DOCSLIB.ORG

The Role of NF-Κb and C/EBP Factors During Pathogen-Mediated

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Role of NF-Κb and C/EBP Factors During Pathogen-Mediated The role of NF-B and C/EBP factors during pathogen-mediated activation of bovine interleukin 8 and beta-defensin in mammary epithelial cells Inaugural dissertation for the academic degree Doctor rerum naturalium of Mathematisch-Naturwissenschaftlichen Fakultät Universität Rostock By Shuzhen Liu (M. Sc.), born on March-02-1972, in Shanxi Province, China From the Forschungsinstitut für die Biologie landwirtschaftlicher Nutztiere in Dummerstorf Rostock 2009 URN: urn:nbn:de:gbv:28-diss2009-0185-5 Dean: Prof. Dr. Hendrik Schubert Reviewers: 1. Prof. Dr. Hans-Martin Seyfert Research Unit molecular biology, Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany 2. Prof. Dr. Dieter G. Weiss Division of animal Physiology, Institute of Cell Biology and Biosystems Technology, University of Rostock, Albert-Einstein-Strasse 3, 18059 Rostock, Germany 3. PD Dr. Ulrike Gimsa Research Unit Behavioural Physiology, Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany Date of defense: October 19th, 2009 Table of Contents TABLE OF CONTENTS 1. INTRODUCTION..................................................................................................................1 1.1 Mastitis as a challenge in general immunology..................................................................1 1.2 Innate immunity of the bovine mammary gland ................................................................2 1.3 Toll-like receptors (TLRs): main receptors perceiving the pathogen presence..................3 1.4 Interleukin 8 (IL-8): a chemokine ......................................................................................4 1.4.1 Inducible expression of IL-8 .......................................................................................4 1.4.2 Molecular regulation of the human IL-8 expression ...................................................5 1.5 Antimicrobial peptides: -defensins...................................................................................6 1.5.1 Classification and structures of defensins ...................................................................6 1.5.2-defensin in mammal.................................................................................................6 1.5.3 Inducible expression of -defensins............................................................................7 1.5.4 Roles of -defensins in mammalian immunity............................................................7 1.5.5 Regulation of -defensins expression..........................................................................8 1.6 Nuclear factors (NF-B).....................................................................................................8 1.6.1 Inhibition of NF-B.....................................................................................................9 1.6.2 Activation of NF-B....................................................................................................9 1.6.3 Phosphorylation and acetylation of NF-B...............................................................10 1.6.4 Transcriptional coactivators and interactive proteins of NF-B ...............................11 1.7 CCAAT-enhancer binding proteins (C/EBP)...................................................................11 1.7.1 Role of C/EBP in inflammatory and immune response ...........................................12 1.7.2 Autoregulation and phosphorylation of the C/EBP family factors............................13 1.7.3 Interaction of C/EBP with NF-B.............................................................................13 1.8 Chromatin remodeling......................................................................................................14 1.8.1 Overview of chromatin remodeling...........................................................................14 1.8.2 The link between acetylation and transcription.........................................................14 1.9 DNA Methylation.............................................................................................................15 1.9.1 Methods for DNA methylation analysis....................................................................15 1.9.2 Roles of DNA methylation........................................................................................16 1.10 Goals:..............................................................................................................................17 2. MATERIALS AND METHODS .........................................................................................18 i Table of Contents 2.1 Materials ...........................................................................................................................18 2.1.1 Instruments ................................................................................................................18 2.1.2 Reagents ....................................................................................................................18 2.1.3 Kits ............................................................................................................................19 2.1.4 Strains and plasmids ..................................................................................................19 2.1.5 Enzymes ....................................................................................................................19 2.1.6 Antibodies..................................................................................................................20 2.1.7 Tissue samples...........................................................................................................20 2.1.8 Cells and cell