Bacterial Transfer of Large Functional Genomic DNA Into Human Cells
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P1 Bacteriophage and Tol System Mutants
P1 BACTERIOPHAGE AND TOL SYSTEM MUTANTS Cari L. Smerk A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE August 2007 Committee: Ray A. Larsen, Advisor Tami C. Steveson Paul A. Moore Lee A. Meserve ii ABSTRACT Dr. Ray A. Larsen, Advisor The integrity of the outer membrane of Gram negative bacteria is dependent upon proteins of the Tol system, which transduce cytoplasmic-membrane derived energy to as yet unidentified outer membrane targets (Vianney et al., 1996). Mutations affecting the Tol system of Escherichia coli render the cells resistant to a bacteriophage called P1 by blocking the phage maturation process in some way. This does not involve outer membrane interactions, as a mutant in the energy transucer (TolA) retained wild type levels of phage sensitivity. Conversely, mutations affecting the energy harvesting complex component, TolQ, were resistant to lysis by bacteriophage P1. Further characterization of specific Tol system mutants suggested that phage maturation was not coupled to energy transduction, nor to infection of the cells by the phage. Quantification of the number of phage produced by strains lacking this protein also suggests that the maturation of P1 phage requires conditions influenced by TolQ. This study aims to identify the role that the TolQ protein plays in the phage maturation process. Strains of cells were inoculated with bacteriophage P1 and the resulting production by the phage of viable progeny were determined using one step growth curves (Ellis and Delbruck, 1938). Strains that were lacking the TolQ protein rendered P1 unable to produce the characteristic burst of progeny phage after a single generation of phage. -
Vectors (M13, Fd, F1); Yacs, Bacs, Pacs, Bibacs
Paper No. : 04 Genetic engineering and recombinant DNA technology Module : 21 Single stranded DNA vectors (M13, fd, f1); YACs, BACs, PACs, BIBACs Principal Investigator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director The Energy and Resouurces Institute (TERI), New Delhi Co-Principal Investigator: Prof S K Jain, Professor, of Medical Biochemistry Jamia Hamdard University, New Delhi Paper Coordinator: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia Islamia, New Delhi Content Writer: Dr. Ashutosh Rai, SERB-National Post Doctoral Fellow, ICAR- Indian Institute of Vegetable Research, Varanasi-221305 Content Reviwer: Dr. Sharmistha Barthakur,Principal Scientist, National Research Centre on Plant Biotechnology, New Delhi – 110012, INDIA Genetic engineering and recombinant DNA technology Biotechnology Single stranded DNA vectors (M13, fd, f1); YACs, BACs, PACs, BIBACs Description of Module Subject Name Biotechnology Paper Name Genetic Engineering and Recombinant DNA Technology Module Name/Title Single stranded DNA vectors (M13, fd, f1); YACs, BACs, PACs, BIBACs Module Id 21 Pre-requisites Objectives Single stranded DNA vectors (M13, f1, fd), Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), P1 derived Artificial Chromosomes (PACs), Binary Bacterial Artificial Chromosomes (BIBACs), Summary Keywords Bacteriophage M13, Bacteriophage f1, Bacteriophage fd, YACs, BACs, PACs, BIBACs Genetic engineering and recombinant DNA technology Biotechnology Single stranded DNA vectors (M13, fd, f1); YACs, BACs, PACs, BIBACs A. Single stranded DNA vectors (M13, fd, f1); YACs, BACs, PACs, BIBACs Cloning vectors have been utilized in recombinant DNA technology not only for replication fucntions, but now a days these are a wonderful tool for various kinds of expression studies, sequencing and mutagenesis related applications. The basic requirements like origin of replication, a partition function, suitable selectable markers for easy and fast identification of clones without any necessity of expression. -
Gibson Assembly Cloning Guide, Second Edition
