...Introduction The profound importance for microorganisms to operate at a maximum efficiency has lead to adaptations allowing for groups of processes to be functional when resources are available, while on the contrary remaining “dormant” when not in need. This has been accomplished at the molecular level by configuring clusters of genes together on the genome into operons that elicit a processive response in the presence of a specific metabolite. The Lac operon is responsible for the cleaving of the disaccharide lactose into two products. A myriad of components control the expression of the Lac operon when two conditions are met. First, the substrate, lactose, must be present. Second, no better substrate for example, glucose, is present (2). The three structural genes in the Lac operon are lacZ, lacY, and lacA. The gene lacZ encodes the tetramer, ß-galactosidase, which is responsible for hydrolyzing the ß-1,4 glycosidic linkage between galactose and glucose in lactose. The transport of lactose into the cell via the enzyme lactose permease is encoded by the gene lacY. The lacA gene encodes the enzyme, galactoside transacetylase, a trimer that transfers an acetyl group from acetyl-CoA to galactosides. Activation of these genes is dependent on the activity of a promoter and three operators based on the nutritional and environmental conditions available to the cell. The lac operon is a negatively controlled inducible operon that utilizes the product of the regulator gene lacI, to...
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...Introduction: The purpose of this experiment was to successfully transform pGLO plasmid into E.Coli cells. In the first segment of this laboratory exercise, one had to carry out a restriction digest. Restriction digestion is the process of cutting DNA molecules into smaller pieces with special enzymes such as, BamH1 and EcoR1. One had to determine which of the two plasmids, A or B, were pGLO or pWEB. A plasmid is a small circular DNA strand in the cytoplasm of a bacterium (Isite, 2013). In order to determine that, one had to use BamH1 and EcoR1 on two tubes each one with plasmid A and the other with plasmid B to observe the cuts made by each enzyme. The hypothesis for this segment of the laboratory exercise states that pGLO was plasmid B, and pWEB was plasmid A. PGLO is a plasmid which contains green fluorescent protein (GFP). GFP is found in the Aquarius Victoria jelly fish. These jelly fish have a bioluminescent protein that emits blue light. GFP converts the blue light into green light, and that is why these jelly fish emit green light. The pGLO was inserted into E.coli by using transformation. Transformation is the process of transferring genetic material between microbial cells (Tu 2008). Bacterial cells need to be in a state of competency prior to transformation (Isite 2013). Some bacteria naturally achieve this stage when nutrients and oxygen are low (Isite 2013). In the laboratory, bacteria were artificially induced with calcium chloride to be competent for transformation...
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...exercise your meals should be higher in carbohydrates and less in fat and days energy output is normal meals should consist of higher fats and lower carbohydrates while protein intake is high on all days (Simmons 2014). Reintroducing your body to healthy foods is important because your body uses the nutrients as fuel. The next fasting approach is the Alternate-Day Diet, which just as the name states, is fasting on alternate days. “Food is consumed for 24 hours, then restricted for 24 hours (water is available at all times) for every 2-day cycle” (Simmons 2014). On fasting days, calories are minimalized to a couple hundred calories and on the other day you would eat as normal. This diet is particularly best for weight loss due to longer periods of food restriction. The next fasting approach is called the Warrior Diet, which is based on the concept of fasting for 20 hours and promotes eating one large, healthy meal at the end of the day (Simmons). “It claims that this pattern of eating is in sync with humans’ circadian rhythm and will promote general health while ‘removing harmful toxins from the body’” (Simmons 2014). The fasting period allows you to consume small snacks such as fruits and veggies and some high protein snacks or shakes. The common factor among each one of the fasting diets is calorie restriction, which eventually leads to weight loss. Because meals are limited to a certain timeframe, ideally the calorie consumption is going to be less when following a proper diet...
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...traits are influenced by things like their genotype or their specific environment. Genomic markers can allow for a more direct comparison of closely related individuals (Ansari and Khan, 2012). In our case we focus on DNA extraction. The two basic parts of a DNA extraction procedure include the breaking of the cell walls to expose the DNA and the use of enzymes to remove contaminants. The DNA is analyzed for purity by taking the absorbance. The pure DNA is then visualized by gel electrophoresis. The DNA extraction of plant seeds is difficult because of their cell wall. The method used to break the cell wall includes grinding the seeds with liquid nitrogen. The addition of DNAzol is used to isolate genomic DNA (Chomczynski et al. 1997). Restriction enzymes are necessary to fragment patterns of the DNA and in turn making it easier to analyze the DNA through gel electrophoresis. BACKGROUND The purpose of our experiment is to extract the DNA from pepper seeds to be able to compare and contrast the similarities in their DNA. The extraction of DNA from a plant is slightly more difficult than that of mammals. This is due to the presence of a rigid cell wall. To overcome this problem, the pepper seeds are grinded using liquid nitrogen (Manen, Jean-François, 2005). The liquid nitrogen facilitates the pulverization of the seeds by flash freezing them and causes the outer shell to become fragile. Plant DNAzol is a guanidine thiocyanate and detergent...
