E. coli Plasmid Vectors M E T H O D S I N M O L E C U L A R B I O L O G YTM John M. Walker, SERIES EDITOR 250.MAP Kinase Signaling Protocols,edited by Rony Seger, 2004 221.Generation of cDNA Libraries: Methods and Protocols, 249.Cytokine Protocols,edited by Marc De Ley, 2004 edited by Shao-Yao Ying, 2003 248.Antibody Engineering: Methods and Protocols, edited 220.Cancer Cytogenetics: Methods and Protocols, edited byJohn byBenny K. C. Lo, 2004 Swansbury, 2003 247.Drosophila Cytogenetics Protocols, edited by Daryl S. 219.Cardiac Cell and Gene Transfer: Principles, Protocols, and Henderson, 2004 Applications,edited by Joseph M. Metzger, 2003 246.Gene Delivery to Mammalian Cells: Volume 2: Viral Gene 218.Cancer Cell Signaling: Methods and Protocols, edited Transfer Techniques, edited by William C. Heiser, 2004 byDavid M. Terrian, 2003 245.Gene Delivery to Mammalian Cells: Volume 1: Nonviral Gene 217.Neurogenetics:Methods and Protocols, edited by Nicholas T. Transfer Techniques, edited by William C. Heiser, 2004 Potter, 2003 244.Protein Purification Protocols, Second Edition,edited by 216.PCR Detection of Microbial Pathogens: Methods and Pro- Paul Cutler, 2004 tocols,edited by Konrad Sachse and Joachim Frey, 2003 243.Chiral Separations: Methods and Protocols, edited by Gerald 215.Cytokines and Colony Stimulating Factors: Methods and Gübitz and Martin G. Schmid, 2004 Protocols,edited by Dieter Körholz and Wieland Kiess, 2003 242.Atomic Force Microscopy: Methods and Applications, edited 214.Superantigen Protocols, edited by Teresa Krakauer, 2003 byPier Carlo Braga and Davide Ricci, 2004 213.Capillary Electrophoresis of Carbohydrates, edited by Pierre 241.Cell Cycle Checkpoint Control Protocols, edited by Howard Thibault and Susumu Honda, 2003 B. Lieberman, 2004 212.Single Nucleotide Polymorphisms: Methods and Protocols, 240.Mammalian Artificial Chromosomes: Methods and Protocols, edited by Pui-Yan Kwok, 2003 edited by Vittorio Sgaramella and Sandro Eridani, 2003 211.Protein Sequencing Protocols, Second Edition, edited by 239.Cell Migration in Inflammation and Immunity: Methods and Bryan John Smith, 2003 Protocols,edited by Daniele D’Ambrosio and Francesco 210.MHC Protocols, edited by Stephen H. Powis and Robert W. Sinigaglia, 2003 Vaughan, 2003 238.Biopolymer Methods in Tissue Engineering, edited by 209.Transgenic Mouse Methods and Protocols, edited by Marten Anthony P. Hollander and Paul V. Hatton, 2003 Hofker and Jan van Deursen, 2003 237.G Protein Signaling: Methods and Protocols, edited by Alan 208.Peptide Nucleic Acids: Methods and Protocols, edited V. Smrcka, 2003 byPeter E. Nielsen, 2002 236.Plant Functional Genomics:Methods and Protocols, edited 207.Recombinant Antibodies for Cancer Therapy: Methods and byErich Grotewold, 2003 Protocols, edited by Martin Welschof and Jürgen Krauss, 2002 235.E. coliPlasmid Vectors:Methods and Applications, edited by 206.Endothelin Protocols, edited by Janet J. Maguire and Anthony Nicola Casali and Andrew Preston, 2003 P. Davenport, 2002 234.p53 Protocols, edited by Sumitra Deb and Swati Palit Deb, 205.E. coliGene Expression Protocols, edited by Peter E. 2003 Vaillancourt, 2002 233.Protein Kinase C Protocols, edited by Alexandra C. Newton, 204.Molecular Cytogenetics: Protocols and Applications,edited 2003 byYao-Shan Fan, 2002 232.Protein Misfolding and Disease:Principles and Protocols, 203.In Situ Detection of DNA Damage: Methods and Protocols, edited by Peter Bross and Niels Gregersen, 2003 edited by Vladimir V. Didenko, 2002 231.Directed Evolution Library Creation:Methods and Protocols, 202.Thyroid Hormone Receptors: Methods and Protocols, edited edited by Frances H. Arnold and George Georgiou,2003 byAria Baniahmad, 2002 230.Directed Enzyme Evolution:Screening and Selection Methods, 201.Combinatorial Library Methods and Protocols, edited by edited by Frances H. Arnold and George Georgiou,2003 Lisa B. English, 2002 229.Lentivirus Gene Engineering Protocols, edited by Maurizio 200.DNA Methylation Protocols, edited by Ken I. Mills and Bernie Federico, 2003 H. Ramsahoye, 2002 228.Membrane Protein Protocols:Expression, Purification, and 199.Liposome Methods and Protocols, edited by Subhash C. Basu Characterization, edited by Barry S. Selinsky, 2003 and Manju Basu, 2002 227.Membrane Transporters: Methods and Protocols, edited 198.Neural Stem Cells: Methods and Protocols, edited by Tanja byQing Yan, 2003 Zigova, Juan R. Sanchez-Ramos, and Paul R. Sanberg, 2002 226.PCR