Gibson Assembly® CLONING GUIDE 2ND EDITION RESTRICTION DIGESTFREE, SEAMLESS CLONING Applications, tools, and protocols for the Gibson Assembly® method: • Single Insert • Multiple Inserts • Site-Directed Mutagenesis #DNAMYWAY sgidna.com/gibson-assembly Foreword Contents Foreword The Gibson Assembly method has been an integral part of our work at Synthetic Genomics, Inc. and the J. Craig Venter Institute (JCVI) for nearly a decade, enabling us to synthesize a complete bacterial genome in 2008, create the first synthetic cell in 2010, and generate a minimal bacterial genome in 2016. These studies form the framework for basic research in understanding the fundamental principles of cellular function and the precise function of essential genes. Additionally, synthetic cells can potentially be harnessed for commercial applications which could offer great benefits to society through the renewable and sustainable production of therapeutics, biofuels, and biobased textiles. In 2004, JCVI had embarked on a quest to synthesize genome-sized DNA and needed to develop the tools to make this possible. When I first learned that JCVI was attempting to create a synthetic cell, I truly understood the significance and reached out to Hamilton (Ham) Smith, who leads the Synthetic Biology Group at JCVI. I joined Ham’s team as a postdoctoral fellow and the development of Gibson Assembly began as I started investigating methods that would allow overlapping DNA fragments to be assembled toward the goal of generating genome- sized DNA. Over time, we had multiple methods in place for assembling DNA molecules by in vitro recombination, including the method that would later come to be known as Gibson Assembly. -
Persistent Virus and Addiction Modules: an Engine of Symbiosis
UC Irvine UC Irvine Previously Published Works Title Persistent virus and addiction modules: an engine of symbiosis. Permalink https://escholarship.org/uc/item/5ck1g026 Journal Current opinion in microbiology, 31 ISSN 1369-5274 Author Villarreal, Luis P Publication Date 2016-06-01 DOI 10.1016/j.mib.2016.03.005 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Available online at www.sciencedirect.com ScienceDirect Persistent virus and addiction modules: an engine of symbiosis Luis P Villarreal The giant DNA viruses are highly prevalent and have a particular host would occasionally survive but still retain a bit of affinity for the lytic infection of unicellular eukaryotic host. The the selfish virus DNA. Thus although parasitic selfish giant viruses can also be infected by inhibitory virophage which (virus-like) information is common in the genomes of all can provide lysis protection to their host. The combined life forms, its presence was explained as mostly defective protective and destructive action of such viruses can define a remnants of past plague sweeps that provides no func- general model (PD) of virus-mediated host survival. Here, I tional benefit to the host (e.g. junk). Until recently, this present a general model for role such viruses play in the explanation seemed satisfactory. In the last twenty years, evolution of host symbiosis. By considering how virus mixtures however, various observation-based developments have can participate in addiction modules, I provide a functional compelled us to re-evaluate this stance. Both comparative explanation for persistence of virus derived genetic ‘junk’ in genomics and metagenomics (sequencing habitats) has their host genomic habitats. -
Construction of a Cdna Library
Learning Objectives : • Understand the basic differences between genomic and cDNA libraries • Understand how genomic libraries are constructed • Understand the purpose for having overlapping DNA fragments in genomic libraries and how they are generated • Understand how cDNA libraries are constructed and the use of reverse transcriptase for their construction • Understand the rationale for library screening • Understand the method of plaque hybridization • Understand the four methods for library screening and when they are put into use Molecular cloning in bacterial cells…. This strategy can be applied to genomic DNA as well as cDNA Library construction • two types of libraries • a genomic library contains fragments of genomic DNA (genes) • a cDNA library contains DNA copies of cellular mRNAs • both types are usually cloned in bacteriophage vectors Construction of a genomic library vector DNA (bacteriophage lambda) • lambda has a linear double- stranded DNA genome • the left and right arms are essential for the phage replication cycle • the internal fragment is dispensable Bam HI sites “left arm” “right arm” internal fragment (dispensable for phage growth) NNGGATCCNN human genomic DNA (isolated from Bam HI sites: many cells) NNCCTAG GNN cut with Bam HI cut with Sau 3A (4-base cutter) (6-base cutter) which has ends compatible with Bam HI: NNN GATCNNN internal fragment NNNCTAG NNN remove internal fragment isolate ~20 kb fragments “left arm” “right arm” “left arm” “right arm” combine and treat with DNA ligase “left arm” “right arm” package -