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...Cloning and expression of α-Amylase gene from Bacillus subtilis in Pichia pastoris and Escherichia coli. Introduction Enzyme is a type of catalyst that present in living organisms used for many biotechnological functions in various industrial processing. It has a special characteristic that allows the chemical reaction to speed up without being altered, thus significantly improve the industrial productivity (Roy et al. 2012). Among various enzymes available in market, α-amylase has received a special attention in commercial production due to its widely used applications. α-Amylase contributed to 50% of the world enzyme production and has a great importance in many industries such as in food processing, laundry and also in pharmaceutical (Asgher et al. 2007). α-Amylase enzyme acts on α-1,4 glycosidic bonds in starch substrate backbone leading to the formation of soluble maltodextrins, glucose and maltose (Vidyalakshmi et al. 2009). This characteristic is extremely useful especially in industries that require the hydroxylation of starch such as the production of sugar syrups. The α-amylase enzyme can be obtained from various sources such as plants animals and microbes (Ahmed et al. 2011). However, the naturally occurring enzyme is still insufficient to support all the industrial production and therefore, it is crucial to find a new alternative sources, which is cost-efficient and high yield capacity to meet the supply demand (Yin et al. 2003). In industries, the microbial...
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...Biogenesis of the T1-S1 Linker of Voltage-Gated K+ Channels Running Title: The T1-S1 Linker of nascent Kv1.3 Key words: Protein folding, nascent peptides, potassium channel biogenesis, T1 domain, S1 transmembrane segment 1 Abstract In the model derived from the crystal structure of Kv1.2, a 6-transmembrane voltagegated potassium channel, the linker between a cytosolic tetramerization domain, T1, and the first transmembrane segment, S1, is projected radially outward from the channel’s central axis. This T1-S1 linker was modeled as two polyglycine helices to accommodate the residues between T1 and S1 [Long et al. (2005) Science 309, 897-903], however, the structure of this linker is not known. Here, we investigate whether a compact secondary structure of the T1-S1 linker exists at an early stage of Kv channel biogenesis. We have used a mass-tagging accessibility assay to report the biogenesis of secondary structure for three consecutive regions of Kv1.3, a highly homologous isoform of Kv1.2. The three regions include the T1-S1 linker and its two flanking regions, α5 of the T1 domain and S1. Both α5 and S1 manifest compact structures (helical) inside the ribosomal exit tunnel, whereas the T1-S1 linker does not. Moreover, the location of the peptide in the tunnel influences compaction. 2 Introduction Voltage-gated K+ (Kv) channels are critical to the normal functioning of excitable and non-excitable cells (1,2). Defects in their biophysical properties, biogenesis...
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...PINE MANOR COLLEGE CH 301 Question Bank 1. Restriction enzymes: A) act at the membrane to restrict the passage of certain molecules into the cell. B) are highly specialized ribonucleases that degrade mRNA soon after its synthesis. C) are sequence-specific DNA endonucleases. D) are very specific proteases that cleave peptides at only certain sequences. E) catalyze the addition of a certain amino acid to a specific tRNA. Answer- C 2. Certain restriction enzymes produce cohesive (sticky) ends. This means that they: A) cut both DNA strands at the same base pair. B) cut in regions of high GC content, leaving ends that can form more hydrogen bonds than ends of high AT content. C) make a staggered double-strand cut, leaving ends with a few nucleotides of single-stranded DNA protruding. D) make ends that can anneal to cohesive ends generated by any other restriction enzyme. E) stick tightly to the ends of the DNA they have cut. Ans: C 3. The PCR reaction mixture does not include: A) all four deoxynucleoside triphosphates. B) DNA containing the sequence to be amplified. C) DNA ligase. D) heat-stable DNA polymerase. E) oligonucleotide primer(s). Ans: C 4. The compound that consists of ribose linked by an N-glycosidic bond to N-9 of adenine is: A) a deoxyribonucleoside. B) a purine nucleotide. C) a pyrimidine nucleotide. D) adenosine monophosphate...
