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.
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References
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 97:5516–5521
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 99:12415–12420
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 94:14759–14763
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 89:8794–8797
Zagursky RJ, Hays JB (1983) Expression of the phage lambda recombination genes exo and bet under lacPO control on a multi-copy plasmid. Gene 23:277–292
Murphy KC (1998) Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180:2063–2071
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 70:2215–2219
Kovall R, Matthews BW (1997) Toroidal structure of lambda-exonuclease. Science 277:1824–1827
Weissbach A, Korn D (1962) The effect of lysogenic induction on the deoxyribonucleases of Escherichia coli K12 lambda. J Biol Chem 237:C3312–C3314
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 281:25205–25214
Kmiec E, Holloman WK (1981) Beta protein of bacteriophage lambda promotes renaturation of DNA. J Biol Chem 256:12636–12639
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 98:6742–6746
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 44:521–533
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 12:2939–2947
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 40:191–197
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 73:56–65
Acknowledgments
The authors thank Jens von Einem, Benedikt B. Kaufer, and Felix Wussow for their help in optimizing the presented method. We gratfully recognize Jenifer Klabis for her technical contributions to the isolation of the GS1783 E. coli strain.
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Tischer, B.K., Smith, G.A., Osterrieder, N. (2010). En Passant Mutagenesis: A Two Step Markerless Red Recombination System. In: Braman, J. (eds) In Vitro Mutagenesis Protocols. Methods in Molecular Biology, vol 634. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-652-8_30
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DOI: https://doi.org/10.1007/978-1-60761-652-8_30
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