Download Glowing Fish (and other uses for green fluorescent protein)

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EUROPEAN BIOINFORMATICS INSTITUTE PROTEIN OF THE MONTH
The green fluorescent protein (GFP) was first isolated from a species of jellyfish, Aequorea
victoria, which was named after a coastal city on Vancouver Island where it can be found in the
shores of the north Pacific. This colourless, transparent hydromedusa can produce bright green
flashes along the rim of its bell-shaped body in response to external stimuli.
GFP acts as a companion protein to the chemiluminescent protein aequorin, transforming the
blue light emitted from aequorin into green light. Most bioluminescent animals lack the GFP
transformation system and emit blue light, which travels the farthest through seawater.
However, many animals in coastal waters, where A. victoria is found, tend to emit light in the
blue-green range, possibly to compensate for the abundance of particulate matter found in the
water.
The GFP is unique amongst natural pigments for its ability to autocatalyse its own chromophore,
requiring only oxygen to complete its synthesis. In this way a single protein acts as both
substrate and enzyme. Other natural pigments require multiple enzymes for their production.
Biotechnology has taken advantage of this unique feature of GFP, putting it to use as an in vivo
marker of gene expression and protein localisation. Since the discovery of GFP there have been
52 homologous proteins deposited into sequence databases that have been isolated from other
Cnidarians, including various corals and anemones.
One of the most striking finds is that only a few come from bioluminescent organisms; the
majority were isolated from fluorescent and coloured animals that had no bioluminescence. This
means that in most cases GFP-like proteins are not part of a bioluminescent system, but are
simply part of animal colouration. Furthermore, ranges of different colours were found. These
proteins are often classified according to their colour: green (GFP), yellow (YFP), red or
orange-red (RFP), and non-fluorescent purple-blue chromoproteins (CP). These different
proteins are responsible for much of the legendary variety of reef colours, with their abundance
of coral fluorescence.
Interestingly, both fluorescent and non-fluorescent proteins can be found in the same organism.
In the snake-locks sea anemone, Anemonia sulcata, the tentacles exhibit three different hues in
daylight: green on the upper side, orange on the underside, and a red-purple on the tips. After
UV-irradiation, the tentacles exhibit bright green and orange fluorescence, while the red-purple
tips are non-fluorescent.
The diversity of GFP-like proteins has had an impact on biotechnology. These proteins expand
the colour range available, permitting multicolour labelling. Some of these proteins have
interesting features as well. The stony 'green open brain' coral, Trachyphyllia geoffroyi (above),
has a fluorescent protein named Kaede, after the Japanese word for maple leaf. Kaede emits
green fluorescence after synthesis that changes to a stable red fluorescence after UV irradiation,
making this protein useful as an optical cell marker.
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Why so many colours?
The question arising when one sees the myriad of colours in coral reefs is: is this diversity
reflected in the function of these proteins or is it random variation? So far the function of GFPrelated colouration remains elusive. In general, bioluminescence in sea creatures is related to
illuminating areas to find food, signalling for a possible mate, emitting clouds of illuminated
material for self-defence, or mimicking sparkling sunlight to hide their shadow from predators
lurking below. There is evidence that jellyfish may use bioluminescence for defence; some
species of jellyfish release clouds of bioluminescence into the surrounding water to blindingly
distract predators. The localisation of bioluminescence in jellyfish is species-specific, raising the
possibility that it could also function in finding a mate.
Non-bioluminescent colouration could have different functional roles. Coral fluorescence may
function in photoprotection by scattering light, or by converting it into less harmful wavelengths.
It could also serve to enhance photosynthesis in low-light conditions by supplying light of the
correct wavelength to zooxanthellae, symbiotic algae that supply more than 90% of the coral’s
energy budget through photosynthesis. The function of the non-fluorescent chromoproteins
remains the most elusive. Chromoproteins often occur alongside fluorescent proteins and can
sometimes be found in the same physiological distribution in different genera (e.g. tentacle tips),
indicating a distinctive functional role for these proteins. Specialised functions would then
support the natural selection of colour diversity, which appears to arise easily with few sequence
changes.
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Glowing Fish
(and other uses for green fluorescent protein)
Copyright 2003 by Edward Willett
In January, a U.S. company hopes to offer for sale the first pet fish genetically
engineered to glow under ultraviolet light. This has alarmed some people, but scientists
have been creating glowing organisms for several years now--and their ability to do so
has revolutionized many fields of science.
