Plasmids 101
Total Page:16
File Type:pdf, Size:1020Kb
Load more
Recommended publications
-
RANDY SCHEKMAN Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, USA
GENES AND PROTEINS THAT CONTROL THE SECRETORY PATHWAY Nobel Lecture, 7 December 2013 by RANDY SCHEKMAN Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, USA. Introduction George Palade shared the 1974 Nobel Prize with Albert Claude and Christian de Duve for their pioneering work in the characterization of organelles interrelated by the process of secretion in mammalian cells and tissues. These three scholars established the modern field of cell biology and the tools of cell fractionation and thin section transmission electron microscopy. It was Palade’s genius in particular that revealed the organization of the secretory pathway. He discovered the ribosome and showed that it was poised on the surface of the endoplasmic reticulum (ER) where it engaged in the vectorial translocation of newly synthesized secretory polypeptides (1). And in a most elegant and technically challenging investigation, his group employed radioactive amino acids in a pulse-chase regimen to show by autoradiograpic exposure of thin sections on a photographic emulsion that secretory proteins progress in sequence from the ER through the Golgi apparatus into secretory granules, which then discharge their cargo by membrane fusion at the cell surface (1). He documented the role of vesicles as carriers of cargo between compartments and he formulated the hypothesis that membranes template their own production rather than form by a process of de novo biogenesis (1). As a university student I was ignorant of the important developments in cell biology; however, I learned of Palade’s work during my first year of graduate school in the Stanford biochemistry department. -
Where Is That Light Coming From?
2004 Deep-Scope Expedition Where is That Light Coming From? FOCUS TEACHING TIME Bioluminescence One 45-minute class period, plus time for student research GRADE LEVEL 9-12 (Chemistry & Life Science) SEATING ARRANGEMENT Classroom style or groups of 3-4 students FOCUS QUESTION What is the basic chemistry responsible for bio- MAXIMUM NUMBER OF STUDENTS luminescence, and how does this process benefit 30 deep-sea organisms? KEY WORDS LEARNING OBJECTIVES Chemiluminescence Students will be able to explain the role of lucif- Bioluminescence erins, luciferases, and co-factors in biolumines- Fluorescence cence, and the general sequence of the light-emit- Phosphorescence ting process. Luciferin Luciferase Students will be able to discuss the major types of Photoprotein luciferins found in marine organisms. Counter-illumination Lux operon Students will be able to define the “lux operon?” BACKGROUND INFORMATION Students will be able to discuss at least three Deep-sea explorers face many challenges: ways that bioluminescence may benefit deep-sea extreme heat and cold, high pressures, and organisms, and give an example of at least one almost total darkness. The absence of light poses organism that actually receives each of the ben- particular challenges to scientists who want to efits discussed. study organisms that inhabit the deep ocean envi- ronment. Even though deep-diving submersibles MATERIALS carry bright lights, simply turning these lights on ❑ None creates another set of problems: at least some mobile organisms are likely to move away from -
Protein Tagging and Detection with Engineered Self-Assembling Fragments of Green fluorescent Protein
LETTERS Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein Ste´phanie Cabantous, Thomas C Terwilliger & Geoffrey S Waldo Existing protein tagging and detection methods are powerful the same amount of refolded superfolder GFP 1–10. In addition to the but have drawbacks. Split protein tags can perturb protein folding reporter GFP mutations, GFP 1–10 OPT contains S30R, solubility1–4 or may not work in living cells5–7. Green Y145F, I171V and A206V substitutions from superfolder GFP and fluorescent protein (GFP) fusions can misfold8 or exhibit seven new mutations: N39I, T105K, E111V, I128T, K166T, I167V and altered processing9. Fluorogenic biarsenical FLaSH or S205T. This protein is B50% soluble when expressed in E. coli at ReASH10 substrates overcome many of these limitations but 37 1C (data not shown). Ultraviolet-visible spectra of 10 mg/ml require a polycysteine tag motif, a reducing environment and solutions of the nonfluorescent GFP 1–10 OPT lacked the 480 nm cell transfection or permeabilization10. An ideal protein tag absorption band of the red-shifted GFP19 (data not shown), suggest- http://www.nature.com/naturebiotechnology would be genetically encoded, would work both in vivo and ing that the addition of GFP 11 triggers a folding step required to in vitro, would provide a sensitive analytical signal and would generate the cyclized chromophore19. Purified GFP 1–10 OPT, super- not require external chemical reagents or substrates. One way folder GFP and folding reporter GFP were each studied by analytical to accomplish this might be with a split GFP11, but the GFP gel filtration loaded at 10 mg/ml. -
