European Bioinformatics Institute Protein of the Month

BIO 208 pre lab reading

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.

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 GFP-related 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.

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 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.)

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 Encodes resistance to ampicillin for antibiotic selection

orf Open reading frame allows plasmid to be copied by bacterial DNA replication enzymes

GFP Gene encoding green fluorescent protein

araC 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

Terms and Concepts

Antibiotic The pGreen plasmid also contains the gene for ampicillin

selection 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 GFP was originally isolated from the bioluminescent jellyfish,

Protein 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

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)