3 Main Classification of Vectors (With Diagram)
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Fosmid Library End Sequencing Reveals a Rarely Known Genome Structure of Marine Shrimp Penaeus Monodon
eScholarship Title Fosmid library end sequencing reveals a rarely known genome structure of marine shrimp Penaeus monodon Permalink https://escholarship.org/uc/item/126680ch Journal BMC Genomics, 12(1) ISSN 1471-2164 Authors Huang, Shiao-Wei Lin, You-Yu You, En-Min et al. Publication Date 2011-05-17 DOI http://dx.doi.org/10.1186/1471-2164-12-242 Supplemental Material https://escholarship.org/uc/item/126680ch#supplemental Peer reviewed eScholarship.org Powered by the California Digital Library University of California Huang et al. BMC Genomics 2011, 12:242 http://www.biomedcentral.com/1471-2164/12/242 RESEARCHARTICLE Open Access Fosmid library end sequencing reveals a rarely known genome structure of marine shrimp Penaeus monodon Shiao-Wei Huang1, You-Yu Lin1, En-Min You1, Tze-Tze Liu2, Hung-Yu Shu2, Keh-Ming Wu3, Shih-Feng Tsai3, Chu-Fang Lo1, Guang-Hsiung Kou1, Gwo-Chin Ma4, Ming Chen1,4,5, Dongying Wu6,7, Takashi Aoki8, Ikuo Hirono8 and Hon-Tsen Yu1* Abstract Background: The black tiger shrimp (Penaeus monodon) is one of the most important aquaculture species in the world, representing the crustacean lineage which possesses the greatest species diversity among marine invertebrates. Yet, we barely know anything about their genomic structure. To understand the organization and evolution of the P. monodon genome, a fosmid library consisting of 288,000 colonies and was constructed, equivalent to 5.3-fold coverage of the 2.17 Gb genome. Approximately 11.1 Mb of fosmid end sequences (FESs) from 20,926 non-redundant reads representing 0.45% of the P. monodon genome were obtained for repetitive and protein-coding sequence analyses. -
Numerous Uncharacterized and Highly Divergent Microbes Which Colonize Humans Are Revealed by Circulating Cell-Free DNA
Numerous uncharacterized and highly divergent microbes which colonize humans are revealed by circulating cell-free DNA Mark Kowarskya, Joan Camunas-Solerb, Michael Kerteszb,1, Iwijn De Vlaminckb, Winston Kohb, Wenying Panb, Lance Martinb, Norma F. Neffb,c, Jennifer Okamotob,c, Ronald J. Wongd, Sandhya Kharbandae, Yasser El-Sayedf, Yair Blumenfeldf, David K. Stevensond, Gary M. Shawd, Nathan D. Wolfeg,h, and Stephen R. Quakeb,c,i,2 aDepartment of Physics, Stanford University, Stanford, CA 94305; bDepartment of Bioengineering, Stanford University, Stanford, CA 94305; cChan Zuckerberg Biohub, San Francisco, CA 94158; dDepartment of Pediatrics, Stanford University School of Medicine, Stanford University, Stanford, CA 94305; ePediatric Stem Cell Transplantation, Lucille Packard Children’s Hospital, Stanford University, Stanford, CA 94305; fDivision of Maternal–Fetal Medicine, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford University, Stanford, CA 94305; gMetabiota, San Francisco, CA 94104; hGlobal Viral, San Francisco, CA 94104; and iDepartment of Applied Physics, Stanford University, Stanford, CA 94305 Contributed by Stephen R. Quake, July 12, 2017 (sent for review April 28, 2017; reviewed by Søren Brunak and Eran Segal) Blood circulates throughout the human body and contains mole- the body (18, 19); combining this observation with the average cules drawn from virtually every tissue, including the microbes and genome sizes of a human, bacterium, and virus (Gb, Mb, and viruses which colonize the body. Through massive shotgun sequenc- kb, respectively) suggests that approximately 1% of DNA by ing of circulating cell-free DNA from the blood, we identified mass in a human is derived from nonhost origins. Previous hundreds of new bacteria and viruses which represent previously studies by us and others have shown that indeed approximately unidentified members of the human microbiome. -
A Field Guide to Eukaryotic Transposable Elements
