39 The six LeGO vectors used for OBC are shown in Table 1.…”

Section: Lentiviral Vectorsmentioning

confidence: 99%

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

et al. 2017

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Intratumoral heterogeneity has been identified as one of the strongest drivers of treatment resistance and tumor recurrence. Therefore, investigating the complex clonal architecture of tumors over time has become a major challenge in cancer research. We developed a new fluorescent "optical barcoding" technique that allows fast tracking, identification, and quantification of live cell clones in vitro and in vivo using flow cytometry (FC). We optically barcoded two cell lines derived from malignant glioma, an exemplary heterogeneous brain tumor. In agreement with mathematical combinatorics, we demonstrate that up to 41 clones can unambiguously be marked using six fluorescent proteins and a maximum of three colors per clone. We show that optical barcoding facilitates sensitive, precise, rapid, and inexpensive analysis of clonal composition kinetics of heterogeneous cell populations by FC. We further assessed the quantitative contribution of multiple clones to glioblastoma growth in vivo and we highlight the potential to recover individual viable cell clones by fluorescence-activated cell sorting. In summary, we demonstrate that optical barcoding is a powerful technique for clonal cell tracking in vitro and in vivo, rendering this approach a potent tool for studying the heterogeneity of complex tissues, in particular, cancer.

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Improving the photostability of bright monomeric orange and red fluorescent proteins

Cited by 923 publications

References 32 publications

Toward Light‐Regulated Living Biomaterials

Toward Light‐Regulated Living Biomaterials

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

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