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Tet-on
Tetracycline-controlled transcriptional activation is a method of inducible gene expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline). Tetracycline-controlled gene expression is based upon the mechanism of resistance to tetracycline antibiotic treatment found in gram-negative bacteria. In nature, the Ptet promoter expresses TetR (the repressor) and TetA, the protein that pumps tetracycline antibiotic out of the cell. The difference between Tet-On and Tet-Off is not whether the transactivator turns a gene on or off, as the name might suggest; rather, both proteins ''activate'' expression. The difference relates to their respective response to tetracycline or doxycycline (Dox, a more stable tetracycline analogue); Tet-Off activates expression in the ''absence'' of Dox, whereas Tet-On activates in the ''presence'' of Dox. Tet-Off and Tet-On The two most commonly used inducible expr ...
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Gene Expression
Gene expression is the process (including its Regulation of gene expression, regulation) by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, proteins or non-coding RNA, and ultimately affect a phenotype. These products are often proteins, but in non-protein-coding genes such as Transfer RNA, transfer RNA (tRNA) and Small nuclear RNA, small nuclear RNA (snRNA), the product is a functional List of RNAs, non-coding RNA. The process of gene expression is used by all known life—eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea), and viruses—to generate the macromolecule, macromolecular machinery for life. In genetics, gene expression is the most fundamental level at which the genotype gives rise to the phenotype, ''i.e.'' observable trait. The genetic information stored in DNA represents the genotype, whereas the phenotype results from the "interpretation" of that informati ...
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Tet-ON Inducible Transgene Expression Cells
Tetracycline-controlled transcriptional activation is a method of inducible gene expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline). Tetracycline-controlled gene expression is based upon the mechanism of resistance to tetracycline antibiotic treatment found in gram-negative bacteria. In nature, the Ptet promoter expresses TetR (the repressor) and TetA, the protein that pumps tetracycline antibiotic out of the cell. The difference between Tet-On and Tet-Off is not whether the transactivator turns a gene on or off, as the name might suggest; rather, both proteins ''activate'' expression. The difference relates to their respective response to tetracycline or doxycycline (Dox, a more stable tetracycline analogue); Tet-Off activates expression in the ''absence'' of Dox, whereas Tet-On activates in the ''presence'' of Dox. Tet-Off and Tet-On The two most commonly used inducible expr ...
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Mitochondria
A mitochondrion () is an organelle found in the cells of most eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and use aerobic respiration to generate adenosine triphosphate (ATP), which is used throughout the cell as a source of chemical energy. They were discovered by Albert von Kölliker in 1857 in the voluntary muscles of insects. The term ''mitochondrion'', meaning a thread-like granule, was coined by Carl Benda in 1898. The mitochondrion is popularly nicknamed the "powerhouse of the cell", a phrase popularized by Philip Siekevitz in a 1957 ''Scientific American'' article of the same name. Some cells in some multicellular organisms lack mitochondria (for example, mature mammalian red blood cells). The multicellular animal '' Henneguya salminicola'' is known to have retained mitochondrion-related organelles despite a complete loss of their mitochondrial genome. A large number of unicellular organisms, such as microspo ...
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Tetracyclines
Tetracyclines are a group of broad-spectrum antibiotic compounds that have a common basic structure and are either isolated directly from several species of ''Streptomyces'' bacteria or produced semi-synthetically from those isolated compounds. Tetracycline molecules comprise a linear fused tetracyclic nucleus (rings designated A, B, C and D) to which a variety of functional groups are attached. Tetracyclines are named after their four ("tetra-") hydrocarbon rings ("-cycl-") derivation ("-ine"). They are defined as a subclass of polyketides, having an octahydrotetracene-2-carboxamide skeleton and are known as derivative (chemistry), derivatives of polycyclic naphthacene carboxamide. While all tetracyclines have a common structure, they differ from each other by the presence of chloro, methyl, and hydroxyl groups. These chemical modification, modifications do not change their broad antibacterial activity, but do affect pharmacological properties such as half-life and binding to pr ...
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Fetal Bovine Serum
Fetal bovine serum (FBS) is the most widely used serum-supplement for the ''in vitro'' cell culture of eukaryotic cells. It is commonly utilized in biomedical research, pharmaceutical development, and biomanufacturing due to its ability to support a wide variety of cell types. This is due to it having a very low level of antibodies and containing more growth factors, allowing for versatility in many cell culture applications. Fetal bovine serum is derived from the blood drawn from a bovine fetus via a closed system of collection at the slaughterhouse. The globular protein bovine serum albumin (BSA) is a major component of fetal bovine serum. It plays a crucial role in maintaining osmotic balance and transporting molecules within the culture medium. Besides BSA, fetal bovine serum is a rich source of growth and attachment factors, lipids, hormones, nutrients and electrolytes necessary to support cell growth in culture. It is typically added to basal cell culture medium, such as ...