lines.....................................................................................................20 2.2 Molecular cloning methods ..............................................................................................21 2.2.1 Preparation of plasmid DNA .....................................................................................21 2.2.1.1 Mini-or midi-preparation of plasmid DNA.........................................................21 2.2.1.2 Preparation of endotoxin free plasmid DNA by Qiagen kit................................21 2.2.2 RNA techniques.........................................................................................................22 2.2.2.1 RNA extraction from cell culture and frozen tissue............................................22 2.2.2.2 RNA electrophoresis ...........................................................................................22 2.2.3 Polymerase Chain Reaction (PCR)............................................................................23 2.2.3.1 Standard PCR and high fidelity PCR ..................................................................23 2.2.3.2 Reverse transcription PCR (RT-PCR).................................................................23 2.2.3.3 Real-time PCR.....................................................................................................24 2.2.3.3.1 Preparation of standard curve...........................................................................25 2.2.3.3.2 Real-time PCR programs..................................................................................25 2.2.4 Plasmid construction techniques ...............................................................................26 2.2.4.1 DNA electrophoresis ...........................................................................................26 2.2.4.2 Purification of DNA from agarose gel ................................................................26 2.2.4.3 Restriction digestion of DNA fragment ..............................................................26 2.2.4.4 Preparation of vector for cloning.........................................................................27 2.2.4.5 Ligation…….. .....................................................................................................27 2.2.5 Transformation ..........................................................................................................27 2.2.5.1 Preparation of competent cells ............................................................................27 2.2.5.2 Transformation procedure ...................................................................................28 2.2.6 PCR mediated mutagenesis and deletion .................................................................28 2.2.7 Rapid Amplification of 5 cDNA Ends (5 RACE) ...................................................29 2.3 Protein techniques ............................................................................................................30 ii Table of Contents 2.3.1 Determination of total protein concentration (Bradford assay).................................30 2.3.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)..............30 2.3.3 Western blot...............................................................................................................31 2.4 Mammalian cell culture and reporter gene assay .............................................................32 2.4.1 Cell types and media .................................................................................................32 2.4.2 Cell culture and cryopreservation..............................................................................32 2.4.2.1 Cryopreservation of cells.....................................................................................32
Load more

Recommended publications
  • CLONING, EXPRESSION ANALYSIS, and TRANSFORMATION VECTOR CONSTRUCTION of DAM HOMOLOGS in PEACH and POPLAR Yuhui Xie Clemson University, [email protected]
    Clemson University TigerPrints All Theses Theses 1-2011 CLONING, EXPRESSION ANALYSIS, AND TRANSFORMATION VECTOR CONSTRUCTION OF DAM HOMOLOGS IN PEACH AND POPLAR Yuhui Xie Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_theses Part of the Plant Biology Commons Recommended Citation Xie, Yuhui, "CLONING, EXPRESSION ANALYSIS, AND TRANSFORMATION VECTOR CONSTRUCTION OF DAM HOMOLOGS IN PEACH AND POPLAR" (2011). All Theses. 1146. https://tigerprints.clemson.edu/all_theses/1146 This Thesis is brought to you for free and open access by the Theses at TigerPrints. It has been accepted for inclusion in All Theses by an authorized administrator of TigerPrints. For more information, please contact [email protected]. CLONING, EXPRESSION ANALYSIS, AND TRANSFORMATION VECTOR CONSTRUCTION OF DAM HOMOLOGS IN PEACH AND POPLAR _______________________________________________ A Thesis Presented to the Graduate School of Clemson University ________________________________________________ In Partial Fulfillment of the Requirement for the Degree Master of Science Plant and Environmental Sciences _________________________________________________ by Yuhui Xie August 2011 ________________________________________________ Accepted by: Dr. Douglas G. Bielenberg, Committee Chair Dr. Haiying Liang Dr. Hong Luo ABSTRACT Genetic fine mapping and sequencing of the EVG locus in peach [Prunus persica (L.) Batsch] identified six tandem arrayed Dormancy-Associated MADS-box (DAM) genes as candidates for regulating growth cessation and terminal bud formation in the non-dormant evergrowing (evg) mutant. Since the mutant is lacking expression of six genes in the mapped locus, further functional analysis is needed to narrow the list of gene candidates for the non-dormant evg phenotype. Here I report three sets of experiments designed to functionally test DAM genes in peach and their homologs in a model tree, hybrid poplar.