GE54CH23_Feschotte ARjats.cls September 12, 2020 7:34 Annual Review of Genetics A Field Guide to Eukaryotic Transposable Elements Jonathan N. Wells and Cédric Feschotte Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14850; email: [email protected], [email protected] Annu. Rev. Genet. 2020. 54:23.1–23.23 Keywords The Annual Review of Genetics is online at transposons, retrotransposons, transposition mechanisms, transposable genet.annualreviews.org element origins, genome evolution https://doi.org/10.1146/annurev-genet-040620- 022145 Abstract Annu. Rev. Genet. 2020.54. Downloaded from www.annualreviews.org Access provided by Cornell University on 09/26/20. For personal use only. Copyright © 2020 by Annual Reviews. Transposable elements (TEs) are mobile DNA sequences that propagate All rights reserved within genomes. Through diverse invasion strategies, TEs have come to oc- cupy a substantial fraction of nearly all eukaryotic genomes, and they rep- resent a major source of genetic variation and novelty. Here we review the defining features of each major group of eukaryotic TEs and explore their evolutionary origins and relationships. We discuss how the unique biology of different TEs influences their propagation and distribution within and across genomes. Environmental and genetic factors acting at the level of the host species further modulate the activity, diversification, and fate of TEs, producing the dramatic variation in TE content observed across eukaryotes. We argue that cataloging TE diversity and dissecting the idiosyncratic be- havior of individual elements are crucial to expanding our comprehension of their impact on the biology of genomes and the evolution of species. 23.1 Review in Advance first posted on , September 21, 2020. -
A Zebrafish Reporter Line Reveals Immune and Neuronal Expression of Endogenous Retrovirus
bioRxiv preprint doi: https://doi.org/10.1101/2021.01.21.427598; this version posted January 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. A zebrafish reporter line reveals immune and neuronal expression of endogenous retrovirus. Noémie Hamilton1,2*, Amy Clarke1, Hannah Isles1, Euan Carson1, Jean-Pierre Levraud3, Stephen A Renshaw1 1. The Bateson Centre, Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield, UK 2. The Institute of Neuroscience, University of Sheffield, Sheffield, UK 3. Macrophages et Développement de l’Immunité, Institut Pasteur, CNRS UMR3738, 25 rue du docteur Roux, 75015 Paris *Corresponding author: [email protected] Abstract Endogenous retroviruses (ERVs) are fossils left in our genome from retrovirus infections of the past. Their sequences are part of every vertebrate genome and their random integrations are thought to have contributed to evolution. Although ERVs are mainly kept silenced by the host genome, they are found activated in multiple disease states such as auto-inflammatory disorders and neurological diseases. What makes defining their role in health and diseases challenging is the numerous copies in mammalian genomes and the lack of tools to study them. In this study, we identified 8 copies of the zebrafish endogenous retrovirus (zferv). We created and characterised the first in vivo ERV reporter line in any species. Using a combination of live imaging, flow cytometry and single cell RNA sequencing, we mapped zferv expression to early T cells and neurons. -
Phagemid-Based Method of Producing Filamentous Bacteriophage Particles Displaying Antibody Molecules and the Corresponding Bacteriophage Particles
Europäisches Patentamt *EP001433846A2* (19) European Patent Office Office européen des brevets (11) EP 1 433 846 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.7: C12N 15/10, C07K 16/00, 30.06.2004 Bulletin 2004/27 C12N 15/62, C12N 7/00, C12N 15/73 (21) Application number: 04005419.9 (22) Date of filing: 10.07.1991 (84) Designated Contracting States: • Griffiths, Andrew David AT BE CH DE DK ES FR GB GR IT LI LU NL SE Cambridge CB1 4AY (GB) • Jackson, Ronald Henry (30) Priority: 10.07.1990 GB 9015198 Cambridge CB1 2NU (GB) 19.10.1990 GB 9022845 • Holliger, Kaspar Philipp 12.11.1990 GB 9024503 Cambridge CB1 4HT (GB) 06.03.1991 GB 9104744 • Marks, James David 15.05.1991 GB 9110549 Kensington, CA 94707-1310 (US) • Clackson, Timothy Piers (62) Document number(s) of the earlier application(s) in Somerville, MA 02143 (US) accordance with Art. 76 EPC: • Chiswell, David John 97120149.6 / 0 844 306 Buckingham MK18 2LD (GB) 96112510.1 / 0 774 511 • Winter, Gregory Paul Cambridge CB2 1TQ (GB) (71) Applicants: • Bonnert, Timothy Peter • Cambridge Antibody Technology LTD Seattle, WA 98102 (US) Cambridge CB1 6GH (GB) • Medical Research Council (74) Representative: Walton, Seán Malcolm et al London W1B 1AL (GB) Mewburn Ellis LLP York House, (72) Inventors: 23 Kingsway • McCafferty, John London WC2B 6HP (GB) Babraham CB2 4AP (GB) • Pope, Anthony Richard Remarks: Cambridge CB1 2LW (GB) This application was filed on 08 - 03 - 2004 as a • Johnson, Kevin Stuart divisional application to the application mentioned Highfields, Cambridge CB3 7NY (GB) under INID code 62. -