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Pleiotropy
Pleiotropy () is a condition in which a single gene or genetic variant influences multiple phenotypic traits. A gene that has such multiple effects is referred to as a ''pleiotropic gene''. Mutations in pleiotropic genes can impact several traits simultaneously, often because the gene product is used in various cell (biology), cells and affects different biological targets through shared signaling pathways. Pleiotropy can result from several distinct but potentially overlapping mechanisms, including gene pleiotropy, developmental biology, developmental pleiotropy, and selectional pleiotropy. Gene pleiotropy occurs when a gene product interacts with multiple proteins or catalyzes different reactions. Developmental pleiotropy refers to mutations that produce several phenotype, phenotypic effects during development. Selectional pleiotropy occurs when a single phenotype influences evolutionary fitness (biology), fitness in multiple ways (depending on factors such as age and sex). T ...
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Estrogen Receptor
Estrogen receptors (ERs) are proteins found in cell (biology), cells that function as receptor (biochemistry), receptors for the hormone estrogen (17β-estradiol). There are two main classes of ERs. The first includes the intracellular estrogen receptors, namely ERα and ERβ, which belong to the nuclear receptor family. The second class consists of membrane estrogen receptors (mERs), such as GPER (GPR30), ER-X, and Gq-mER, Gq-mER, which are primarily G protein-coupled receptors. This article focuses on the nuclear estrogen receptors (ERα and ERβ). Upon activation by estrogen, intracellular ERs undergo protein targeting, translocation to the nucleus where they bind to specific DNA sequences. As DNA-binding transcription factors, they regulate the activity of various genes. However, ERs also exhibit functions that are independent of their DNA-binding capacity. These non-genomic actions contribute to the diverse effects of estrogen signaling in cells. Estrogen receptors (ERs) b ...
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FLP-FRT Recombination
In genetics, Flp-''FRT'' recombination is a site-specific recombination, site-directed recombination technology, increasingly used to manipulate an organism's DNA under controlled conditions ''in vivo''. It is analogous to Cre-Lox recombination, Cre-''lox'' recombination but involves the recombination of sequences between short flippase recognition target (''FRT'') sites by the recombinase flippase (''Flp'') derived from the 2 μ plasmid of baker's yeast ''Saccharomyces cerevisiae''. The 34bp minimal FRT site sequence has the sequence ::5'3' for which flippase (Flp) binds to both 13-bp 5'-GAAGTTCCTATTC-3' arms flanking the 8 bp spacer, i.e. the site-specific recombination (region of crossover) in reverse orientation. ''FRT''-mediated cleavage occurs just ahead from the asymmetric 8bp core region (5''3') on the top strand and behind this sequence on the bottom strand. Several variant ''FRT'' sites exist, but recombination can usually occur only between two ''identical'' ''F ...
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Cre Recombinase
Cre recombinase is a tyrosine recombinase enzyme derived from the P1 bacteriophage. The enzyme uses a topoisomerase I-like mechanism to carry out site specific recombination events. The enzyme (38 kDa) is a member of the integrase family of site specific recombinase and it is known to catalyse the site specific recombination event between two DNA recognition sites ( LoxP sites). This 34 base pair (bp) loxP recognition site consists of two 13 bp palindromic sequences which flank an 8bp spacer region. The products of Cre-mediated recombination at loxP sites are dependent upon the location and relative orientation of the loxP sites. Two separate DNA species both containing loxP sites can undergo fusion as the result of Cre mediated recombination. DNA sequences found between two loxP sites are said to be " floxed". In this case the products of Cre mediated recombination depends upon the orientation of the loxP sites. DNA found between two loxP sites oriented in the same direction ...
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Plasmids
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 archaea; however plasmids are sometimes present in and eukaryotic organisms as well. Plasmids often carry useful genes, such as those involved in antibiotic resistance, virulence, secondary metabolism and bioremediation. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain additional genes for special circumstances. 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 internet by various vendors ...
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Negative Feedback
Negative feedback (or balancing feedback) occurs when some function (Mathematics), function of the output of a system, process, or mechanism is feedback, fed back in a manner that tends to reduce the fluctuations in the output, whether caused by changes in the input or by other disturbances. Whereas positive feedback tends to instability via exponential growth, oscillation or chaos theory, chaotic behavior, negative feedback generally promotes stability. Negative feedback tends to promote a settling to List of types of equilibrium, equilibrium, and reduces the effects of perturbations. Negative feedback loops in which just the right amount of correction is applied with optimum timing, can be very stable, accurate, and responsive. Negative feedback is widely used in Mechanical engineering, mechanical and electronic engineering, and it is observed in many other fields including biology, chemistry and economics. General negative feedback systems are studied in Control engin ...
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Synthetic Biological Circuit
Synthetic biological circuits are an application of synthetic biology where biological parts inside a Cell (biology), cell are designed to perform logical functions mimicking those observed in electronic circuits. Typically, these circuits are categorized as either genetic circuits, RNA circuits, or protein circuits, depending on the types of biomolecule that interact to create the circuit's behavior. The applications of all three types of circuit range from simply inducing production to adding a measurable element, like green fluorescent protein, to an existing Gene regulatory network, natural biological circuit, to implementing completely new systems of many parts. The goal of synthetic biology is to generate an array of tunable and characterized parts, or modules, with which any desirable synthetic biological circuit can be easily designed and implemented. These circuits can serve as a method to modify cellular functions, create cellular responses to environmental conditions, ...
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