    [Show full text]
  • Enzymes in Cloning Part I
    ˹̀/˺̀/˺̊˼̊ ENZYMES IN CLONING PART I Dr.Sarookhani ˺ ˹̀/˺̀/˺̊˼̊ Cloning --aa definition •• From the Greek --klon,klon, a twig •• An aggregate of the asexually produced progeny of an individual;a group of replicas of all or part of a macromolecule (such as DNA or an antibody) •• An individual grown from a single somatic cell of its parent & genetically identical to it •• Clone: a collection of molecules or cells, all identical to an original molecule or cell Dr.Sarookhani ˻ ˹̀/˺̀/˺̊˼̊ Different types of Cloning 1. Reproductive Cloning 2. Therapeutic Cloning 3. Recombinant DNA Technology or DNA Cloning Dr.Sarookhani ˼ ˹̀/˺̀/˺̊˼̊ DNA CLONING A method for identifying and purifying a particular DNA fragment (clone) of interest from a complex mixture of DNA fragments, and then producing large numbers of the fragment (clone) of interest. Dr.Sarookhani ̊ ˹̀/˺̀/˺̊˼̊ What is genetic engineering • Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype. • Various kinds of genetic modification are possible: inserting a foreign gene from one species into another, forming a transgenic organism; altering an existing gene so that its product is changed; or changing gene expression so that it is translated more often or not at all. Dr.Sarookhani ̋ ˹̀/˺̀/˺̊˼̊ Dr.Sarookhani ̌ ˹̀/˺̀/˺̊˼̊ Dr.Sarookhani ̀ ˹̀/˺̀/˺̊˼̊ Genomic Library Dr.Sarookhani ́ ˹̀/˺̀/˺̊˼̊ Dr.Sarookhani ̂ ˹̀/˺̀/˺̊˼̊ Basic steps in genetic engineering 1.
    [Show full text]
  • Cyclic Digestion and Ligation-Mediated PCR Used For
    www.nature.com/scientificreports OPEN Cyclic Digestion and Ligation- Mediated PCR Used for Flanking Sequence Walking Dong Yu1,2,5, Tianshun Zhou2,4,5, Xuewu Sun2,3, Zhizhong Sun2, Xiabing Sheng1,2, Yanning Tan2, Ling Liu2,4, Ning Ouyang2,4, Ke Xu2, Kaibing Shi2, Guilong Yuan2, Jia Ding2, Meijuan Duan3* & Dingyang Yuan1,2,3,4* Ligation-mediated PCR (LM-PCR) is a classical method for isolating fanking sequences; however, it has a common limitation of reduced success rate owing to the circularization or multimerization of target restriction fragments including the known sequence. To address this limitation, we developed a novel LM-PCR method, termed Cyclic Digestion and Ligation-Mediated PCR (CDL-PCR). The novelty of this approach involves the design of new adapters that cannot be digested after being ligated with the restriction fragment, and cyclic digestion and ligation may be manipulated to block the circularization or multimerization of the target restriction fragments. Moreover, to improve the generality and fexibility of CDL-PCR, an adapter precursor sequence was designed, which could be digested to prepare 12 diferent adapters at low cost. Using this method, the fanking sequences of T-DNA insertions were obtained from transgenic rice and Arabidopsis thaliana. The experimental results demonstrated that CDL-PCR is an efcient and fexible method for identifying the fanking sequences in transgenic rice and Arabidopsis thaliana. Identifcation of fanking sequences has ofen been employed to determine the location of T-DNA insertion in genomic DNA. Methods to obtain fanking sequencea include inverse PCR1, randomly primed PCR2–5, and ligation-mediated PCR6–8. Inverse PCR, the earliest fanking cloning technique, has a low efciency and is limited by the rate of self-ligation and amplifcation range of DNA polymerases9.