Many creatures glow naturally, a phenomenon called bioluminescence. In the dark
depths of the oceans, the majority do. But even though there are plenty of glowing fish
in the seas, the zebra fish has never been one of them--until recently.
Researchers at the National University of Singapore injected the fish’s eggs with a gene
that from a sea anemone which makes the anemone red, then added a jellyfish gene
that causes cells to produce green fluorescent protein, or GFP, which glows green when
hit by blue or ultraviolet light. The fish don’t glow all the time; instead, the production of
GFP is linked to genetic switches in the fish that are activated by the presence certain
toxins, such as estrogen. The goal isn’t to create a novelty pet, but a cheap and
effective pollution detector. The researchers say the zebra fish could be made to
fluoresce as many as five different colors, with each color activated by the presence of a
different pollutant.
Both the Singapore scientists and Yorktown Technologies, the U.S. company that wants
to sell the "GloFish" as pets, say they pose no hazard to the environment if they escape
into the wild: not only are they tropical fish, unsuited to North American waters, but their
fluorescence is actually a hindrance in the wild, requiring energy to maintain and making
them more susceptible to predators (who wouldn’t be harmed by eating the fish--GFP is
completely non-toxic).
The glowing fish have made the news, as did glowing green mice a few years ago and a
glowing green bunny in 2000 (intended as art). But out of the limelight, many more
organisms are glowing, making possible research that would otherwise be either difficult
or impossible.
A Princeton scientist, Osamu Shimomura, discovered GFP in 1962 while studying a
small bioluminescent jellyfish called Aequoria victoria. Within the jellyfish, one protein,
aequorin, glows blue in the presence of calcium. GFP, when struck by blue light, glows
green, giving the jellyfish its overall green glow. Almost 30 years later, Douglas Prasher,
a researcher at the Woods Hole Oceanographic Institute, discovered the gene in
jellyfish that coded for GFP. He ran out of funding before he could test the gene by
inserting it into bacteria. That task fell to Columbia University professor Martin Chalfie.
Most scientists assumed GFP would only glow in the presence of an enzyme or some
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other protein that only existed in the jellyfish. But they were wrong: when the GFP gene
was put into bacteria and a blue light was shone on them, they glowed.
That’s what makes GFP such a powerful research tool. If scientists want to see if they
have successfully inserted a gene into an organism, all they have to do is link it to GFP,
then shine a light on the organism to see if it glows (although the glow is usually only
detectable by highly sensitive cameras). GFP can also be linked to other proteins,
allowing scientists to track the movement of specific proteins within a cell or organism.
GFP and other forms of bioluminescence are being used in hundreds of research
projects today. The GFP gene, for instance, has enabled the study of adult stem-cells-those fabulous non-specific cells that can turn into the specialized cells required by all
the body’s various systems. Stem cells taken from a mouse engineered to contain the
GFP gene can be put into another mouse and their activity tracked by their glow.
Diseases like cystic fibrosis, AIDS and cancer can now be studied in greater detail than
ever before. The progress of diseases in lab animals can be followed in real time using
a sensitive camera, rather than interpolated after the fact by the results of dissection. Of
course, there are also more frivolous uses for bioluminescence--even more frivolous
than glowing pet fish. A company called Prolume markets squirt guns that shoot glowing
water and glowing "alien crystals"; future products touted at their Web site
(www.prolume.com) include glowing food (like a Bud Light that is really a Bud light) and
glowing cosmetics.
Indeed, one can only say that the future of bioluminescence looks bright! (Sorry.)
BIO 208 pre lab reading
Transformation of E. coli with pGLO Plasmid DNA
With pGLO transformation, students transform bacteria with a gene that codes for a green
fluorescent protein (GFP). The natural source for the GFP gene is the bioluminescent jellyfish,
Aequorea victoria. The gene encodes for the GFP which allows the jellyfish to glow in the dark.