A Short History of DNA Technology 1865 - Gregor Mendel the Father of Genetics
A Short History of DNA Technology 1865 - Gregor Mendel The Father of Genetics The Augustinian monastery in old Brno, Moravia 1865 - Gregor Mendel • Law of Segregation • Law of Independent Assortment • Law of Dominance 1865 1915 - T.H. Morgan Genetics of Drosophila • Short generation time • Easy to maintain • Only 4 pairs of chromosomes 1865 1915 - T.H. Morgan •Genes located on chromosomes •Sex-linked inheritance wild type mutant •Gene linkage 0 •Recombination long aristae short aristae •Genetic mapping gray black body 48.5 body (cross-over maps) 57.5 red eyes cinnabar eyes 67.0 normal wings vestigial wings 104.5 red eyes brown eyes 1865 1928 - Frederick Griffith “Rough” colonies “Smooth” colonies Transformation of Streptococcus pneumoniae Living Living Heat killed Heat killed S cells mixed S cells R cells S cells with living R cells capsule Living S cells in blood Bacterial sample from dead mouse Strain Injection Results 1865 Beadle & Tatum - 1941 One Gene - One Enzyme Hypothesis Neurospora crassa Ascus Ascospores placed X-rays Fruiting on complete body medium All grow Minimal + amino acids No growth Minimal Minimal + vitamins in mutants Fragments placed on minimal medium Minimal plus: Mutant deficient in enzyme that synthesizes arginine Cys Glu Arg Lys His 1865 Beadle & Tatum - 1941 Gene A Gene B Gene C Minimal Medium + Citruline + Arginine + Ornithine Wild type PrecursorEnz A OrnithineEnz B CitrulineEnz C Arginine Metabolic block Class I Precursor OrnithineEnz B CitrulineEnz C Arginine Mutants Class II Mutants PrecursorEnz A Ornithine -
Current Topics in Microbiology and Immunology
Current Topics in Microbiology and Immunology Volume 431 Series Editors Rafi Ahmed School of Medicine, Rollins Research Center, Emory University, Atlanta, GA, USA Shizuo Akira Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan Klaus Aktories Faculty of Medicine, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Freiburg, Baden-Württemberg, Germany Arturo Casadevall W. Harry Feinstone Department of Molecular Microbiology & Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA Richard W. Compans Department of Microbiology and Immunology, Emory University, Atlanta, GA, USA Jorge E. Galan Boyer Ctr. for Molecular Medicine, School of Medicine, Yale University, New Haven, CT, USA Adolfo Garcia-Sastre Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA Bernard Malissen Parc Scientifique de Luminy, Centre d‘Immunologie de Marseille-Luminy, Marseille, France Rino Rappuoli GSK Vaccines, Siena, Italy The review series Current Topics in Microbiology and Immunology provides a synthesis of the latest research findings in the areas of molecular immunology, bacteriology and virology. Each timely volume contains a wealth of information on the featured subject. This review series is designed to provide access to up-to-date, often previously unpublished information. 2019 Impact Factor: 3.095., 5-Year Impact Factor: 3.895 2019 Eigenfaktor Score: 0.00081, Article Influence Score: 1.363 2019 Cite Score: 6.0, SNIP: 1.023, h5-Index: 43 More -
Cloning of Gene Coding Glyceraldehyde-3-Phosphate Dehydrogenase Using Puc18 Vector
Available online a t www.pelagiaresearchlibrary.com Pelagia Research Library European Journal of Experimental Biology, 2015, 5(3):52-57 ISSN: 2248 –9215 CODEN (USA): EJEBAU Cloning of gene coding glyceraldehyde-3-phosphate dehydrogenase using puc18 vector Manoj Kumar Dooda, Akhilesh Kushwaha *, Aquib Hasan and Manish Kushwaha Institute of Transgene Life Sciences, Lucknow (U.P), India _____________________________________________________________________________________________ ABSTRACT The term recombinant DNA technology, DNA cloning, molecular cloning, or gene cloning all refers to the same process. Gene cloning is a set of experimental methods in molecular biology and useful in many areas of research and for biomedical applications. It is the production of exact copies (clones) of a particular gene or DNA sequence using genetic engineering techniques. cDNA is synthesized by using template RNA isolated from blood sample (human). GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is one of the most commonly used housekeeping genes used in comparisons of gene expression data. Amplify the gene (GAPDH) using primer forward and reverse with the sequence of 5’-TGATGACATCAAGAAGGTGGTGAA-3’ and 5’-TCCTTGGAGGCCATGTGGGCCAT- 3’.pUC18 high copy cloning vector for replication in E. coli, suitable for “blue-white screening” technique and cleaved with the help of SmaI restriction enzyme. Modern cloning vectors include selectable markers (most frequently antibiotic resistant marker) that allow only cells in which the vector but necessarily the insert has been transfected to grow. Additionally the cloning vectors may contain color selection markers which provide blue/white screening (i.e. alpha complementation) on X- Gal and IPTG containing medium. Keywords: RNA isolation; TRIzol method; Gene cloning; Blue/white screening; Agarose gel electrophoresis. -