The Transgenic Core Facility
Genetic modification of the mouse Ben Davies Wellcome Trust Centre for Human Genetics Genetically modified mouse models • The genome projects have provided us only with a catalogue of genes - little is know regarding gene function • Associations between disease and genes and their variants are being found yet the biological significance of the association is frequently unclear • Genetically modified mouse models provides a powerful method of assaying gene function in the whole organism Sequence Information Human Mutation Gene of Interest Mouse Model Reverse Genetics Phenotype Gene Function How to study gene function Human patients: • Patients with gene mutations can help us understand gene function • Human’s don’t make particularly willing experimental organisms • An observational science and not an experimental one • Genetic make-up of humans is highly variable • Difficult to pin-point the gene responsible for the disease in the first place Mouse patients: • Full ability to manipulate the genome experimentally • Easy to maintain in the laboratory – breeding cycle is approximately 2 months • Mouse and human genomes are similar in size, structure and gene complement • Most human genes have murine counterparts • Mutations that cause disease in human gene, generally produce comparable phentoypes when mutated in mouse • Mice have genes that are not represented in other model organisms e.g. C. elegans, Drosophila – genes of the immune system What can I do with my gene of interest • Gain of function – Overexpression of a gene of interest “Transgenic -
Review on Applications of Genetic Engineering and Cloning in Farm Animals
Journal of Dairy & Veterinary Sciences ISSN: 2573-2196 Review Article Dairy and Vet Sci J Volume 4 Issue 1 - October 2017 Copyright © All rights are reserved by Ayalew Negash DOI: 10.19080/JDVS.2017.04.555629 Review on Applications of Genetic Engineering And Cloning in Farm animals Eyachew Ayana1, Gizachew Fentahun2, Ayalew Negash3*, Fentahun Mitku1, Mebrie Zemene3 and Fikre Zeru4 1Candidate of Veterinary medicine, University of Gondar, Ethiopia 2Candidate of Veterinary medicine, Samara University, Ethiopia 3Lecturer at University of Gondar, University of Gondar, Ethiopia 4Samara University, Ethiopia Submission: July 10, 2017; Published: October 02, 2017 *Corresponding author: Ayalew Negash, Lecturer at University of Gondar, College of Veterinary Medicine and science, University of Gondar, P.O. 196, Gondar, Ethiopia, Email: Abstract Genetic engineering involves producing transgenic animal’s models by using different techniques such as exogenous pronuclear DNA highly applicable and crucial technology which involves increasing animal production and productivity, increases animal disease resistance andmicroinjection biomedical in application. zygotes, injection Cloning ofinvolves genetically the production modified embryonicof animals thatstem are cells genetically into blastocysts identical and to theretrovirus donor nucleus.mediated The gene most transfer. commonly It is applied and recent technique is somatic cell nuclear transfer in which the nucleus from body cell is transferred to an egg cell to create an embryo that is virtually identical to the donor nucleus. There are different applications of cloning which includes: rapid multiplication of desired livestock, and post-natal viabilities. Beside to this Food safety, animal welfare, public and social acceptance and religious institutions are the most common animal conservation and research model. -
Schematic and Time Line for the Generation of Knockout Mice Aurora Burds Connor, Feb 2007