Copycontrol™ Fosmid Autoinduction Solution
CopyControl™ Fosmid Autoinduction Solution Cat. No. AIS107F Available exclusively thru Lucigen. lucigen.com/epibio www.lucigen.com MA263E CopyControl™ Fosmid Autoinduction Solution • 12/2016 1 MA263E CopyControl™ Fosmid Autoinduction Solution 1. Introduction The CopyControl™ Fosmid Autoinduction Solution is designed to induce CopyControl Fosmid clones and clones retrofitted with the EZ-Tn5™ <oriV/KAN-2> Transposon, grown in TransforMax™ EPI300™ E. coli cells, from single-copy number to a higher-copy number of approximately 50 fosmids per cell. The Fosmid Autoinduction Solution induces expression of a mutant trfA gene contained in the TransforMax EPI300 cells. Expression of trfA gene results in initiation of replication from the oriV high copy origin of replication and subsequent amplification of the CopyControl clones to high copy number. The Fosmid Autoinduction protocol improves upon the existing induction protocol by including the autoinduction supplement in the media prior to culture inoculation, removing the need for time-consuming subculturing and the 2-hour incubation required in the standard induction protocol. The Fosmid Autoinduction Solution also contains cell growth enhancers which boost cell numbers and typically provides higher DNA yields than with the standard CopyControl induction protocol. The hands-off autoinduction protocol makes CopyControl Fosmid Autoinduction solution ideal for high-throughput purification protocols in 96-well format. The autoinduction solution is also compatible with larger scale DNA purifications and can be scaled according to the amount of media used. 2. Product Specifications Storage: Store only at –20°C in a freezer without a defrost cycle. Mix thoroughly after thawing. Size and Formulation: CopyControl Fosmid Autoinduction Solution is available in a 50-ml size concentrate of 500X in sterile water. -
Antibody Discovery for Development of a Serotyping Dengue Virus NS1 Capture Assay
Antibody Discovery for Development of a Serotyping Dengue Virus NS1 Capture Assay Kebaneilwe Lebani Master of Biotechnology (Advanced) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 Australian Institute for Bioengineering and Nanotechnology ABSTRACT Dengue virus (DENV) infections are a significant public health burden in tropical and sub-tropical regions of the world. Infections are caused by four different but antigenically related viruses which result in four DENV serotypes. The multifaceted nature of DENV pathogenesis hinders the sensitivity of assays designed for the diagnosis of infection. Different markers can be optimally detected at different stages of infection. Of particular clinical importance is the identification of acute viremia during the febrile phase of infection which is pivotal for management of infection. Non-structural protein 1 (NS1) has been identified as a good early surrogate marker of infection with possible applications in epidemiological surveillance and the development of blood screening assays. This contribution is towards using serotype-specificity to achieve specific and more sensitive diagnostic detection of DENV NS1. The general aim of this work is to isolate immune-reagents that can be used to develop an assay with improved sensitivity of DENV NS1 detection in a diagnostic setting. In this work, we sought to isolate serotype-specific antibodies that discern discreet antigenic differences in NS1 from each DENV serotype. Additionally, we also sought to isolate a pairing antibody that recognises NS1 from all four DENV serotypes (pan-reactive) for tandem capture of the DENV NS1. To achieve this, three naive, immunoglobulin gene libraries (a VH domain, a scFv and a Fab library) were interrogated for binders to recombinant NS1 antigen from all four DENV serotypes using phage display technology and various biopanning approaches. -