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...disorder, affects 1 in every 180,000 babies born annually. Caused by the inability to metabolize the amino acids leucine (leu), isoleucine, and valine in protein, the disease produces a maple syrup smell in the urine of diagnosed persons; thus, earning its name. (The smell is produced due to the build up of these chemicals in the blood) First discovered in 1954, by John Menkes, this recessive trait is produced by a gene defect passed down through various families. It consists of a classic form and several less common forms each varying in its strength and features. Although they differ, all forms of the disorder can be caused by mutations in any of the 6 genes used to build the branched-chain alpha-ketoacid dehydrogenase (BCKDHA) of Chromosome 19. Babies with MSUD often appear normal at birth but within 3-4 days can display other symptoms such as food avoidance, vomiting and fussiness. If left untreated, seizures occur and the baby will naturally slip into a coma shortly ending in death. Treatment of this disorder is managed by a strict diet free of the three harmful amino acids discussed earlier and must be performed early enough to avoid brain damage. Baby formula with these restrictions are available but as the child grows into adolescence, they must always monitor their diets, especially paying attention to avoidance of high proteins such as eggs, nuts and meat. In the most acute mode, MSUD can induce mental retardation as a result of physical stress. Fever, infection or lacks of...
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...diet, increase lifespan in a variety of organisms. Now new research is illuminating how this works at the molecular level. A particular protein is key in regulating at least one aspect of the stress response and may be a good model for anti-aging drugs. "What we have here is an essential protective pathway that now looks like a very effective therapeutic target," said biologist Richard Morimoto of Northwestern University. Most research on this protein, called sirtuin1 (SIRT1), has concentrated on its ability to regulate and protect mitochondria — cellular power generators that are corroded over time by reactive oxygen molecules. But SIRT1 also protects DNA in the cell nucleus. Morimoto’s findings, published Thursday in Science, give a precise mechanical explanation for the effects. Cells have evolved a particular response to stay alive in adverse conditions. When a cell starts getting too hot, too hungry or too oxygen-deprived, certain proteins migrate into the nucleus. There, they latch onto sections of DNA and cause heat-shock proteins to be produced. Heatshock proteins — so named because they were first discovered in cells experiencing high temperatures — cruise around the cell, fixing damaged or improperly folded proteins. "Proteins are very delicate," Morimoto said. "Any change in the environment causes them to misfold." Repairing proteins keeps cells, and the body, in top shape. Animals exposed to only minor stresses — such as a calorie-restricted diet — reap the benefits...
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...areas of science. Purified, high molecular weight DNA is used for many applications such as: * To prepare genomic DNA libraries * To isolate genes to be cloned * To carry out southern blotting and hybridization Many different methods are available for isolation of genomic DNA, and they all involve the following basic stages. * Disruption and lysis of starting material: * Cell walls and cell membranes are broken to release DNA and other cellular components. * Removal of proteins and other contaminants: * Protein contaminations are removed by digestion with proteinase K followed by salting out and denaturing some proteins by SDS. * Recovery of DNA: * DNA is precipitated using ethanol or isopropanol in the presence of monovalent cations All DNA isolation protocols have a common goal in isolating high molecular weight DNA, which meet the following three criteria: 1) Purity should be high enough for further downstream applications such as restriction digestion. 2) DNA should be intact to give accurate and reproducible migration patterns on gel electrophoresis. 3) Yield should be high enough to obtain a required quantity of DNA from a reasonable amount of tissue. Materials and Methods: Materials Bench centrifuge, incubator, Falcon tubes, eppendorf tubes, micropipettes, micropipette tips gloves. DNA extraction Frozen blood samples were thawn 30ml of lysis buffer was added, mixed and kept on crushed ice for 15mins Centrifuged...
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...Function of Restriction Enzymes: Restriction endonucleases cleave the phosphodiester bond between an adjacent phosphate and deoxyribose group in the phosphate backbone of the DNA. The active site of the endonuclease perform this cleavage by binding to the side chain of certain amino acids to the phosphate group through a chemical bond. This dissolves the preexisting bond between the deoxyribose sugar and the phosphate resulting in a breakage with in the DNA chain at a specific location. (3, 7) One characteristic feature of restriction endonucleases is that they cut at a very particular site having a specific DNA sequence. This specific sequence that allows the enzyme to attach is known as the recognition site. Consider the example of the first restriction enzyme discovered, EcoRI....
