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The pGLO plasmid is an engineered
In 1994 Marty Chalfie and his team were able to successfully create bacteria and round worms that expressed the GFP protein. Soon after, Roger Tsien and his team were able to create mutant GFP that can emit a range of colors, not just green.
The three scientists hold the Nobel Prize in Chemistry for 2008 for the discovery and development of the green fluorescent protein, GFP.
plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria and ...
used in biotechnology
Biotechnology is a multidisciplinary field that involves the integration of natural sciences and Engineering Science, engineering sciences in order to achieve the application of organisms and parts thereof for products and services. Specialists ...
as a vector for creating genetically modified organism
A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques. The exact definition of a genetically modified organism and what constitutes genetic engineering varies, with ...
s. The plasmid contains several reporter gene
Reporter genes are molecular tools widely used in molecular biology, genetics, and biotechnology to study gene function, expression patterns, and regulatory mechanisms. These genes encode proteins that produce easily detectable signals, such as ...
s, most notably the green fluorescent protein
The green fluorescent protein (GFP) is a protein that exhibits green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea victo ...
(GFP) and the ampicillin resistance gene. GFP was isolated from the jelly fish ''Aequorea victoria
''Aequorea victoria'', also sometimes called the crystal jelly, is a bioluminescent hydrozoan jellyfish, or hydromedusa, that is found off the west coast of North America.
The species is best known as the source of aequorin (a photoprotein), and ...
''. Because it shares a bidirectional promoter with a gene for metabolizing arabinose
Arabinose is an aldopentose – a monosaccharide containing five carbon atoms, and including an aldehyde (CHO) functional group.
Properties
For biosynthetic reasons, most saccharides are almost always more abundant in nature as the "D"-form, o ...
, the GFP gene is expressed in the presence of arabinose, which makes the transgenic organism express its fluorescence under UV light
Ultraviolet radiation, also known as simply UV, is electromagnetic radiation of wavelengths of 10–400 nanometers, shorter than that of visible light, but longer than X-rays. UV radiation is present in sunlight and constitutes about 10% of t ...
. GFP can be induced in bacteria containing the pGLO plasmid by growing them on +arabinose plates. pGLO is made by Bio-Rad Laboratories.
Structure
pGLO is made up of three genes that are joined together using recombinant DNA technology. They are as follows: *''Bla'', which codes for the enzymebeta-lactamase
Beta-lactamases (β-lactamases) are enzymes () produced by bacteria that provide multi-resistance to beta-lactam antibiotics such as penicillins, cephalosporins, cephamycins, monobactams and carbapenems ( ertapenem), although carbapene ...
giving the transformed bacteria resistance to the beta-lactam family of antibiotics (such as of the penicillin
Penicillins (P, PCN or PEN) are a group of beta-lactam antibiotic, β-lactam antibiotics originally obtained from ''Penicillium'' Mold (fungus), moulds, principally ''Penicillium chrysogenum, P. chrysogenum'' and ''Penicillium rubens, P. ru ...
family)
*''araC'', a promoter region that regulates the expression of ''GFP'' (specifically, the ''GFP'' gene will be expressed only in the presence of arabinose
Arabinose is an aldopentose – a monosaccharide containing five carbon atoms, and including an aldehyde (CHO) functional group.
Properties
For biosynthetic reasons, most saccharides are almost always more abundant in nature as the "D"-form, o ...
)
*''GFP'', the green fluorescent protein, which gives a green glow if cells produce this type of protein
Like most other circular plasmids, the pGLO plasmid contains an origin of replication
The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semi ...
(ori), which is a region of the plasmid where replication will originate.
The pGLO plasmid was made famous by researchers in France
France, officially the French Republic, is a country located primarily in Western Europe. Overseas France, Its overseas regions and territories include French Guiana in South America, Saint Pierre and Miquelon in the Atlantic Ocean#North Atlan ...
who used it to produce a green fluorescent rabbit
Rabbits are small mammals in the family Leporidae (which also includes the hares), which is in the order Lagomorpha (which also includes pikas). They are familiar throughout the world as a small herbivore, a prey animal, a domesticated ...
named Alba
''Alba'' ( , ) is the Scottish Gaelic name for Scotland. It is also, in English-language historiography, used to refer to the polity of Picts and Scots united in the ninth century as the Kingdom of Alba, until it developed into the Kingd ...
.
Other features on pGLO, like most other plasmids, include a selectable marker and an MCS (multiple cloning site
A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites—a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring onl ...
) located at the end of the GFP gene. The plasmid is 5371 base pair
A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...
s long. In supercoiled
DNA supercoiling refers to the amount of twist in a particular DNA strand, which determines the amount of strain on it. A given strand may be "positively supercoiled" or "negatively supercoiled" (more or less tightly wound). The amount of a st ...
form, it runs on an agarose gel in the 4200–4500 range.
Discovery of GFP
The GFP gene was first observed by Osamu Shimomura and his team in 1962 while studying the jellyfish ''Aequorea victoria
''Aequorea victoria'', also sometimes called the crystal jelly, is a bioluminescent hydrozoan jellyfish, or hydromedusa, that is found off the west coast of North America.
The species is best known as the source of aequorin (a photoprotein), and ...
'' that have a ring of blue light under their umbrella''.'' Shimomura and his team isolated the protein aequorin
Aequorin is a calcium-activated photoprotein isolated from the hydrozoan ''Aequorea victoria''. Its bioluminescence was studied decades before the protein was isolated from the animal by Osamu Shimomura in 1962. In the animal, the protein occur ...
from thousands of jellyfish until they gathered enough for a full analysis of the protein. It was through the study of aequorin that Shimomura discovered small amounts of GFP which glows green when aequorin emits blue light. After successfully discovering how GFP works with aequorin in the jellyfish, he set it aside to study bioluminescence in other organisms.
In 1994 Marty Chalfie and his team were able to successfully create bacteria and round worms that expressed the GFP protein. Soon after, Roger Tsien and his team were able to create mutant GFP that can emit a range of colors, not just green.
The three scientists hold the Nobel Prize in Chemistry for 2008 for the discovery and development of the green fluorescent protein, GFP.
References
Genetically modified organisms Plasmids Fluorescence techniques {{genetics-stub