Protocols, Second Edition,edited by John M. S. Bartlett 197.Mitochondrial DNA: Methods and Protocols, edited by William and David Stirling, 2003 C. Copeland, 2002 225.Inflammation Protocols, edited by Paul G. Winyard and Derek 196.Oxidants and Antioxidants: Ultrastructure and Molecular A. Willoughby, 2003 Biology Protocols, edited by Donald Armstrong, 2002 224.Functional Genomics: Methods and Protocols, edited 195.Quantitative Trait Loci: Methods and Protocols,edited byMichael J. Brownstein and Arkady B. Khodursky, 2003 byNicola J. Camp and Angela Cox, 2002 223.Tumor Suppressor Genes: Volume 2: Regulation, Function, 113.DNA Repair Protocols: Eukaryotic Systems, edited by Daryl and Medicinal Applications, edited by Wafik S. El-Deiry, 2003 S. Henderson, 1999 222.Tumor Suppressor Genes: Volume 1: Pathways and Isolation 112.2-D Proteome Analysis Protocols,edited by Andrew J. Link, 1999 Strategies,edited by Wafik S. El-Deiry, 2003 111.Plant Cell Culture Protocols, edited by Robert D. Hall, 1999 M E T H O D S I N M O L E C U L A R B I O L O G YTM E. coliPlasmid Vectors Methods and Applications Edited by Nicola Casali University of California at Berkeley, School of Public Health, Berkeley, CA and Andrew Preston University of Cambridge, Department of Clinical Veterinary Medicine, Cambridge, UK Humana Press Totowa, New Jersey © 2003 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology™is a trademark of The Humana Press Inc. All papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. (cid:39) ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Cover illustration provided by Nicola Casali. Cover design by Patricia F. Cleary. Production Editor: Wendy S. Kopf. For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341; E-mail: [email protected]; Website: http://humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press, provided that the base fee of US $20.00 per copy, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [1-58829- 151-0/03 $20.00]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging in Publication Data E. coli plasmid vectors : methods and applications / edited by Nicola Casali and Andrew Preston. p. cm. -- (Methods in molecular biology ; v. 235) Includes bibliographical references and index. ISBN 1-58829-151-0 (alk. paper); (e-ISBN) 1-59259-409-3 ISSN 1064-3745 1. Plasmids--Laboratory manuals. I. Casali, Nicola II. Preston, Andrew, Ph.D. III. Methods in molecular biology (Totowa, N.J.) ; v. 235. QH452.6.E13 2003 572.8'69--dc21 2002191946 Preface Plasmids are autonomously replicating extrachromosomal DNA mol- ecules that are stably inherited and can be present at many copies per cell. These features, coupled to the ease with which plasmids can be manipulated to carry fragments of foreign DNA, have led to their exploitation as one of the critical workhorses of modern molecular biology. E. coli Plasmid Vectors focuses on the manipulation of plasmids in Escherichia coli. The well-characterized genetics of this bacterium have estab- lished its role as the universal cloning host, and recombinant DNA manipulation is almost exclusively performed in this organism. Despite the astonishing advances in molecular biology technologies and applications witnessed in the last decade, the ability to clone a DNA fragment of interest into a recombinant plasmid vector, and to maintain and manipulate it in anE. colihost, remains the foundation of many genetic analyses. E. coli Plasmid Vectors introduces relevant aspects of plasmid biology and describes the development of plasmid vectors. It also provides advice on choosing the right vector and a suitable host strain. The middle segment covers methods that are required to clone DNA into plasmid vectors, transform E. coli, and analyze recombinant clones. Protocols for the construction and screening of libraries are included, as well as specific techniques required for specialized cloning vehicles, such as cosmids, bacterial artificial chromosomes (BACs), (cid:104) vectors, and phagemids. The final section gives protocols for a variety of commonly used downstream applications. The value of E. coli-derived plas- mid vectors in providing the means to study diverse organisms is evident from chapters describing the mutagenesis of foreign genes for reintroduction into the homologous host, the production of recombinant proteins, and the uses of reporter genes. Commercial kits dominate many of these areas of molecular biology. Where pertinent, chapters