Protocol for Epifos™ Fosmid Library Production
EpiFOS™ Fosmid Library Production Kit Cat. No. FOS0901 Connect with Epicentre on our blog (epicentral.blogspot.com), Facebook (facebook.com/EpicentreBio), and Twitter (@EpicentreBio). www.epicentre.com Lit. # 149 • 8/2012 1 EPILIT149 Rev. A EpiFOS™ Fosmid Library Production Kit 1. Overview of the EpiFOS Fosmid Library Production Process Fosmid vectors1-3 provide an improved method for cloning and the stable maintenance of cosmid-sized (35-45 kb) libraries in E. coli. The stability of such large constructs in vivo is facilitated by the pEpiFOS™-5 vector that maintains the clones at single copy in the cell. The EpiFOS Fosmid Library Production Kit will produce a complete and unbiased primary fosmid library. The kit utilizes a novel strategy of cloning randomly sheared, end-repaired DNA. Shearing the DNA leads to the generation of highly random DNA fragments in contrast to more biased libraries that result from fragmenting the DNA by partial restriction digests. The steps involved (protocols for steps 2-7 are included in this manual): 1. Purify DNA from the desired source (the kit does not supply materials for this step). 2. Shear the DNA to approximately 40-kb fragments. 3. End-repair the sheared DNA to blunt, 5′-phosphorylated ends. 4. Size-resolve the end-repaired DNA by Low Melting Point (LMP) agarose gel electrophoresis. 5. Purify the blunt-end DNA from the LMP agarose gel. 6. Ligate the blunt-end DNA to the Cloning-Ready pEpiFOS-5 vector. 7. Package the ligated DNA and plate on EPI100™-T1R Plating Strain. Grow clones overnight. pEpiFOS-5 is a 7518 bp. -
Cdna Libraries and Expression Libraries
Solutions for Practice Problems for Recombinant DNA, Session 4: cDNA Libraries and Expression Libraries Question 1 In a hypothetical scenario you wake up one morning to your roommate exclaiming about her sudden hair growth. She has been supplementing her diet with a strange new fungus purchased at the local farmer’s market. You take samples of the fungus to your lab and you find that this fungus does indeed make a protein (the harE protein) that stimulates hair growth. You construct a fungal genomic DNA library in E. Coli with the hope of cloning the harE gene. If you succeed you will be a billionaire! You obtain DNA from the fungus, digest it with a restriction enzyme, and clone it into a vector. a) What features must be present on your plasmid that will allow you to use this as a cloning vector to make fungal genomic DNA library. Your vector would certainly need to have a unique restriction enzyme site, a selectable marker such as the ampicillin resistance gene, and a bacterial origin of replication. Other features may be required depending upon how you plan to use this library. b) You clone your digested genomic DNA into this vector. The E. coli (bacteria) cells that you will transform to create your library will have what phenotype prior to transformation? Prior to transformation, the E. coli cells that you will transform will be sensitive to antibiotic. This allows you to select for cells that obtained a plasmid. c) How do you distinguish bacterial cells that carry a vector from those that do not? Cells that obtained a vector will have obtained the selectable marker (one example is the ampicillin resistance gene). -
Gene Cloningcloningcloning
GeneGeneGene CloningCloningCloning 20042004 SeungwookSeungwookKim Kim Chem.Chem. && Bio.Bio. Eng.Eng. Reference z T.A. Brown, Gene Cloning, Chapman and Hall z S.B. Primrose, Molecular Biotechnology, Blackwell 1 Why Gene Cloning is Important? z A century ago, Gregor Mendel : { Basic assumption (each heritable property of an organism) is controlled by a factor (gene). z In 1900, Mandel's law Æ the birth of genetics. z what these genes are and exactly how they work The Early Development of Genetics z In 1903, Sutton, W { Proposed that genes reside on chromosomes 2 z In 1910, Morgan, TH { Experimental backing on that --> development of the techniques for gene mapping (To establish the structure or structural details or location) { By 1922, a comprehensive analysis of the relative positions of over 2000 genes on the four chromosomes of the fruit fly. (Drosophilia melanogaster) z In 1944, Avery, MacLeod and McCarty z In 1952, Hershey and Chase { Experimental results were shown that DNA is the genetic material. { Conventional idea : genes were made of protein z In 1952-1966, Delbruck, Chargaff, Crick and Monod { The structure of DNA was elucidated. { The genetic code was cracked. { The process of transcription and translation were described. 