Schematic and Time Line for the Generation of Knockout Mice Aurora Burds Connor, Feb 2007 Making the DNA construct Time Line (in your lab) The Transgenic Facility also has a “Beginner’s Guide to Gene Targeting” on the website in Methods. We are also happy to assist with advice and reagents to help you make an effective targeting construct. It is recommended that you contact Aurora ([email protected]) early in the process. Acquire the genomic DNA for your gene or locus of interest You can either screen a 129/Sv genomic library 1-3 months or use genomic databases, public BACs and PCR for a C57BL/6 background Make a DNA construct. DNA from the genomic locus (orange) flanks the DNA to be inserted (blue) and it is placed in a bacterial plasmid. This construct is designed to add new pieces of DNA into the mouse genome at a 3-6 months desired locus. In a knockout experiment, the new DNA will replace the normal gene. Linearize the construct and give it to the Rippel ES Cell Facility at MIT Generating Targeted ES Cells Time Line (done by the Facility) Day 0 Insert the DNA into embryonic stem cells (ES cells) via electroporation. In the cells, the homologous pieces of DNA recombine. This inserts new DNA from the construct into the desired locus, usually replacing a portion of the original genome. Place the ES cells in selective media, allowing for the growth of cells containing the DNA construct. - The DNA construct has a drug-resistance marker Day 1-10 - Very few of the cells take up the construct – cells that do not will die because they are not resistant to the drug added to the media. -
Production of Lentiviral Vectors Using Novel, Enzymatically Produced, Linear DNA
Gene Therapy (2019) 26:86–92 https://doi.org/10.1038/s41434-018-0056-1 ARTICLE Production of lentiviral vectors using novel, enzymatically produced, linear DNA 1 2,3 4 4 4 Rajvinder Karda ● John R. Counsell ● Kinga Karbowniczek ● Lisa J. Caproni ● John P. Tite ● Simon N. Waddington1,5 Received: 17 October 2018 / Revised: 27 November 2018 / Accepted: 5 December 2018 / Published online: 14 January 2019 © The Author(s) 2019. This article is published with open access Abstract The manufacture of large quantities of high-quality DNA is a major bottleneck in the production of viral vectors for gene therapy. Touchlight Genetics has developed a proprietary abiological technology that addresses the major issues in commercial DNA supply. The technology uses ‘rolling-circle’ amplification to produce large quantities of concatameric DNA that is then processed to create closed linear double-stranded DNA by enzymatic digestion. This novel form of DNA, Doggybone™ DNA (dbDNA™), is structurally distinct from plasmid DNA. Here we compare lentiviral vectors production from dbDNA™ and plasmid DNA. Lentiviral vectors were administered to neonatal mice via intracerebroventricular fi 1234567890();,: 1234567890();,: injection. Luciferase expression was quanti ed in conscious mice continually by whole-body bioluminescent imaging. We observed long-term luciferase expression using dbDNA™-derived vectors, which was comparable to plasmid-derived lentivirus vectors. Here we have demonstrated that functional lentiviral vectors can be produced using the novel dbDNA™ configuration for delivery in vitro and in vivo. Importantly, this could enable lentiviral vector packaging of complex DNA sequences that have previously been incompatible with bacterial propagation systems, as dbDNA™ technology could circumvent such restrictions through its phi29-based rolling-circle amplification. -
1 Producing Gene Deletions in Escherichia Coli by P1 Transduction with Excisable Antibiotic 2 Resistance Cassettes 3 4 AU
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Nottingham Trent Institutional Repository (IRep) 1 TITLE: 2 Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic 3 Resistance Cassettes 4 5 AUTHORS AND AFFILIATIONS: 6 Athanasios Saragliadis1, Thomas Trunk1, Jack C. Leo1 7 8 1Evolution and Genetics, Department of Biosciences, University of Oslo, Norway 9 10 Corresponding Author: 11 Jack C. Leo ([email protected]) 12 Tel: +47-22859027 13 14 E-mail Addresses of Co-authors: 15 Athanasios Saragliadis ([email protected]) 16 Thomas Trunk ([email protected]) 17 18 KEYWORDS: 19 Antibiotic resistance cassette; deletion mutagenesis; FLP recombinase; P1 transduction; 20 translocation and assembly module; trimeric autotransporter adhesin 21 22 SUMMARY: 23 Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion 24 constructs as a basis for making deletion mutants in other E. coli strains. Such deletion 25 mutations can be mobilized and inserted into the corresponding locus of a recipient strain using 26 P1 bacteriophage transduction. 