Using PATIMDB to Create Bacterial Transposon Insertion Mutant Libraries

Jonathan M. Urbach1, Tao Wei2, Nicole Liberati1, Daniel Grenfell‐Lee3, Jacinto Villanueva4, Gang Wu1, Frederick M. Ausubel1

1 Massachusetts General Hospital, Boston, Massachusetts, 2 Eli Lilly and Company, Indianapolis, Indiana, 3 Microbia Precision Engineering, Lexington, Massachusetts, 4 Mariposa Bioscience Inc., Burlingame, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 19.7
DOI:  10.1002/0471142727.mb1907s86
Online Posting Date:  April, 2009
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Abstract

PATIMDB is a software package for facilitating the generation of transposon mutant insertion libraries. The software has two main functions: process tracking and automated sequence analysis. The process tracking function specifically includes recording the status and fates of multiwell plates and samples in various stages of library construction. Automated sequence analysis refers specifically to the pipeline of sequence analysis starting with ABI files from a sequencing facility and ending with insertion location identifications. The protocols in this unit describe installation and use of PATIMDB software. Curr. Protoc. Mol. Biol. 86:19.7.1‐19.7.34. © 2009 by John Wiley & Sons, Inc.

Keywords: transposon; mutant; library; PATIMDB; software; process tracking; pipeline

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Installing and Configuring the PATIMDB Application and Helper Applications
  • Basic Protocol 2: Mutant Library Setup
  • Basic Protocol 3: Creating Plate Sets and Performing Processes with the PATIMDB Application
  • Basic Protocol 4: Performing Automated Sequence Analysis with the PATIMDB
  • Basic Protocol 5: Retrieving Plate and Mutant Reports
  • Support Protocol 1: Installing Java 2 Platform Standard Edition on Windows Computers
  • Support Protocol 2: Installing Java 2 Platform Standard Edition on Linux Computers
  • Support Protocol 3: Updating to J2SE 5.0 on Mac OS X Computers
  • Support Protocol 4: Installing MySQL Server and Client on Package‐Based Linux Systems
  • Support Protocol 5: Installing MySQL Server and Client on Generic Linux‐, Mac OS X–, and Unix‐Based Computers
  • Support Protocol 6: Installing MySQL Server and Client on Windows
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Installing and Configuring the PATIMDB Application and Helper Applications

  Materials
  • Hardware
    • Windows, Mac OS X, or Linux Computer with J2SE installed (See Support Protocol protocol 61, protocol 72 or protocol 83)
    • Computer running the MySQL server and client applications (See Support Protocol protocol 94, protocol 105 or protocol 116)
  • Software
    • PATIMDB Application, available from the Ausubel Group at Massachusetts General Hospital, http://ausubellab.mgh.harvard.edu/patimdb_application
    • MySQL Connector/J, Version 3.0.15 or later, available from MySQL AB, http://dev.mysql.com/downloads/connector/j/
    • Phred, version 0.000925.c or later, available from the University of Washington (http://www.phrap.org) for academic licenses and from the Codon Code Corporation (http://www.phrap.com) for commercial licenses
    • NCBI BLAST, version 2.2.14 or later, available from the NCBI, ftp://ftp.ncbi.nih.gov/blast/executables/LATEST (NCBI BLAST packages include the formatdb application)

Basic Protocol 2: Mutant Library Setup

  Materials
  • Hardware
    • Windows, Mac OS X, or Linux Computer with the PATIMDB installed ( protocol 1)
    • Computer hosting the PATIMDB Database
  • Data
    • Genome sequence of your transposon insertion library host organism in GenBank (preferred) or FASTA format
    • Gene descriptions in GenBank or tabular format (see below)
    • 15‐ to 30‐base segment of the transposon sequence that abuts the transposon junction and can be detected in the sequencing reads

Basic Protocol 3: Creating Plate Sets and Performing Processes with the PATIMDB Application

  Materials
  • Hardware
    • A computer running Windows 2000, XP, or Vista
  • Software
    • Java 2 Platform Standard Edition (J2SE), version 5.0 or later, available from http://java.sun.com/javase

Basic Protocol 4: Performing Automated Sequence Analysis with the PATIMDB

  Materials
  • Hardware
    • A computer running Linux kernel version 2.4 or later
  • Software
    • Java 2 Platform Standard Edition (J2SE), version 5.0 or later, available from http://java.sun.com/javase

Basic Protocol 5: Retrieving Plate and Mutant Reports

  Materials
  • Hardware
    • A computer running Mac OS X 10.4 or later (J2SE version 5.0 is not available for earlier versions of Mac OS X)
  • Software
    • J2SE 5.0, available through the Mac OS X Software Update utility, or from http://developer.apple.com/java/download/

Support Protocol 1: Installing Java 2 Platform Standard Edition on Windows Computers

  Materials
  • Hardware
    • A computer running Linux kernel version 2.4 or later
  • Software
    • MySQL Server and Command Line Client Programs, available from MySQL AB http://dev.mysql.com/downloads/mysql/5.0.html
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Figures

Videos

Literature Cited

Literature Cited
   Cameron, D.E., Urbach, J.M., and Mekalanos, J.J. 2008. A defined transposon mutant library and its use in identifying motility genes in Vibrio cholerae. Proc. Natl. Acad. Sci. U.S.A. 105:8736‐8741.
   Caetano‐Anollés, G. and Bassam, B.J. 1993. DNA amplification fingerprinting using arbitrary oligonucleotide primers. Appl. Biochem. Biotechnol. 42:189‐200.
   Garsin, D.A., Urbach, J., Huguet‐Tapia, J.C., Peters, J.E., and Ausubel, F.M. 2004. Construction of an Enterococcus faecalis Tn917‐mediated‐gene disruption library offers insight into Tn917 insertion patterns. J. Bacteriol. 186:7280‐7289.
   Jacobs, M.A., Alwood, A., Thaipisuttikul, I., Spencer, D., Haugen, E., Ernst, S., Will, O., Kaul, R., Raymond, C., Levy, R., Chun‐Rong, L., Guenthner, D., Bovee, D., Olson, M.V., and Manoil, C. 2003. Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U.S.A. 100:14339‐14344.
   Liberati, N.T., Urbach, J.M., Miyata, S., Lee, D.G., Drenkard, E., Wu, G., Villanueva, J., Wei, T., and Ausubel, F.M. 2005. An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proc. Natl. Acad. Sci. U.S.A. 103:2833‐2838.
   Liberati, N.T., Urbach, J.M., Thurber, T.K., Wu, G., and Ausubel, F.M. 2008. Comparing insertion libraries in two Pseudomonas aeruginosa strains to assess gene essentiality. In Methods in Molecular Biology Vol 416: Microbial Gene Essentiality: Protocols and Bioinformatics (S.Y. Gerdes, J.‐I. Kato, B.D. Peyser, R. Irizarry, F.A. Spencer, T. Baba, and H. Mori, eds.) pp 153‐169. Humana Press, Totowa, N.J.
   Vastrik, I. 2008. Installing a local copy of the reactome Web site and database. Curr. Protoc. Bioinform. 9:10.1‐9.10.9.
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