Systemic Infection Induced by Campylobacter Jejuni: Development of a Mouse Model and Elucidation of Molecular Mechanisms Samantha Terhorst Iowa State University
Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2012 Systemic infection induced by Campylobacter jejuni: Development of a mouse model and elucidation of molecular mechanisms Samantha Terhorst Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Microbiology Commons Recommended Citation Terhorst, Samantha, "Systemic infection induced by Campylobacter jejuni: Development of a mouse model and elucidation of molecular mechanisms" (2012). Graduate Theses and Dissertations. 12653. https://lib.dr.iastate.edu/etd/12653 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Systemic infection induced by Campylobacter jejuni: Development of a mouse model and elucidation of molecular mechanisms by Samantha Ashley Terhorst A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Toxicology Program of Study Committee: Qijing Zhang, Major Professor Byron Brehm-Stecher Paul Plummer Orhan Sahin Iowa State University Ames, Iowa 2012 Copyright © Samantha Ashley Terhorst, 2012. All rights reserved. ii TABLE OF CONTENTS ABSTRACT ..................................................................................................................... -
Protein Expression and Purification Series
Bio-Rad Explorer™ Protein Expression and Purifi cation Series Catalog Numbers 1665040EDU Centrifugation Purifi cation Process 1665045EDU Handpacked Purifi cation Process 1665050EDU Prepacked Purifi cation Process 1665070EDU Assessment Module explorer.bio-rad.com Please see each individual module for storage conditions. Duplication of any part of this document permitted for classroom use only. Please visit explorer.bio-rad.com to access our selection of language translations for Biotechnology Explorer Kit curricula. For technical service, call your local bio-rad offi ce, or in the U.S., call 1-800-4BIORAD (1-800-424-6723) Protein Expression and Purifi cation Series Dear Educator One of the great promises of the biotechnology industry is the ability to produce biopharmaceuticals to treat human disease. Genentech pioneered the development of recombinant DNA technology to produce products with a practical application. In the mid-1970s, insulin, used to treat diabetics, was extracted from the pancreas glands of swine and cattle that were slaughtered for food. It would take approximately 8,000 pounds of animal pancreas glands to produce one pound of insulin. Rather than extract the protein from animal sources, Genentech engineered bacterial cells to produce human insulin, resulting in the world’s fi rst commercial genetically engineered product. Producing novel proteins in bacteria or other cell types is not simple. Active proteins are often comprised of multiple chains of amino acids with complex folding and strand interactions. Commandeering a particular cell to reproduce the native form presents many challenges. Considerations of cell type, plasmid construction, and purifi cation strategy are all part of the process of developing a recombinant protein. -
GFP Plasmid) 35X
Název: Biotechnology and Fluorescent protein Školitel: Ana Maria Jimenez Jimenez, Iva Blažková Datum: 19.7.2013 Reg.č.projektu: CZ.1.07/2.3.00/20.0148 Název projektu: Mezinárodní spolupráce v oblasti "in vivo" zobrazovacích technik INTRODUCTION GFP - Green fluorescent protein A protein composed of 238 amino acid residues (26.9 kDa) exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range GFP was isolated from the jellyfish Aequorea victoria. In cell and molecular biology, the GFP gene is frequently used as a reporter of expression The GFP gene has been introduced and expressed in many bacteria, yeast and other fungi, fish, plant and mammalian cells. Green fluorescent protein (GFP) Nobel prize in Chemistry (2008): for the discovery and development of the green fluorescent protein, GFP Osamu Shimomura Martin Chalfie Roger Y. Tsien Now GFP is found in laboratories all over the world where it is used in every conceivable plant and animal GFP Types Fluorescent proteins enable the creation of highly specific biosensors to monitor a wide range of intracellular phenomena. Mutagenesis of A. victoria GFP has resulted in fluorescent proteins that range in color from blue to yellow Transluminator Fluoresence spectrophotometry Fluorescence In-vivo Xtreme Detection Fluorescence microscopy In-vivo Xtreme cm Polyacrylamide gel In-vivo Xtreme 1/16 1/8 1/4 1/2 1 10000 y = 9003x + 732,46 9000 R² = 0,9984 Max-Backgroumd 8000 7000 Rostoucí koncentrace Mean-Background 6000 5000 4000 3000 y = 5748,2x + 490,64 R² = 0,9995 2000 Fluorescence intensity [a.u.] 1000 0 0 0,2 0,4 0,6 0,8 1 1,2 concentration [µg/ml] In-vivo Xtreme GFP encapsulated GFP in liposome GFP water in liposome Max.int: 8800 a. -
A Brief History of the Family of Sonja Escherich-Eisenmenger-Weber