include overviews of the methods that underpin these kits, give specific protocols for representative techniques, and include practi- cal advice and tips for troubleshooting problems. In doing so, E. coliPlasmid Vectors provides not only a basic guide for those new to the field, but also a valuable resource for more experienced researchers. Nicola Casali Andrew Preston v Contents Preface .............................................................................................................v Contributors.....................................................................................................ix 1 The Function and Organization of Plasmids Finbarr Hayes.........................................................................................1 2 Choosing a Cloning Vector Andrew Preston...................................................................................19 3 Escherichia coli Host Strains Nicola Casali.........................................................................................27 4 Chemical Transformation of E. coli W. Edward Swords..............................................................................49 5 Electroporation of E. coli Claire A. Woodall.................................................................................55 6 DNA Transfer by Bacterial Conjugation Claire A. Woodall.................................................................................61 7 Cosmid Packaging and Infection of E. coli Mallory J. A. White and Wade A. Nichols.........................................67 8 Isolation of Plasmids from E. coli by Alkaline Lysis Sabine Ehrt and Dirk Schnappinger..................................................75 9 Isolation of Plasmids from E. coli by Boiling Lysis Sabine Ehrt and Dirk Schnappinger..................................................79 10 High-Purity Plasmid Isolation Using Silica Oxide Stefan Grimm and Frank Voß-Neudecker.........................................83 11 High-Throughput Plasmid Extraction Using Microtiter Plates Michael A. Quail...................................................................................89 12 Isolation of Cosmid and BAC DNA from E. coli Daniel Sinnett and Alexandre Montpetit...........................................99 13 Preparation of Single-Stranded DNA from Phagemid Vectors W. Edward Swords............................................................................103 14 Using Desktop Cloning Software to Plan, Track, and Evaluate Cloning Projects Robert H. Gross.................................................................................107 15 Cloning in Plasmid Vectors Carey Pashley and Sharon Kendall.................................................121 vii viii Contents 16 Extraction of DNA from Agarose Gels Nicholas Downey...............................................................................137 17 Cloning PCR Products with T-Vectors Wade A. Nichols.................................................................................141 18 Construction of Genomic Libraries in (cid:104)-Vectors Yilun Wang, Zheng Cao, Darryl Hood, and James G. Townsel..................................................................153 19 Rapid Screening of Recombinant Plasmids Sangwei Lu.........................................................................................169 20 Restriction Analysis of Recombinant Plasmids Joanne Goranson-Siekierke and Jarrod L. Erbe...........................175 21 Screening Recombinant DNA Libraries Wade A. Nichols.................................................................................183 22 Sequencing Using Fluorescent-Labeled Nucleotides Allison F. Gillaspy.............................................................................195 23 Site-Directed Mutagenesis Using the Megaprimer Method Zhidong Xu, Alessia Colosimo, and Dieter C. Gruenert...............203 24 Site-Directed Mutagenesis by Inverse PCR Clifford N. Dominy and David W. Andrews....................................209 25 Creating Nested DNA Deletions Using Exonuclease III Rosamund Powles and Lafras M. Steyn.........................................225 26 Transposon and Transposome Mutagenesis of Plasmids, Cosmids, and BACs Alistair McGregor...............................................................................233 27 In Vitro Transcription and Translation Farahnaz Movahedzadeh, Susana González Rico, and Robert A. Cox.........................................................................247 