    [Show full text]
  • James Alan Tunaley
    Structure and Activity Investigations of the Cell Fate Determinant, SpoIIE, from Bacillus subtilis. James Alan Tunaley Thesis Submitted for the Degree of Doctor of Philosophy University of York Department of Chemistry September 2013 Abstract For many years the Gram positive bacterium Bacillus subtilis has been a model organism for prokaryotic cell and molecular biology. The asymmetric cell division which B. subtilis undergoes during sporulation is a simple system by which to study the process of cell differentiation. Sporulation is governed by a series of genetic temporal and spatial controls. Gene regulation brought about by a series of σ factors and transcriptional regulators is coupled to key morphological stages or checkpoints. σF initiates the first step in a cascade of complex genetic control which eventually produces a resilient endospore. The activation of σF, the first compartment-specific sigma factor, in the forespore and its regulation through interaction between three proteins; SpoIIAA, SpoIIAB and SpoIIE, is of particular interest. SpoIIE, a protein phosphatase which binds to the asymmetric division septum, is a crucial factor in the selective activation of σF in the forespore. Of three putative domains in SpoIIE only the C-terminal PP2C phosphatase domain has been structurally characterised. The central domain, domain II, of SpoIIE has been assigned a role in interaction with the cell division machinery; however mutational studies have shown that, in addition, this domain is also responsible for the regulation of phosphatase activity. This work describes the isolation and characterisation of three new fragments of SpoIIE containing elements of the central cytoplasmic domain of SpoIIE. These include a fragment found to accurately represent the N-terminal solubility limit of domain II which shows a high degree of oligomeric character.
    [Show full text]
  • Dissertation M.Sc Lila Oubraham
    Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by M.Sc Lila Oubraham born in: Algiers, Algeria Oral-examination: 11.05.2016 Nuclease-mediated gene manipulation of factors implicated in zebrafish neurogenesis Referees: Prof. Dr. Uwe Strähle Prof. Dr. Nicholas S. Foulkes Summary Complex and differential gene expression programs give rise to several cell types that constitute the different parts of the organism. This cell fate determination is controlled by a group of proteins, named transcription regulators (TRs). Our group investigates the molecular mechanisms underlying neurogenesis using the zebrafish as a model system. To that purpose, a genome-wide analysis of TR gene expression was performed in our laboratory, and hundreds of these regulators were identified. On the basis of these initial studies, a number of TRs were selected for further characterization. For this project, two model systems for zebrafish neurogenesis were chosen: The embryonic spinal cord and the adult telencephalon. The spinal cord is considered as relatively simple and is used to understand the neural differentiation and function in vertebrates during development. Based on morpholinos knockdown experiments, two closely related genes sox1a and sox1b encoding transcription factors, were shown to play a role in the specification of a newly observed sub-type of interneurons, named V2c, in the ventral spinal cord of the zebrafish. Nevertheless, the epistatic relationship between these genes has still to be investigated. On the other hand, in the adult brain, new neurons are continuously generated from neural progenitor cells.
    [Show full text]
  • Assignment 1 Part I
    1 Assignment 1 Note: Part I of the assignment is to be submitted through Blackboard (See instructions on Blackboard). Parts II, III and IV are to be submitted as a hard copy during your lab session. Part I Dilutions and Concentrations When doing your calculations, do not round off your intermediate numbers. Only round off the final answer. Your answers must be submitted to two significant figures after the decimal. For example 2.00, 0.020, or 0.0020. It is strongly recommended that you submit your answers using the web browser Firefox. You will be allowed two submissions! Use the following information to answer questions 1-6 You prepare a solution by adding the following ingredients in the order indicated: 600 mL H2O, 125 mL 1.6 M LiCl, 50 mL 20 % (m/v) MgCl2, and 25 mL 10g/L NaCl. The properties of each ingredient are as follows: LiCl: MW 200g/mole, density 1.2g/mL, density of 1.6 M soln. 1.05g/mL MgCl2: MW 150g/mole, density 1.3g/mL, density of 20% soln. 1.1g/mL NaCl: MW 35g/mole, density 1.15g/mL, density of 10g/L soln. 1.03g/mL Final solution: density: 1.25g/mL 1. What is the final molarity of MgCl2 in the solution? (0.083M) 2. What is the volume in milliliters of one part? (25 mL) 3. What is the percentage (m/m) of NaCl in the final solution? (0.025%) 4. What is the percentage (m/v) of LiCl in the final solution? (5%) 5. What is the number of parts of solvent in the final solution? (24 parts) 6.