A Brief History of the family of Sonja Escherich-Eisenmenger-Weber © Copyright 1990 Ernst Weber, Tryon, North Carolina Part I Pfaundlers, Oetz and Piburg Part II Theodor Eseherich and Child Care in Vienna Part III Sonya Escherich-Eisenmenger, 1895 until 1936 Privately Printed By M.A. DESIGNS Tryon, NC An Introduction to Sonya & Ernst Weber An address by James M. Flack (son-in-law of Sonya Weber) at the Cosmos Club in Washington, D.C. Tuesday, October 14, 1975 on the occasion of the establishment of the Sonya & Ernst Weber Scholarship Fund. Dr. Bugliarello, Friends, Associates and Family of Sonya and Ernst Weber. It is my honor and privilege to speak on behalf of the dose members of the Weber family. Dr. Bugliarello, we are grateful to you and to Polytechnic for establishing the Sonya and Ernst Weber Scholarship Fund. The criterion for the awards - based on excellence and regardless of need is most appropriate. This is consonant with the lives of the Webers. They have searched for the good - for things of real value. And when things of value were discovered, they were able to recognize, cherish, nurture and reward them to assure their continuity and growth. Sonya and Ernst Weber are well known - both nationally and internationally, each in her and his own field. They have each been showered with honors for their achievements and contributions to a better way of life in our time and for the future to come - Ernst in the field of science as a physicist, engineer, educator and administrator; Sonya also in the field of science as a doctor of physical medicine and physical fitness. -
Molecular Biology and Applied Genetics
MOLECULAR BIOLOGY AND APPLIED GENETICS FOR Medical Laboratory Technology Students Upgraded Lecture Note Series Mohammed Awole Adem Jimma University MOLECULAR BIOLOGY AND APPLIED GENETICS For Medical Laboratory Technician Students Lecture Note Series Mohammed Awole Adem Upgraded - 2006 In collaboration with The Carter Center (EPHTI) and The Federal Democratic Republic of Ethiopia Ministry of Education and Ministry of Health Jimma University PREFACE The problem faced today in the learning and teaching of Applied Genetics and Molecular Biology for laboratory technologists in universities, colleges andhealth institutions primarily from the unavailability of textbooks that focus on the needs of Ethiopian students. This lecture note has been prepared with the primary aim of alleviating the problems encountered in the teaching of Medical Applied Genetics and Molecular Biology course and in minimizing discrepancies prevailing among the different teaching and training health institutions. It can also be used in teaching any introductory course on medical Applied Genetics and Molecular Biology and as a reference material. This lecture note is specifically designed for medical laboratory technologists, and includes only those areas of molecular cell biology and Applied Genetics relevant to degree-level understanding of modern laboratory technology. Since genetics is prerequisite course to molecular biology, the lecture note starts with Genetics i followed by Molecular Biology. It provides students with molecular background to enable them to understand and critically analyze recent advances in laboratory sciences. Finally, it contains a glossary, which summarizes important terminologies used in the text. Each chapter begins by specific learning objectives and at the end of each chapter review questions are also included. -
Issue 2, September 2011
Quarterly Newsletter of the Belgian Society for Microbiology Issue no. 2, September 2011 Contents Welcome by the president of BSM Page 1 As indicated in the program, the morning session Membership Page 2 consists of 4 plenary lectures, while in the News from FEMS Page 2 afternoon 2 parallel sessions (bacteriology, BSM Symposium 2011 Page 3 virology) are programmed with time reserved for Theodor Escherich Page 5 short oral communications from selected Report on MRM Symposium Page 7 abstracts. PhD Corner Page 9 Call for contributions Page 10 Besides this, there will be also ample time to Composition of the BSM board Page 10 discuss during the posters session. As such this meeting is also meant as an opportunity to meet microbiology colleagues and to exchange ideas. More details of the symposium, and how to Welcome register, you will find on p 3. Concomitantly with the start of the new academic year, we send you Issue 2 of the E-Newsletter of the We also started with a series of short overviews Belgian Society for Microbiology. Since the first E- on historical data in microbiology (see p 4), and Newsletter, the BSM board was further active in continue with the PhD corner, and report on FEMS several respects. sponsored activities. The BSM Board has further worked on finalizing the BSM was also for the first time host of the FEMS th program of the symposium (Brussels, 16th November Council meeting held in Leuven 16-17 2011), the yearly most important activity of BSM. September, which was attended by 37 delegates (22 different nationalities) representing different This year’s topic is “Live, death and survival of Micro- microbiological societies unified under the organisms” emphasizing that thanks to the umbrella of FEMS, which altogether represent continuous progress in microbiological sciences, more than 30000 European microbiologists (see strategies used by micro-organisms to live and http://www.fems-microbiology.org).