The minimals

As most synthetic biologists and iGEM teams work with Escherichia Coli, the use of other model systems can cause confusion. We hope to ease the legibility of our project descriptions by creating eukaryopedia, an overview about transcription factors and cell lines we used in our studies, as well as general molecular biology issues that affect our work. We hope it can help you find guidance in the jungle that mammalian molecular biology is at the moment.

Contents

Essentials 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 Sensing

Cellular signaling

Cells 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

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Nuclear Receptos

Nuclear receptors are a class of receptors localized within cells which can sense the presence of lipid soluble ligands (eg steroid hormones). Upon ligand binding these receptors undergo a change in conformation and translocate to the nucleus. A unique feature of these proteins, amongst other receptor classes, is the ability to directly bind to DNA segments known as response elements. These receptors are best viewed as transcription factors which can be activated by extracellular cues[2][3]. The binding ultimately leads to defined changes in gene expression (both activation and repression), thereby controlling the development, homeostasis, and metabolism of the organism. Nuclear receptors bear high homology to each other and are modular into distinct domains: N-terminal regulatory domain, DNA-binding domain, a Hinge region, Ligand binding domain (LBD) and a C-terminal domain.

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Ligand Binding Domains

Ligand binding domain is a conserved sequence and structure amongst the various nuclear receptors whose structure usually referred to as an alpha helical sandwich fold. Three anti parallel alpha helices (the "sandwich filling") are flanked by two alpha helices on one side and three on the other (the "bread"). The ligand cavity has an internal localization just below three anti parallel alpha helical sandwich "filling". 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.

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Model Organisms

Drosophila 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.

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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.

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Homo Sapiens

Homo 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.

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In The Laboratory (Techniques And Reagents)

Two Hybrid Screening

Two-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.

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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.

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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]

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Dose response curve

Dose 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]

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Drugs

CPT

Camptothecin (CPT) is a cytotoxic quinoline alkaloid and a topoisomerase I inhibitor isolated from the Camptotheca acuminata (Camptotheca or the Happy tree). It was discovered during a screen for natural anti-cancer drugs in 1966 but it is not not used in cancer therapy due to its severe side effects, but there were various derivatives developed to increase the benefits of this drug while decreasing its negative effects [52]. The two CPT analogues have been approved for cancer chemotherapy today are topotecan and irinotecan. CPT acts by binding to the topoisomerase I-DNA complex using hydrogen bonds and thereby preventing DNA-religation, inducing DNA damage and ultimately causing the cell to die [53].

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Hygromycin

The aminocyclitol antibiotic hygromycin B, that is produced in Streptomyces hygoscopicus, inhibits protein synthesis by interfering into aminoacyl-tRNA recognition and ribosomal translocation. It shows effects in prokaryotes and eukaryotes alike. Hygromycine can be used as a selection marker. The resistance gene encodes for a hygromycin B phosphotransferase, which inactivates the antibiotic by phosphorylation [68]. In the iGEM 2009 project of Heidelberg hygromycin B was used for selection of cells which performed a stable integration of the transfected Plasmid.

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Zeocin

Zeocin shows a high effectiveness in a wide range of organisms. Mammalian, insect and yeast cells are effected as well as prokaryotic cells. It damages DNA by intercalating and causing breaks and therefore cell death. The zeocin resistance gene encodes for protein which binds zeocin and prohibits DNA destructionhttp://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Culture/Transfection/Selection/Zeocin.html 71.

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Neomycin

The aminoglycoside antibiotic neomycin is produced by Streptomyces fradiae [69]. Neomycin is a selectionmarker for many different cell types. The resistance gene encodes for a phosphotransferase which inactivates neomycine by phosphorylating it [70].

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Cellular components as tools

GPI

Glycosylphosphatidylinositol (GPI) is a glycolipid. During the posttranslational modification in eukaryotic cells, it becomes attached to hydrophobic C-termini of proteins that have a special singnal peptide on them. This signalpeptide leads their translation into the ER, where the hydrophobic C-terminus will be replaced by a GPI anchor. Because of its hydrophobic nature it attaches the bound protein to the cell membrane [54].

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Sar-1

Sar-1 GTP-binding proteins direct the transport of molecules inside of veiscles from the ER to the golgi and the other way round. Being an anchor for COPII molecules that cause the budding of vesicles off the membranes, it needs a domain to attach to the ER membrane [55]. The C terminus of the Sar-1 protein fullfills this task. Therefore one can use the C terminus as an ER targeting sequence for other proteins.

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Myrpalm

This localization signal is located at the N-terminal end of the amino acid chain. The myrpalm signaling sequence causes a myristilation and palmitolyation of the targeted protein. Both modifications lead to a binding to the cell membrane [56].

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NLS

Nuclear localisation signals are peptidesequences that are able to bind to nuclear import receptors. These cause an import of newly synthesized protein through nuclear pores. This feature is caused by several positively charged amino acids. Nuclear localization signals can be located almost anywhere in the peptide chain [57]. We used a nuclear localization signal at the C-terminal end of the protein.

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GFP

Green Fluorescent Protein (GFP) was first discovered by Shimomura et al. in the Aequorea jellyfish. They described a slightly green colour of a GFP-containing solution that in the sunlight [63]. The same group of scientists investigated the protein in more detail, and have since discovered many characteristics, including the excitation and emission wavelengths . The most important accomplishment was the cloning of the GFP gene into other organisms to make them fluorescent [64], [65]. Many scientists have since worked on GFP and introduced mutations to enhance fluorscence levels or change the spectra. Nowadays flourescent proteins exist in different colours exist increasing their range of application even more.

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References