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Team:Debrecen-Hungary/minimals
From 2010.igem.org
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Welcome To The MinimalsFrom year to year (and from one to jamboree to another) the world of synthetic biology exponentially expands. Some iGEMers may find niches of complex biological systems and use special model organisms or laboratory tools. Our philosophy is that a good project is one that can be kept simple and short.
ContentsEssentials Of Lipid Sensing Cellular signaling - Nuclear Receptors - Ligand binding domains Model Organisms Drosophila Melanogaster - Caenorhabditis elegans - Homo sapiens In The Laboratory (Techniques And Reagents) Two hybrid screening - Luciferase - Cos-1 cells - Dose response curve Essentials Of Lipid SensingCellular signalingCells have an innate ability to “listen” and correctly react to their local or even distant environment. Through time it has been observed that a complex systems of communication governs essential cellular activates and coordinates cell actions.[1] Today, it is well known that processes such as development, growth, tissue repair or death, metabolic shifts and immunity are all governed, at the molecular level, by signaling. By understanding cell signaling, diseases may be treated effectively and, theoretically, artificial tissues may be created. Cells sense information from their local surroundings through a class of proteins known as receptors. Chemicals that activate (or inhibit) receptors are often named hormones, growth factors, cytokines or even neurotransmitters yet their proper term is receptor ligands. Water soluble ligands have cell membrane penetration and thus mostly interact with trans-membranous receptors, whereas ligands with high lipid solubility easily penetrate the cell membrane Nuclear Receptors
Ligand Binding DomainsLigand binding domain (LBD) is a well conserved domain amongst various nuclear receptors whose structure usually referred to as an alpha helical sandwich fold. The LBD shows some diversity among nuclear receptors as it is a site for receptor-specific events. It possesses transactivation ability and contains a ligand-binding pocket as well as the main interaction surfaces for other proteins. The DNA-binding domain (DBD) contains two Zinc-finger motifs and is linked to the LBD by a highly flexible hinge region. This segment of the nuclear receptors holds the ability to recognize and bind to preferred or specific DNA-motifs, the response elements. The Ligand binding domain together with the DNA binding domain contributes to the interface of the receptor by binding accessory proteins (coactivator and corepressor) and dimerization of receptors. The LBD also contains the activation function 2 (AF-2) whose action is dependent on the presence of bound ligand [8]. The change in receptor configuration which occurs upon ligand binding exposes the AF-2 domain, which promotes transcriptional activity by a wide variety of mechanisms. Model OrganismsDrosophila Melanogaster
Drosophila Melanogaster, also known as the common fruit fly, is one of the most frequently used model organisms in biological sciences, including studies in genetics, physiology, microbial pathogenesis and life history evolution.[9] The ecdysone receptor is a nuclear receptor found in D.Melanogaster, where it controls development and contributes to other processes such as reproduction. Its ligands are ecdysteroid which are secreted by the organism’s prothoracic gland. Caenorhabditis elegans
Caenorhabditis elegans is a free-living, transparent nematode (roundworm), about 1 mm in length,[10] which lives in temperate soil environments C. elegans is intensively studied as a model organism in biology for a variety of reasons. The developmental fate of every single somatic cell (959 in the adult hermaphrodite; 1031 in the adult male) has been mapped out.[11] [12] The C.elegans genome harbors 284 nuclear receptors [10] (a striking figure), which have been shown to control traits such as sex determination, larva development, life span, neuronal growth and identity and much more. As far as nuclear receptors go, they are a gold mine. Homo SapiensHomo sapiens are the only living species in the Homo genus of bipedal primates in the great ape family. Nuclear receptors number up to 47 in humans, yet only few have been well characterized. They constitute the focus of medicinal reproductive technologies, hormonal medicine (endocrinology), immunology, drug interaction and much more. In The Laboratory (Techniques And Reagents)Two Hybrid ScreeningTwo-hybrid screening is a technique in molecular biology which can be used to investigate protein interaction with other proteins or DNA[13] [14] by testing for biochemical interactions such as binding. The premise behind the test is the activation of reporter gene by a transcription factor binding to DNA response elements located upstream (aka upstream activating sequence or UAS. The transcription factor being investigated is split to two separate functional fragments. The binding domain is the DNA binding domain responsible for associating with the UAS. The activating domain is responsible for transcriptional activation. When simplified it may be viewed as a biological system at which the input is the transcription factor concentration and the output is the transcriptional activity generated. Many versions of the technique have been implemented including one for the study of DNA binding affinity changes in receptors as a cause of ligand binding (the one hybrid screening). Yeast Gal 4 is a common DBD used for this techniques purpose. Commly used reporter genes include the product of the LacZ gene (Beta galactosidase) and Luciferase. Luciferase
Luciferase is an enzyme class able to produce bioluminescence by oxidizing the substrate luciferin. "Firefly luciferase" as a laboratory reagent usually refers to P. pyralis luciferase. Its emission can be measured photometrically and hence used to deduce the protein enzyme concentration through standardized methods.
Cos-1 cells
Cos-1 cells (acronym for CV-1 simian origin, SV-40 viral positive) is cell line derived from the African green monkey kidney cells. It is also often used to transfect cells in tissue culture conditions to produce recombinant proteins for molecular biology, biochemistry, and cell biology experiments. Two forms of COS cell lines commonly used are COS-1 and COS-7. The cell line was obtained by immortalizing the original CV-1 cells with SV-40 virus genome. This allows the production of large T antigen but has a defect in genomic replication.[15] Dose response curveDose response curve depicts a change in a measured effect on an organism caused by differing levels of exposure to a chemical in standardized measuring conditions. It may apply to either individuals or to populations. The curve is usually displayed in a simple X-Y graph (X being logarithm of dose, Y for effect). The half maximal effective concentration (EC50), a common feature of drug potency, is the chemical’s concentration which induces a response halfway between the baseline and maximum.[16]
References
1. ^ Witzany, Guenther (2010). Biocommunication and Natural Genome Editing. Springer. ISBN 9789048133185. 
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