In Vitro Mutagenesis Protocols pp 421-430

Part of the Methods in Molecular Biology book series (MIMB, volume 634)

En Passant Mutagenesis: A Two Step Markerless Red Recombination System

  • B. Karsten Tischer
  • Gregory A. Smith
  • Nikolaus Osterrieder
Protocol

Abstract

Bacterial artificial chromosomes are used to maintain and modify large sequences of different origins in Escherichia coli. In addition to RecA-based shuttle mutagenesis, Red recombination is commonly used for sequence modification. Since foreign sequences, such as antibiotic resistance genes as well as frt- or loxP-sites are often unwanted in mutant BAC clones, we developed a Red-based technique that allows for the scarless generation of point mutations, deletions, and insertion of smaller and larger sequences. The method employs a sequence duplication that is inserted into the target sequence in the first recombination step and the excision of the selection marker by in vivo I-SceI cleavage and the second Red recombination. To allow for convenient and highly efficient mutagenesis without the use of additional plasmids, the E. coli strain GS1783 with a chromosomal encoded inducible Red- and I-SceI-expression was created.

Key words

Red recombination BAC Markerless En passant mutagenesis I-SceI 

References

  1. 1.
    Almazan F, Gonzalez JM, Penzes Z, Izeta A, Calvo E, Plana-Duran J, Enjuanes L (2000) Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci USA 97:5516–5521PubMedCrossRefGoogle Scholar
  2. 2.
    Domi A, Moss B (2002) Cloning the vaccinia virus genome as a bacterial artificial chromosome in Escherichia coli and recovery of infectious virus in mammalian cells. Proc Natl Acad Sci USA 99:12415–12420PubMedCrossRefGoogle Scholar
  3. 3.
    Messerle M, Crnkovic I, Hammerschmidt W, Ziegler H, Koszinowski UH (1997) Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci USA 94:14759–14763PubMedCrossRefGoogle Scholar
  4. 4.
    Shizuya H, Birren B, Kim UJ, Mancino V, Slepak T, Tachiiri Y, Simon M (1992) Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc Natl Acad Sci USA 89:8794–8797PubMedCrossRefGoogle Scholar
  5. 5.
    Zagursky RJ, Hays JB (1983) Expression of the phage lambda recombination genes exo and bet under lacPO control on a multi-copy plasmid. Gene 23:277–292PubMedCrossRefGoogle Scholar
  6. 6.
    Murphy KC (1998) Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180:2063–2071PubMedGoogle Scholar
  7. 7.
    Sakaki Y, Karu AE, Linn S, Echols H (1973) Purification and properties of the gamma-protein specified by bacteriophage lambda: an inhibitor of the host RecBC recombination enzyme. Proc Natl Acad Sci USA 70:2215–2219PubMedCrossRefGoogle Scholar
  8. 8.
    Kovall R, Matthews BW (1997) Toroidal structure of lambda-exonuclease. Science 277:1824–1827PubMedCrossRefGoogle Scholar
  9. 9.
    Weissbach A, Korn D (1962) The effect of lysogenic induction on the deoxyribonucleases of Escherichia coli K12 lambda. J Biol Chem 237:C3312–C3314Google Scholar
  10. 10.
    Wu Z, Xing X, Bohl CE, Wisler JW, Dalton JT, Bell CE (2006) Domain structure and DNA binding regions of beta protein from bacteriophage lambda. J Biol Chem 281:25205–25214PubMedCrossRefGoogle Scholar
  11. 11.
    Kmiec E, Holloman WK (1981) Beta protein of bacteriophage lambda promotes renaturation of DNA. J Biol Chem 256:12636–12639PubMedGoogle Scholar
  12. 12.
    Ellis HM, Yu D, DiTizio T, Court DL (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci USA 98:6742–6746PubMedCrossRefGoogle Scholar
  13. 13.
    Colleaux L, D’Auriol L, Betermier M, Cottarel G, Jacquier A, Galibert F, Dujon B (1986) Universal code equivalent of a yeast mitochondrial intron reading frame is expressed into E. coli as a specific double strand endonuclease. Cell 44:521–533PubMedCrossRefGoogle Scholar
  14. 14.
    Perrin A, Buckle M, Dujon B (1993) Asymmetrical recognition and activity of the I-SceI endonuclease on its site and on intron-exon junctions. EMBO J 12:2939–2947PubMedGoogle Scholar
  15. 15.
    Tischer BK, von Einem J, Kaufer B, Osterrieder N (2006) Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. Biotechniques 40:191–197PubMedCrossRefGoogle Scholar
  16. 16.
    Lee EC, Yu D, Martinez de Velasco J, Tessarollo L, Swing DA, Court DL, Jenkins NA, Copeland NG (2001) A highly efficient Esche­ri­chia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73:56–65PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • B. Karsten Tischer
    • 2
  • Gregory A. Smith
    • 1
  • Nikolaus Osterrieder
    • 2
    • 3
  1. 1.Department of Microbiology-ImmunologyNorthwestern UniversityChicagoUSA
  2. 2.Institut für Virologie, Freie Universität BerlinBerlinGermany
  3. 3.Department of Microbiology and ImmunologyCornell UniversityIthacaUSA

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