The protein absorbs energy when exposed to ultraviolet light and gives off some of this energy in
the form of visible green light. Genes can be moved into bacteria by the use of small circular
pieces of DNA called plasmids. Plasmids may confer beneficial traits to a bacterium, such as
resistance to ampicillin or other antibiotics. The pGLO plasmid encodes for resistance to
ampicillin as well as a gene regulation system to control the expression of GFP. Expression of
GFP by transformed bacteria will result in green glowing colonies. Those bacteria that do not
take up the plasmid DNA will not produce GFP.
pGLO plasmid vector
bla
orf
GFP
araC
Encodes resistance to ampicillin for antibiotic selection
Open reading frame allows plasmid to be copied by bacterial DNA replication enzymes
Gene encoding green fluorescent protein
Encodes a repressor to prevent transcription (and expression) of the GFP gene. The
arabinose sugar binds the repressor to allow GFP expression. This allows the research to
turn on and off GFP expression by addition or removal of arabinose from the bacterial
growth medium.
Transformation
E. coli bacterium
r
plasmids
E. coli
DNA
Bacterial cell wall and cell
membrane
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Terms and Concepts
Antibiotic
selection
The pGreen plasmid also contains the gene for ampicillin
resistance. This gene encodes for beta-lactamase (bla) which is secreted
by the bacteria harboring the plasmid. The beta-lactamase
inactivates the ampicillin contained on the LB agar plates thereby
allowing the bacteria to grow. Non-transformed cells (do not
the plasmid) are killed by ampicillin.
Arabinose
A sugar used as an energy source by bacteria. Bacteria express
genes needed for arabinose uptake and metabolism only in the presence of
arabinose
Beta-lactamase
see antibiotic selection
Cloning
A population of cells produced from a single cell contains identical clones.
The process of producing a clonal population is called “cloning”. One can
produce large quantities of a specific DNA sequence or gene
Colony
A group of identical bacterial cells (clones) growing on an agar
plate. The colony contains millions of individual cells.
Competent cells
E. coli that have been treated with calcium chloride more readily
accept plasmid DNA. Ca+2 ions are thought to neutralize the
phospholipids of the cell membrane allowing the DNA to pass
through the bacterial cell wall and enter the cell.
E. coli
The host organism in this experiment is E. coli strain K12. It is not
pathogenic. However, Standard Microbiological Practices should be used.
These practices include decontaminated work surfaces, and proper
disposal of bacterial waste in a biohazard bag. Mouth pipetting, eating,
and drinking are prohibited.
Gene Regulation
Genes involved in the transport and breakdown of arabinose are
highly regulated. The genes encoding the bacterial enzymes
needed are only transcribed in the presence of arabinose. When
the arabinose runs out, the genes are turned off.
Green Fluorescent
Protein
GFP was originally isolated from the bioluminescent jellyfish,
Aequorea victoria. The gene for GFP has been cloned and plasmid
vectors containing the gene are in wide use. The 3D structure of
GFP causes it to give off energy when exposed to UV light in the
form of visible green light
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Heat Shock
The heat shock increases the permeability of the bacterial cell
membrane to exogenous DNA. The time of the heat shock is
critical and is optimized for the tube type volume
LB media
Liquid and solid media are made from an extract of yeast and an
enzymatic digest of meat byproducts which provides a mixture of
carbohydrates, amino acids, nucleotides, salts, and vitamins – nutrients for
bacterial growth. LB agar (made from seaweed) provides a support on
which bacterial colonies can grow
Plasmid
A small circular DNA molecule capable of autonomous replication
Many plasmids also contain antibiotic resistance genes and must
contain an origin of replication.
pGLO
pGLO is a plasmid which contains the GFP gene and the
ampicillin resistance gene which encodes for beta-lactamase
Recovery
The 10 minute incubation following the addition of LB broth
allows the cells to grow and express the ampicillin resistance
protein, beta-lactamase.
Sterile technique
It is important not to introduce contaminating bacteria into an experiment.
Because contaminating bacteria are found everywhere including fingertips
and bench tops, it is important to avoid these surfaces. Wear gloves. Do
not touch the portion of sterile toothpicks, inoculating loops, the pipette
tip, and agar plates that will come into contact with your bacteria.
Transformation
Occurs when a bacterium takes up and expresses a new piece of genetic
material (DNA). Genes are often on a vector
UV lamps
Ultraviolet radiation can cause damage to eyes and skin. Short wave UV is
more damaging than long wave (used in this experiment). Wear protective
goggles.
Vector
An autonomously replicating DNA molecule into which
foreign DNA fragments are inserted and then propagated
in a host cell (a plasmid is a type of vector)