27 28 ABSTRACT: 29 A first approach to study the function of an unknown gene in bacteria is to create a knock-out 30 of this gene. Here, we describe a robust and fast protocol for transferring gene deletion 31 mutations from one Escherichia coli strain to another by using generalized transduction with 32 the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on 33 gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized 34 from a donor strain and introduced into a recipient strain of interest to quickly and easily 35 generate a gene deletion mutant. -
Transient Gene Expression Following DNA Transfer to Plant Cells: the Phenomenon; Its Causes and Some Applications
Transient Gene Expression Following DNA Transfer to Plant Cells: The Phenomenon; Its Causes and Some Applications. A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Cellular and Molecular Biology in the University of Canterbury. RICHARD JOHN WELD 2000 1 ..... ACKNOWLEDGEMENTS ..... I would like to thank Dr Jack Heinemann, Dr Colin Eady and Dr Sandra Jackson for their supervision of this project, for their criticism and their encouragement. I would also like to thank Dr Ross Bicknell for his advice and support. I acknowledge the generosity of Dr Jim Haseloff for the gift of plasmid pBINmgfp5-ER, Dr Ed Morgan for the gift of Nicotiana plumbaginifolia suspension cells and advice on their culture, Dr Steve Scofield for the gift ofpSLJll 01, Dr Nicole Houba-Herin for the gift ofpNT103 andpNT804, Dr Jerzy Paszkowski for the gift ofpMDSBAR, Dr Andrew Gleave for the gift ofpART8 and pART7, Dr n Yoder for the gift ofpAL144 and Dr Bernie Carroll for information on the construction of pSLJ3 621. This project was made possible by funds provided by a FfRST Doctoral Fellowship provided through the New' Zealand Institute for Crop and Food Research and a University of Canterbury Doctoral Scholarship. I would like to offer my grateful thanks to all those staff and students of the Plant and Microbial Sciences Department and those staff and students at the New Zealand Institute for Crop and Food Research at Lincoln who have assisted me in various ways during the course of my research and to those who have offered their friendship, support and encouragement. -
Cleavage of the Bacteriophage P1 Packaging Site (Pac) Is Regulated by Adenine Methylation (DNA Adenine Methyltransferase/Methylation/Phage DNA Packaging) N
Proc. Natl. Acad. Sci. USA Vol. 87, pp. 8070-8074, October 1990 Biochemistry Cleavage of the bacteriophage P1 packaging site (pac) is regulated by adenine methylation (DNA adenine methyltransferase/methylation/phage DNA packaging) N. STERNBERG AND J. COULBY E. 1. duPont de Nemours & Co. Inc., Central Research and Development Department, P.O. Box 80328, Wilmington, DE 19880-0328 Communicated by John Roth, June 25, 1990 (received for review February 9, 1990) ABSTRACT The packaging of bacteriophage P1 DNA is cleavage process to be regulated in the cell, so as to permit initiated when the phage packaging site (pac) is recognized and the production of terminally redundant viral molecules. cleaved and continues until the phage head is full. We have previously shown that pac is- a 162-base-pair segment of P1 MATERIAL AND METHODS DNA that contains seven DNA adenine methyltransferase methylation sites (5'-GATC). We show here that cleavage of Bacterial and Phage Strains. Escherichia coli strain N99 is pac is methylation sensitive. Both in vivo and in vitro experi- supo (13) and strain NS2626 is N99Tn9::dam. Strain NS2626 ments indicate that methylated pac is cleavable, whereas un- was constructed by P1-mediated transduction of the Tn9 methylated pac is not. Moreover, DNA isolated from P1 phage insertion mutation in the E. coli dam gene from strain and containing an uncut pac site was a poor substrate for in GM3808 (14) to strain N99. Strain NS2342 is N99 (Aimm434- vitro cleavage until it was methylated by the Escherichia coli P1:20b) (3). Strain NS2635 is NS2626 (Aimm434-P1:20b).