E. coli Genome Manipulation by P1 Transduction

Lynn C. Thomason1, Nina Costantino1, Donald L. Court1

1 National Cancer Institute at Frederick, Frederick, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 1.17
DOI:  10.1002/0471142727.mb0117s79
Online Posting Date:  May, 2014
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Abstract

This unit describes the procedure used to move portions of the E. coli genome from one genetic variant to another. Fragments of ∼100 kb can be transferred by the P1 bacteriophage. The phage is first grown on a strain containing the elements to be moved, and the resulting phage lysate is used to infect a second recipient strain. The lysate will contain bacterial DNA as well as phage DNA, and genetic recombination, catalyzed by enzymes of the recipient strain, will incorporate the bacterial fragments into the recipient chromosome. Curr. Protoc. Mol. Biol. 79:1.17.1‐1.17.8. © 2007 by John Wiley & Sons, Inc.

Keywords: transduction; E. coli bacteriophage P1; genetic recombination

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of P1 Liquid Lysate and P1 Transduction
  • Support Protocol 1: Titration of the P1 Lysate
  • Alternate Protocol 1: Preparation of P1 Plate Lysates
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of P1 Liquid Lysate and P1 Transduction

  Materials
  • LB broth (unit )
  • E. coli nonlysogenic donor strain (containing the genetic marker to be transduced)
  • 20% glucose stock solution
  • 1 M CaCl 2
  • 109 to 1010 pfu/ml P1 stock, a virulent P1 mutant preferred (e.g., P1vir)
  • Chloroform (CHCl 3)
  • E. coli recipient strain (to be transduced)
  • Tris‐magnesium (TM) buffer: 10 mM MgSO 4/10 mM Tris⋅Cl, pH 7.4 ( )
  • P1 salts solution: 10 mM CaCl 2/5 mM MgSO 4
  • 1 M sodium citrate
  • Selective plates (see unit ) with 5 mM sodium citrate (recommended); e.g., minimal plates if selecting for prototrophy or rich LB plates containing the appropriate antibiotic (depending on drug cassette used):
    • 30 µg/ml ampicillin
    • 30 µg/ml kanamycin
    • 10 µg/ml chloramphenicol
    • 12.5 µg/ml tetracycline (6.25 µg/ml if sodium citrate is added)
    • 50 µg/ml spectinomycin
  • 50‐ml Erlenmeyer flasks, preferably baffled
  • 18 × 150–mm glass culture tubes
  • Shaking water bath or test‐tube roller at appropriate temperature (usually 32° to 37°C)
  • 35‐ml plastic centrifuge tubes
  • Refrigerated low‐speed centrifuge with Sorvall SA‐600 rotor (or equivalent)
  • 5‐ml screw‐cap vials
NOTE: All material coming in contact with E. coli must be sterile.

Support Protocol 1: Titration of the P1 Lysate

  • Standard E. coli K12 strain (e.g., C600 or MG1655)
  • LB plates and top agar (unit ), each containing 2.5 mM CaCl 2
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Figures

Videos

Literature Cited

   Baba, T. , Ara, T. , Hasegawa, M. , Takai, Y. , Okumura, Y. , Baba, M. , Datsenko, K. A. , Tomita, M. , Wanner, B. L. , and Mori, H. 2006. Construction of Escherichia coli K‐12 in‐frame, single‐gene knockout mutants: The Keio collection. Mol. Syst. Biol. 2:2006.0008.
   Franklin, N.C. 1969. Mutation in gal U gene of E. coli blocks phage P1 infection. Virology 38:189‐191.
   Lennox, E.S. 1955. Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190‐206.
   Sandulache, R. , Prehm, P. , and Kamp, D. 1984. Cell wall receptor for bacteriophage Mu G(+). J. Bacteriol. 160:299‐303.
   Silhavy, T.J. , Berman, M.L. , and Enquist, L.W. 1984. Preparation of a P1Tn9clr100 lysate. In Experiments with Gene Fusions, pp. 109‐110. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Singer, M. , Baker, T.A. , Schnitzler, G. , Deischel, S.M. , Goel, M. , Dove, W. , Jaacks, K.J. , Grossman, A.D. , Erickson, J.W. , and Gross, C.A. 1989. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli . Microbiol. Rev. 53:1‐24.
   Yarmolinsky, M.B. and Sternberg, N. 1988. Bacteriophage P1. In The Bacteriophages, Vol. 1. ( R. Calendar , ed.) pp. 291‐438. Plenum Press, New York.
Key References
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