PGLO
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PGLO
The pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating genetically modified organisms. The plasmid contains several reporter genes, most notably the green fluorescent protein (GFP) and the ampicillin resistance gene. GFP was isolated from the jelly fish ''Aequorea victoria''. Because it shares a bidirectional promoter with a gene for metabolizing arabinose, the GFP gene is expressed in the presence of arabinose, which makes the transgenic organism express its fluorescence under UV light. 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 enzyme beta-lactamase giving the transformed bacteria resistance to the beta-lactam family of antibiotics (such as of the penicillin family) *araC, a promoter region that regulates ...
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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 the most common being an organism altered in a way that "does not occur naturally by mating and/or natural recombination". A wide variety of organisms have been genetically modified (GM), from animals to plants and microorganisms. Genes have been transferred within the same species, across species (creating transgenic organisms), and even across kingdoms. New genes can be introduced, or endogenous genes can be enhanced, altered, or knocked out. Creating a genetically modified organism is a multi-step process. Genetic engineers must isolate the gene they wish to insert into the host organism and combine it with other genetic elements, including a promoter and terminator region and often a selectable marker. A number of techniques are a ...
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Genetically Modified Organisms
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 the most common being an organism altered in a way that "does not occur naturally by mating and/or natural recombination". A wide variety of organisms have been genetically modified (GM), from animals to plants and microorganisms. Genes have been transferred within the same species, across species (creating transgenic organisms), and even across kingdoms. New genes can be introduced, or endogenous genes can be enhanced, altered, or knocked out. Creating a genetically modified organism is a multi-step process. Genetic engineers must isolate the gene they wish to insert into the host organism and combine it with other genetic elements, including a promoter and terminator region and often a selectable marker. A number of techniques are av ...
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Green Fluorescent Protein
The green fluorescent protein (GFP) is a protein that exhibits bright 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 victoria'' and is sometimes called ''avGFP''. However, GFPs have been found in other organisms including corals, sea anemones, zoanithids, copepods and lancelets. The GFP from ''A. victoria'' has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower green portion of the visible spectrum. The fluorescence quantum yield (QY) of GFP is 0.79. The GFP from the sea pansy (''Renilla reniformis'') has a single major excitation peak at 498 nm. GFP makes for an excellent tool in many forms of biology due to its ability to form an internal chromophore without requiring any accessory cofactors, gene products, or enzymes / substrates other than mo ...
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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; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that benefit the survival of the organism and confer selective advantage such as antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation. Synthetic plasmids are available for procurement over the inter ...
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Rabbit
Rabbits, also known as bunnies or bunny rabbits, are small mammals in the family Leporidae (which also contains the hares) of the order Lagomorpha (which also contains the pikas). ''Oryctolagus cuniculus'' includes the European rabbit species and its descendants, the world's 305 breeds of domestic rabbit. ''Sylvilagus'' includes 13 wild rabbit species, among them the seven types of cottontail. The European rabbit, which has been introduced on every continent except Antarctica, is familiar throughout the world as a wild prey animal and as a domesticated form of livestock and pet. With its widespread effect on ecologies and cultures, the rabbit is, in many areas of the world, a part of daily life—as food, clothing, a companion, and a source of artistic inspiration. Although once considered rodents, lagomorphs like rabbits have been discovered to have diverged separately and earlier than their rodent cousins and have a number of traits rodents lack, like two extra incis ...
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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 occurs together with the green fluorescent protein to produce green light by resonant energy transfer, while aequorin by itself generates blue light. Discussions of "jellyfish DNA" that can make "glowing" animals often refer to transgenic animals that express the green fluorescent protein, not aequorin, although both originally derive from the same animal. Apoaequorin, the protein portion of aequorin, is an ingredient in the dietary supplement Prevagen. The US Federal Trade Commission (FTC) has charged the maker with false advertising for its memory improvement claims. Discovery Work on aequorin began with E. Newton Harvey in 1921. Though Harvey was unable to demonstrate a classical luciferase-luciferin reaction, he showed that water could ...
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Agarose Gel
Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar. The proteins may be separated by charge and/or size (isoelectric focusing agarose electrophoresis is essentially size independent), and the DNA and RNA fragments by length. Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix. Agarose gel is easy to cast, has relatively fewer charged groups, and is particularly suitable for separating DNA of size range most often encountered in laboratories, which accounts for the popularity of its use. The separated DNA may be viewed with stain, most commonly under UV light, and the DNA fragments can be extracted from the gel with relative ease. Mos ...
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DNA Supercoil
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 strand’s supercoiling affects a number of biological processes, such as compacting DNA and regulating access to the genetic code (which strongly affects DNA metabolism and possibly gene expression). Certain enzymes, such as topoisomerases, change the amount of DNA supercoiling to facilitate functions such as DNA replication and transcription. The amount of supercoiling in a given strand is described by a mathematical formula that compares it to a reference state known as "relaxed B-form" DNA. Overview In a "relaxed" double-helical segment of B-DNA, the two strands twist around the helical axis once every 10.4–10.5 base pairs of sequence. Adding or subtracting twists, as some enzymes do, imposes strain. If a DNA segment under twist s ...
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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 and RNA. Dictated by specific hydrogen bonding patterns, "Watson–Crick" (or "Watson–Crick–Franklin") base pairs (guanine–cytosine and adenine–thymine) allow the DNA helix to maintain a regular helical structure that is subtly dependent on its nucleotide sequence. The Complementarity (molecular biology), complementary nature of this based-paired structure provides a redundant copy of the genetic information encoded within each strand of DNA. The regular structure and data redundancy provided by the DNA double helix make DNA well suited to the storage of genetic information, while base-pairing between DNA and incoming nucleotides provides the mechanism through which DNA polymerase replicates DNA and RNA polymerase transcribes DNA in ...
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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 only once within a given plasmid. The purpose of an MCS in a plasmid is to allow a piece of DNA to be inserted into that region. An MCS is found in a variety of vectors, including cloning vectors to increase the number of copies of target DNA, and in expression vectors to create a protein product. In expression vectors, the MCS is located downstream of the promoter. Creating a multiple cloning site In some instances, a vector may not contain an MCS. Rather, an MCS can be added to a vector. The first step is designing complementary oligonucleotide sequences that contain restriction enzyme sites along with additional bases on the end that are complementary to the vector after digesting. Then the oligonucleotide sequences can be annealed and li ...
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Alba (rabbit)
Alba was the name of a genetically modified "glowing" rabbit created as an artistic work by contemporary artist Eduardo Kac, produced in collaboration with French geneticist Louis-Marie Houdebine. Houdebine used the GFP gene found in the jellyfish, ''Aequorea victoria'', that fluoresces green when exposed to blue light. This is a protein used in many standard biological experiments involving fluorescence. When Alba was exposed to such light, she would literally glow green — though photos by Kac showing the entire organism, including its hair, glowing a uniform green have had their veracity challenged. Eduardo Kac has described Alba as an animal that does not exist in nature. In an article published in ''The Boston Globe'', Houdebine admitted creating Alba for Kac and stated that Alba has a 'particularly mellow and sweet disposition.' This article generated a global media scandal, which caused Houdebine to distance himself from Kac's work. All subsequent media articles present ...
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