HistoryDavenport, the man credited with the isolation and purification of this substance, as well as determining that it was a protein, was Japanese scientist Osamu Shimomura in 1962 . Following its discovery and identification, many years passed before its potential applications were fully understood. In 1987, Douglas Prasher conceived of the idea that GFP might be able to be utilized as a biological tracer, but unfortunately was only able to clone GFP once in 1992 before running out of funding. In 1994, however, American research scientist Martin Chalfie along with his collaborators published a paper entitled "Green Fluorescent Protein as a Marker for Gene Expression" which would have an extremely significant impact on the way GFP was utilized . Later that same year, Roger Tsien, in collaboration with Prasher and another colleague, began the task of characterizing and manipulating the fluorescence of GFP in order to improve its function as a biological tool . Their research resulted in not only a GFP with a better quantum yield, but one that was less resistant to photobleaching, possessed only one absorbance peak, and whose excitation peak was much better suited for detection using a TRITC filter set. Additionally, they discovered a mutant that fluoresced at a different wavelength, initiating further investigation into the creation of different "colored" mutants. Following these discoveries, focus continued to be placed upon improving GFP and investigating variants, with the discovery of Enhanced GFP (eGFP) in 1995 , the subsequent discovery of yellow GFP (yGFP) , and others. By 1998, Tsien himself published a review article investigating the already astounding numbers of applications for GFP and its variants, including use as a marker for gene expression, protein targeting, and the potential for use as a biosensor. In 2011, Remington released another review detailing the biosensor applications and a much more refined understanding of the photochemical properties of GFP.
Protein Structure and Chemical Mechanism6]. This activation is demonstrated in a short tutorial which emphasizes the autocatalytic nature of this
With respect to activation, wild type GFP from Aequorea victoria is readily capable of absorbing blue light, with the maximum absorbance wavelength occurring at 395 nm, and a second, smaller peak absorbance wavelength occurring at 470 nm. Its subsequent emission occurs in the green portion of the visible spectrum with a peak wavelength occurring at 509 nm, and a smaller peak also occurring at 540 nm, giving the protein its name .
In Modern Synthetic Biology
As indicated above, there are a plethora of ways in which GFP serves as a valuable tool in the life sciences, and due to the far-reaching nature of synthetic biology, the number of applications in this field is high. The following represents a few of the many potential applications:
- Monitoring of gene expression
- Fluorescent tagging (molecular marker)
The self-catalyzed nature and relative stability of the activation of the GFP chromophore make it a very successful biosensor for a number of different cellular parameters. The first is pH, as the activation process is sensitive to protonation and deprotonation. Secondly, it can be used to detect enzymatic activity such as cleavage of a substrate. Additionally, it can be used to detect levels of radical oxygen species or other small molecules such as calcium cations. A short review by a number of researchers at Rensselaer Polytechnic Institute last year explains these various applications. A large portion of the research that involves GFP as a biosensor relies on the use of fluorescence resonance energy transfer (FRET), between either two GFPs or GFP and a second fluorophore, as an analytical tool .
- Tumor labeling
Similar selective expression in cell types has also been used to track the growth and movement of metastatic cancer cells throughout the body. Cells designed to express constant levels of GFP can be tracked throughout the body without invasive procedures. In addition, this is being used to study clean excisions of localized tumors .
- In animals
GFP has also been used to indicate that animals are clones by providing an easy method to check for this without having to perform DNA sequencing. An animal of interest that started out in a lab but ended up in popular culture is the zebra fish, which is now available for the public to purchase. The various strains glow green (GFP) yellow (Yellow Fluorescent Protein) and red (Red Fluorescent Protein), as pictured at right.
iGEMWithin the iGEM registry, it is clear that the use of GFP as a reporter is one of its most popular applications. Well over half of the reporters listed on the Reporters page are conjugated with GFP. Other efforts include a submission in 2004 which made available the first GFP mutant BioBrick GFPmut3b, submitted by the Antiquity group and designed by Jennifer Braff. In 2008, the Cambridge team made an attempt to standardize additional GFP mutants, and was successful in creating BioBricks for Superfolder GFP and P7 GFP. Recently, in 2012, the Georgia Tech iGEM team branched out and released a complex BioBrick version of split GFP that allows a bacterium to produce two complementary halves of GFP that do not combine or become biologically active unless in the presence of a particular chemical signal, exemplifying its rising popularity as a biosensor.
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Review article exploring the physical qualities and existing applications of GFP
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First paper to explore applications of GFP as a reporter for gene expression
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First step towards refining GFP as a biological tool
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Tsien's 1998 review with extensive descriptions of GFP structure, chromophore activation, applications
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GFP as a reporter
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GFP as a molecular marker
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GFP and FRET
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GFP in optogenetics
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In vivo cancer cell imaging with GFP