CH391L/S14/GFP

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Contents

Introduction

GFP expression in Arabidopsis plant owu.edu
Green Fluorescent Protein (GFP) is a small 27 kD, 238
Ribbon structure of GFP (blue) and chromophore (green) Conncoll.edu
amino acid protein that was discovered in the jellyfish Aequorea victoria. It has since become a valuable asset to researchers in the life sciences. The ease with which the gene coding for GFP can be inserted into various bacterial plasmid vectors has rendered it extremely popular as a reporter protein. In this capacity it can provide information with respect to integration and expression of a particular gene of interest when inserted downstream from a gene. It can also serve as a marker if inserted into the genetic code of a protein before its natural stop codon, allowing identification of protein location and distribution within the cell without affecting normal protein function. Additionally, it has become popular as a biosensor- providing information about metabolic activity, oxygen availability, pH and more. Efforts to expand these applications appear promising, and already multiple derivatives of GFP have been created that span the visible spectrum, enabling even more complex cellular analyses to be performed [1].

History

File:FPbeachTsien.jpg
Multiple GFP derivatives expressed in culture Tsien Lab
While a green fluorescent "substance" was first observed and described in 1955 by British scientist D. Davenport, 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 [2]. 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 [3]. 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 [4], the subsequent discovery of yellow GFP (yGFP) [5], 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 Mechanism

Mechanisms of chromophore activation showing two proposed cyclization reaction schemes. Following cyclization, HBI is able to absorb light very efficiently which then results in fluorescence of the molecule. Conncoll.edu
These photochemical properties of GFP are a direct result of its protein structure. The 238 residues are assembled into beta sheets, which in turn come together to form a tertiary structure that resembles a cylinder. Within this cylinder, tethered to residues 64 and 170 (designated by R groups in the image at left), is the chromophore responsible for both absorbing light and giving GFP its characteristic green fluorescent emission, 4-(p-hydroxybenzylidene)imidazolidin-5-one (HBI). Suggested mechanisms of chromophore activation are displayed in the figure to the right, with the unique cylindrical shape of the protein protecting HBI from unwanted environmental contact that could result in a quenching event [6]. This activation is demonstrated in a short tutorial which emphasizes the autocatalytic nature of this
GFP chromophore Conncoll.edu
activation. This is important because it means that no cofactors or enzymes are required for GFP activation, and thus it is species-independent, which expands the range of its use.

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 [6].

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
This is perhaps the oldest and most familiar application, involving the insertion of the GFP gene into the genetic sequence of the system being studied. This is facilitated most often via plasmid vector delivery. Once the GFP genetic code has been inserted downstream of the promoter of the gene coding for the protein of interest, any GFP expression will indicate that the gene of interest is being transcribed as well. Moreover, this expression can be quantified, and depending on particular factors influencing the promoter can be used as to measure the effects of certain manipulations meant to affect gene expression [7].
Cell showing localization of GFP tagged alpha tubulin Evrogen
  • Fluorescent tagging (molecular marker)
In this case the gene for the protein of interest has GFP inserted before the stop codon, such that the protein itself is transcribed with a GFP 'tag' attached. Observation of GFP fluorescence, then, indicates exactly where the protein of interest is localized within the cell and enables real-time tracking of the protein [8]. This can also be used to track RNA, as shown in the image to the right.
"Tracking mRNA: The green lines show individual mRNA molecules labeled with molecular beacons moving around the chromatin (blue) within a mammalian cell nucleus. Red dots show dense locations where mRNA cannot move freely; yellow dots show more open spaces within the chromatin." Sanjay Tyagi, University of Medicine and Dentistry of New Jersey The Scientist
  • Biosensors

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 [9].

  • Optogenetics
A relatively new area of investigation that frequently utilizes GFP is optogenetics. With this technique, through careful genetic engineering of animal lines, expression of membrane localized opsin proteins fused with GFP occurs in specific cell types. Upon the addition of light of the given wavelength, the opsins are activated and can induce neuronal firing [10]. Experiments have been done investigating the effectiveness of this induced firing on manipulating behavior, as in this video.
Zebra fish glow in various colors National Geographic
  • 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 [11].

  • 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.

iGEM

Within 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.
Common GFP variants

References

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  1. Error fetching PMID 21714025: [Remington2011]
    Review article exploring the physical qualities and existing applications of GFP
  2. Error fetching PMID 8303295: [Chalfie1994]
    First paper to explore applications of GFP as a reporter for gene expression
  3. Error fetching PMID 7809066: [Tsien1994]
    First step towards refining GFP as a biological tool
  4. Error fetching PMID 7854443: [Tsien1995]
    Enhanced GFP
  5. Error fetching PMID 8703075: [Remington1996]
    yGFP
  6. Error fetching PMID 9759496: [Tsien1998]
    Tsien's 1998 review with extensive descriptions of GFP structure, chromophore activation, applications
  7. Error fetching PMID 8777060: [Kitts1995]
    GFP as a reporter
  8. Error fetching PMID 12588095: [Matejczyk2002]
    GFP as a molecular marker
  9. Error fetching PMID 10087619: [Heim1999]
    GFP and FRET
  10. Error fetching PMID 16116447: [Deisseroth2005]
    GFP in optogenetics
  11. Error fetching PMID 24092439: [Hoffman2014]
    In vivo cancer cell imaging with GFP
All Medline abstracts: PubMed | HubMed