3 The Advent of Gene Cloning z In the late 1960's ; The experimental techniques were not sophisticated. z In 1971 ~ 1973 ; A new experimental techniques were developed. { Recombinant DNA technology or Genetic engineering based on the process of gene cloning { This led to rapid and efficient DNA sequencing techniques that enabled the structures of individual genes to be determined. z In the 1990s ; started with massive genome sequencing projects including the human project. -
Molecular Cloning and Functional Expression of Gibberellin 2- Oxidases, Multifunctional Enzymes Involved in Gibberellin Deactivation
Proc. Natl. Acad. Sci. USA Vol. 96, pp. 4698–4703, April 1999 Plant Biology Molecular cloning and functional expression of gibberellin 2- oxidases, multifunctional enzymes involved in gibberellin deactivation STEPHEN G. THOMAS,ANDREW L. PHILLIPS, AND PETER HEDDEN* Institute of Arable Crops Research (IACR)-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, United Kingdom Communicated by Jake MacMillan FRS, University of Bristol, Bristol, United Kingdom, February 16, 1999 (received for review December 22, 1998) ABSTRACT A major catabolic pathway for the gibberel- centration of bioactive GAs, the genes for these enzymes have lins (GAs) is initiated by 2b-hydroxylation, a reaction cata- not yet been isolated and it has not been possible to study their lyzed by 2-oxoglutarate-dependent dioxygenases. To isolate a regulation. GA 2b-hydroxylase cDNA clone we used functional screening Gibberellin 2b-hydroxylase activity is abundant in seeds of a cDNA library from developing cotyledons of runner bean during the later stages of maturation, particularly in legume (Phaseolus coccineus L.) with a highly sensitive tritium-release seeds, which accumulate large amounts of 2b-hydroxylated b assay for enzyme activity. The encoded protein, obtained by GAs (6–8). Indeed, GA8, the first 2 -hydroxyGA to be heterologous expression in Escherichia coli, converted GA9 to identified, was extracted from seeds of runner bean (Phaseolus b GA51 (2 -hydroxyGA9) and GA51-catabolite, the latter pro- coccineus, originally classified as P. multiflorus) (9). In certain duced from GA51 by further oxidation at C-2. The enzyme thus species, including legumes, further metabolism of 2b- is multifunctional and is best described as a GA 2-oxidase. -
10. E. Coli Cloning Vector Pbr322
10. E. coli cloning vector pBR322 Important questions based on it: A. (a) Name the organism in which the vector shown is inserted to get the copies of the desired gene. (b) Mention the area labelled in the vector responsible for controlling the copy number of the inserted gene. (c) Name and explain the role of a selectable marker in the vector shown. (AI 2010) B. (a) Identify the selectable markers in the diagram of E. coli vector shown below. (b) How is the coding sequence of -galactosidase considered a better marker than the ones identified by you in the diagram? Explain. (Delhi 2009) A. Explain the importance of (a) ori, (b) ampR and (c) rop in the E. coli vector shown below. (AI 2008) B. Draw pBR322 cloning vector. Label ‘ori’, ‘rop’ and any one antibiotic resistance site on it and state their functions. (AI 2015C) C. Draw a schematic diagram of the E. coli cloning vector pBR322 and mark the following in it: (a) ori (b) rop (c) ampicillin resistance gene (d) tetracycline resistance gene (e) restriction site BamHI (f) restriction site EcoR I (AI 2014C) D. Draw a schematic sketch of pBR322 plasmid and label the following in it: (a) Any two restriction sites. (b) Ori and rop genes. (c) An antibiotic resistant gene. (Delhi 2012) E. Identify A, B, C and D in the given diagram. (a) A-ori, B-ampR, C-tetR, D-HindIII (b) A-HindIII, B-tetR, C-ampR, D-ori (c) A-ampR, B-tetR, C-HindIII, D-ori (d) A-tetR, B-HindIII, C-ori, D-ampR (COMEDK) F.