    [Show full text]
  • WO 2017/054721 Al 6 April 2017 (06.04.2017) P O P C T
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/054721 Al 6 April 2017 (06.04.2017) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12N 15/82 (2006.01) C12N 15/113 (2010.01) kind of national protection available): AE, AG, AL, AM, A01H 5/00 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, (21) Number: International Application DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/CN2016/100533 HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (22) International Filing Date: KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, 28 September 2016 (28.09.201 6) MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (25) Filing Language: English SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, (26) Publication Language: English TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 2015 1063 1450.5 (84) Designated States (unless otherwise indicated, for every 29 September 2015 (29.09.2015) CN kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (71) Applicant: INSTITUTE OF GENETICS AND DEVEL¬ TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, OPMENTAL BIOLOGY, CHINESE ACADEMY OF TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, SCIENCES [CN/CN]; No.
    [Show full text]
  • Building a Cassette-Based System for Combined Eukaryotic Gene Expression
    AN ABSTRACT OF THE THESIS OF Raviteja Madhira for the degree of Honors Baccalaureate of Science in Biochemistry and Biophysics presented on August 26, 2009. Title: Building a cassette-based system for combined eukaryotic gene expression. Abstract Approved:________________________________________________________ Michael K. Gross Gene regulatory networks control the state of a cell. Models of such networks have been produced for sea urchin development and suggest that cell type may be defined by distinct network kernels. Such kernels are composed of a set of sequence specific DNA-binding transcription factors (SSTFs) that maintain each other’s expression through cell type-specific cis- regulatory modules. Ectopic expression of some individual SSTFs leads to trans-differentiation, or the conversion of one cell type to another. Our hypothesis is that trans-differentiation will be more efficient and targeted if specific SSTF combinations, corresponding to those of a network kernel, are expressed ectopically. A transient, polycistronic, cassette-based expression system was designed and built. This system allow for Lbx1-dependent network kernels to be transiently introduced into heterologous cells. Transfecting a cell with a specific combination of SSTFs may trigger establishment of the corresponding endogenous network kernel and thereby lead to trans- differentiation. Five SSTF ORFs (Lbx1, Lmx1b, Pax2, Pax8, and Isl1) were PCR amplified with gene-specific primers that included AsiSI and PacI restriction sites. A base bacterial vector was created with a custom polylinker to receive the PCR-generated minimal ORFs. This custom polylinker was constructed so that PCR-generated minimal ORFs could readily be transferred to other vector systems by classic recombinant methods.
    [Show full text]
  • Download (8MB)
    Kentner, Jeffrey Louis (2015) Engineering the zinc finger recombinase for use in targeted genomic editing. PhD thesis. https://theses.gla.ac.uk/6910/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] Engineering the Zinc Finger Recombinase for use in Targeted Genomic Editing Jeffrey Louis Kentner Honours Bachelor of Science Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Institute of Molecular, Cell and Systems Biology College of Medical, Veterinary and Life Sciences University of Glasgow November 2015 Abstract The zinc finger recombinase (ZFR) is a chimeric enzyme system for use in targeted genomic editing. The ZFR is comprised of a recombinase catalytic domain, which is able to catalyse recombination reactions between DNA molecules, and a zinc finger array DNA- binding domain, which is able to target the enzyme to a desired genetic sequence. Currently the ZFR is in an early stage of development and will require several crucial improvements before it can be adopted as a useful genome editing tool by researchers.