28 Vectors for the Expression of Recombinant Proteins in E. coli Sally A. Cantrell.................................................................................257 29 Expression of Recombinant Proteins from lac Promoters Charles R. Sweet................................................................................277 30 Plasmid-Based Reporter Genes: Assays for (cid:96)-Galactosidase and Alkaline Phosphatase Activities Minghsun Liu......................................................................................289 31 Plasmid-Based Reporter Genes: Assays for Green Fluorescent Protein Sergei R. Doulatov.............................................................................297 Index............................................................................................................ 305 Contributors DAVID W. ANDREWS • Department of Biochemistry, McMaster University, Hamilton, ON, Canada SALLY A. CANTRELL • Division of Infectious Diseases, School of Public Health, University of California, Berkeley, CA ZHENG CAO • Department of Anatomy and Physiology, Meharry Medical College, Nashville, TN NICOLA CASALI • Division of Infectious Diseases, School of Public Health, University of California, Berkeley, CA ALESSIA COLOSIMO • University of Rome and CSS-Mendel Institute, Rome, Italy ROBERT A. COX • National Institute for Medical Research, London, UK CLIFFORD N. DOMINY • SYN-X Pharma Inc, Toronto, ON, Canada SERGEI R. DOULATOV • Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA NICHOLAS DOWNEY • Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA SABINE EHRT • Department of Microbiology, Weill Medical College of Cornell University, New York, NY JARROD L. ERBE • Department of Biology, Wisconsin Lutheran College, Milwaukee, WI ALLISON F. GILLASPY • Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK JOANNE GORANSON-SIEKIERKE • Department of Microbiology, University of Colorado Health Sciences Center, Denver, CO STEFAN GRIMM • Max-Planck-Institute for Biochemistry, Martinsried, Germany ROBERT H. GROSS • Department of Biological Sciences and Center for Biological and Biomedical Computing, Dartmouth College, Hanover, NH DIETER C. GRUENERT • Department of Medicine, University of Vermont, Colchester, VT FINBARR HAYES • Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, Manchester, UK DARRYL HOOD • Department of Pharmacology, Meharry Medical College, Nashville, TN ix x Contributors SHARON KENDALL • Department of Pathology and Infectious Diseases, Royal Veterinary College, London, UK MINGHSUN LIU • Department of Medicine, School of Medicine, University of Washington, Seattle, WA SANGWEI LU • Division of Infectious Diseases, School of Public Health, University of California, Berkeley, CA ALISTAIR MCGREGOR • Department of Molecular Genetics, University of Cincinnati, Cincinnati, OH ALEXANDRE MONTPETIT • Service d’hématologie-oncologie, Centre de Cancérologie Charles Bruneau, Centre de Recherche, Hôpital Ste-Justine, Département de pédiatrie, Université de Montréal, Montréal, Canada FARAHNAZ MOVAHEDZADEH • Department of Pathology and Infectious Diseases, Royal Veterinary College, London, UK WADE A. NICHOLS • Department of Biological Sciences, Illinois State University, Normal, IL CAREY PASHLEY • Department of Medical Microbiology, St. Bartholomew’s and Royal London School of Medicine and Dentistry, London, UK ROSAMUND POWLES • Division of Medical Microbiology, University of Cape Town, Rondebosch, South Africa ANDREW PRESTON • Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge, UK MICHAEL A. QUAIL • Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, UK SUSANA GONZÁLEZ RICO • Sección de Bacteriología, Instituto de Medicina Tropical, Universidad Central de Venezuela, Caracas, Venezuela DIRK SCHNAPPINGER • Department of Microbiology, Weill Medical College of Cornell University, New York, NY DANIEL SINNETT • Service d’Hématologie-Oncologie, Centre de Cancérologie Charles Bruneau, Centre de Recherche, Hôpital Ste-Justine, Département de pédiatrie, Université de Montréal, Montréal, Canada LAFRAS M. STEYN • Division of Medical Microbiology, University of Cape Town, Rondebosch, South Africa CHARLES R. SWEET • Division of Infectious Disease, University of Massachusetts Medical Center, Worcester, MA W. EDWARD SWORDS • Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, NC JAMES G. TOWNSEL • Department of Anatomy and Physiology, Meharry Medical College, Nashville, TN