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...thuringiensis (B. thuringiensis) C CaCl2: calcium chloride cDNA: copy DNA CD4 cells: referring to human white blood cells, which contain the cell surface recognition protein CD4 cm: centimeter CF: cystic fibrosis CD: compact disc CDC: Center for Disease Control and Prevention cGMPs: good manufacturing practices CHO: Chinese hamster ovary cm: centimeter CPDP: Comprehensive Product Development Plan CO2: carbon dioxide COOH: carboxyl [group] CT [scan]: computed tomography CuSO4: cupric sulfate (anhydrous) D dATP: deoxyadenosine triphosphate, the cell’s source of cytosine (C) for DNA molecules D: diopter DEAE: diethylaminoethyl DNA: deoxyribonucleic acid dH20: distilled water ddNTP: dideoxynucleotide dNTP: deoxynucleotide triphosphates (the dATP, dCTP, dGTP, and dTTPs) DMV: Department of Motor Vehicles DPA: diphenylamine [solution] DTT: dithiothreitol DVM: Doctor of Veterinary Medicine E EDTA: ELISA: enzyme-linked immunosorbent assay EPA: Environmental Protection Agency EtBr: ethidium bromide EtOH: ethanol F FAA: Federal Aviation Administration FDA: Food and Drug Administration FLPC: fast-performance liquid chromatography G g: gram GC: gas chromatograph gDNA: genomic DNA GFP: green fluorescent protein GMO: genetically modified organism gp: glycosylated protein GM-CSF: granulocyte-macrophage colony-stimulating factor GTE: glucose/TRIS/EDTA H H20: water H202: hydrogen peroxide...
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... Disrupting the peptidoglycan will result the release of the content inside the cell and the death of the cell. In addition to inhibiting DNases, the EDTA in the lysis solution aids the lysozyme to access the peptidoglycan by removing the Mg2+ from the lipopolysaccharide (LPS) layer, which will disrupt the membrane of the bacteria (1). The isolation step requires the removal of other macromolecules, such as protein and RNA, in the lysate. Protein can be dissociated from nucleic acids with phenol and chloroform. Proteolytic enzymes including pronase and proteinase K are often added to further remove protein. RNA, on the other hand, can be removed by RNase. Lastly, the DNA can be precipitated out from alcohol. In order for the nucleic acid to precipitate out of alcohol, some sort of cations, such as sodium and ammonium, are used to shield the negative charge of nucleic acid. As a result, nucleic acid will have a reduced solubility in solution and become insoluble pellet. The DNA extraction procedure provides a sample of purified DNA extract that can be used for restriction enzyme digest in upcoming experiment. Plasmid isolation protocol (mini-prep) is somewhat like genomic DNA isolation but with some variation to the content of lysis buffer, method of macromolecule removal and DNA collection. The focus of mini-prep is that plasmid isolation requires the separation from not only the cellular components but also from the genomic DNA. To achieve that, the protocol exploits the size...
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...579 Atomic force microscopy and other scanning probe microscopies Helen G Hansma and Lía Pietrasanta The highlight of the past year is the unfolding and refolding of the muscle protein titin in the atomic force microscope. A related highlight in the intersection between experiment and theory is a recent review of the effects of molecular forces on biochemical kinetics. Other advances in scanning probe microscopy include entropic brushes, molecular sandwiches and applications of atomic force microscopy to gene therapy. Address Department of Physics, University of California, Santa Barbara, CA 93106, USA Current Opinion in Chemical Biology 1998, 2:579–584 http://biomednet.com/elecref/1367593100200579 © Current Biology Ltd ISSN 1367-5931 Abbreviations AFM atomic force microscopy/microscope SFM scanning force microscopy/microscope SICM scanning ion conductance microscopy/microscope SPM scanning probe microscopy/microscope STM scanning tunneling microscopy/microscope A new journal, Probe Microscopy, was launched in 1997 as a forum specifically devoted to the science and technology of SPM. AFM and SFM have been also newsworthy items in Science and Nature in the past year [14••,15•–17•,18••,19]. An introduction to AFM is covered well in a recent issue of Current Opinion in Chemical Biology, which describes and illustrates the design and mode of operation of AFM [4••]. The AFM images sample surfaces by raster-scanning a sharp tip back and forth over the surface. The tip is on...
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...Genetics Pregnancy Cholesterol ester storage disease (chylomicronemia) Defects in lipid carrier proteins or enzymes Consumption of a fatty meal Nephrotic syndrome Diagnosis of High Cholesterol in Cats The veterinarian will need to know the cat's complete health history, which will include any recent dietary changes, a detailed list of all of the symptoms the cat is experiencing, when the symptoms first began and any other conditions that may have caused the high cholesterol levels to occur. The veterinarian will physically examine the cat, looking for any signs of cutaneous xanthomata, feeling for abdominal distention and examining the cat's...
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