    [Show full text]
  • Engineering Dual-Glycan Responsive Expression Systems for Tunable Production of Heterologous Proteins in Bacteroides Thetaiotaomicron
    ENGINEERING DUAL-GLYCAN RESPONSIVE EXPRESSION SYSTEMS FOR TUNABLE PRODUCTION OF HETEROLOGOUS PROTEINS IN BACTEROIDES THETAIOTAOMICRON MARSHALL SMITH Bachelor of Science, University of Lethbridge, 2016 A Thesis Submitted to the School of Graduate Studies Of the University of Lethbridge In Partial Fulfilment of the Requirements for the Degree MASTER OF SCIENCE Department of Biological Sciences University of Lethbridge LETHBRIDGE ALBERTA CANADA © Marshall Smith, 2018 ENGINEERING DUAL-GLYCAN RESPONSIVE EXPRESSION SYSTEMS FOR TUNABLE PRODUCTION OF HETEROLOGOUS PROTEINS IN BACTEROIDES THETAIOTAOMICRON MARSHALL SMITH Date of Defence: December 12th 2018 Dr. D. Wade Abbott Research Scientist, Adjunct Professor Ph.D. Co-Supervisor Dr. L. Brent Selinger Professor Ph.D. Co-Supervisor Dr. G. Douglas Inglis Research Scientist, Adjunct Professor Ph.D. Thesis Examination Committee Member Dr. Tony Russell Associate Professor Ph.D. Thesis Examination Committee Member Dr. Igor Kovalchuk Professor Ph.D. Thesis Examination Committee Member Chair, Thesis Examination Committee ii Dedication To my family, for the constant support and encouragement. To my lab mates. This project would have been no fun without you, and I am proud to call you my friends. Thank you. iii Abstract Genetically engineering symbiotic bacteria remains an underexploited opportunity to improve host-health and create new classes of biological devices, such as diagnostics or intestinal delivery systems for therapeutics. Bacteroides thetaiotamicron (B. theta) is a Gram-negative intestinal anaerobe with potential for the capability to produce functional heterologous proteins within a host intestine. To improve the strength and regulatory fidelity of transgene expression in B. theta, I have developed platform expression strains with engineered regulatory proteins under control of promoter elements that respond to dextran and arabinogalactan, two chemically distinct glycans.
    [Show full text]
  • Restriction Enzymes to Cut DNA • Ligate Fragments Into a Cloning Vector • Transform Recombinant DNA Into a Host to Replicate the DNA and Pass Copies Into Progeny
    Genetic Engineering Dr. S.K. Halder What and Why? • Genetic engineering, also called genetic modification, is the direct manipulation of an organism's genome using biotechnology. • New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism. • An organism that is generated through genetic engineering is considered to be a genetically modified organism (GMO). Flash back…. • Genetic engineering as the direct manipulation of DNA by humans outside breeding and mutations has only existed since the 1970s. • The term "genetic engineering" was first coined by Jack Williamson in his science fiction novel Dragon's Island, published in 1951, one year before DNA's role in heredity was confirmed by Alfred Hershey and Martha Chase, and two years before James Watson and Francis Crick showed that the DNA molecule has a double-helix structure.!!! • The first GMOs: bacteria in 1973 and mice in 1974. • Insulin-producing bacteria were commercialized in 1982 and genetically modified food has been sold since 1994. • Glofish, the first GMO designed as a pet, was first sold in the United States December in 2003. DNA cloning Cloning is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. • To obtain large amounts of pure DNA – Procedure • Isolate DNA • Use restriction enzymes to cut DNA • Ligate fragments into a cloning vector • Transform recombinant DNA into a host to replicate the DNA and pass copies into progeny.
    [Show full text]
  • Assignment 1 Part I
    1 Assignment 1 Note: Part I of the assignment is to be submitted through Blackboard (See instructions on Blackboard). Parts II, III and IV are to be submitted as a hard copy during your lab session. Part I Dilutions and Concentrations When doing your calculations, do not round off your intermediate numbers. Only round off the final answer. Your answers must be submitted to two significant figures after the decimal. For example 2.00, 0.020, or 0.0020. It is strongly recommended that you submit your answers using the web browser Firefox. You will be allowed two submissions! Use the following information to answer questions 1-6 You prepare a solution by adding the following ingredients in the order indicated: 600 mL H2O, 125 mL 1.6 M LiCl, 50 mL 20 % (m/v) MgCl2, and 25 mL 10g/L NaCl. The properties of each ingredient are as follows: LiCl: MW 200g/mole, density 1.2g/mL, density of 1.6 M soln. 1.05g/mL MgCl2: MW 150g/mole, density 1.3g/mL, density of 20% soln. 1.1g/mL NaCl: MW 35g/mole, density 1.15g/mL, density of 10g/L soln. 1.03g/mL Final solution: density: 1.25g/mL 1. What is the final molarity of MgCl2 in the solution? (0.083M) 2. What is the volume in milliliters of one part? (25 mL) 3. What is the percentage (m/m) of NaCl in the final solution? (0.025%) 4. What is the percentage (m/v) of LiCl in the final solution? (5%) 5. What is the number of parts of solvent in the final solution? (24 parts) 6.
    [Show full text]