Acta Biologica Hungarica
Volume/Issue:
Volume 58: Issue Issue Supplement-1
Directed homologous recombination for genome engineering in Escherichia coli
Authors:
B. Csörgő Biological Research Center of the Hungarian Academy of Sciences Institute of Biochemistry Szeged Hungary

Search for other papers by B. Csörgő in
Current site
Google Scholar
PubMed Close
and
G. Pósfai Biological Research Center of the Hungarian Academy of Sciences Institute of Biochemistry Szeged Hungary

Search for other papers by G. Pósfai in
Current site
Google Scholar
PubMed Close
Pages:
1–10
Online Publication Date:
24 Oct 2007
Publication Date:
01 Nov 2007
Article Category:
Research Article
DOI:
https://doi.org/10.1556/abiol.58.2007.suppl.1
Keywords:
Suicide plasmid; DSB; Chi; gene replacement; RecA
Restricted access

E. coli K-12 is the workhorse of molecular biology and the platform of choice for production of DNA, metabolites and proteins of industrial interest. To construct strains for the multitude of purposes, efficient genome manipulation methods are required. The suicide plasmid-mediated, homologous recombination-based gene replacement method is a convenient genome engineering tool. In consecutive recombination events, genomic integration of the plasmid, carrying the modified allele, is followed by its excision, resulting in either a modified genome or the original wild-type chromosome. Using the lac operon as a chromosomal target, we systematically investigated the effects of several factors influencing the outcome of the procedure. Recombinogenic activity was proportional to the length of the targeting homologous fragments. Presence of a properly oriented Chi site stabilized broken chromosomal ends and stimulated recombination in the downstream genomic region. Introduction of a double-stranded break in the chromosome had a profound stimulatory effect on recombination of the free DNA ends. These results shed light on some details of the complex events of intra-and intermolecular homologous recombination in the E. coli genome. Taking into account these findings at the assembly of the targeting plasmid constructs, serial genomic modifications can be created with enhanced efficiency and speed.

  • Blattner, F. R., Plunkett, G., III, Bloch, C. A., Perna, N. T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K., Mayhew, G. F., Gregor, J., Davis, W. D., Kirkpatrick, H. A., Goeden, M. A., Rose, D. J., Mau, B., Shao, Y. (1997) The complete genome sequence of Escherichia coli K-12. Science 277 , 1453–1474.

    Shao Y. , 'The complete genome sequence of Escherichia coli K-12 ' (1997 ) 277 Science : 1453 -1474 .

    • Search Google Scholar

  • Burland, V., Plunkett, G., III, Daniels, D. L., Blattner, F. R. (1993) DNA sequence and analysis of 136 kilobases of the Escherichia coli genome: organizational symmetry around the origin of replication. Genomics 16 , 551–561.

    Blattner F. R. , 'DNA sequence and analysis of 136 kilobases of the Escherichia coli genome: organizational symmetry around the origin of replication ' (1993 ) 16 Genomics : 551 -561 .

    • Search Google Scholar

  • Court, D. L., Sawitzke, J. A., Thomason, L. C. (2002) Genetic engineering using homologous recombination. Annu. Rev. Genet. 36 , 361–388.

    Thomason L. C. , 'Genetic engineering using homologous recombination ' (2002 ) 36 Annu. Rev. Genet. : 361 -388 .

    • Search Google Scholar

  • Dabert, P., Smith, G. R. (1997) Gene replacement with linear DNA fragments in wild-type Escherichia coli: enhancement by Chi sites. Genetics 145 , 877–889.

    Smith G. R. , 'Gene replacement with linear DNA fragments in wild-type Escherichia coli: enhancement by Chi sites ' (1997 ) 145 Genetics : 877 -889 .

    • Search Google Scholar

  • Datsenko, K. A., Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97 , 6640–6645.

    Wanner B. L. , 'One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products ' (2000 ) 97 Proc. Natl. Acad. Sci. USA : 6640 -6645 .

    • Search Google Scholar

  • Dixon, D. A., Kowalczykowski, S. C. (1991) Homologous pairing in vitro stimulated by the recombination hotspot, Chi. Cell 66 , 361–371.

    Kowalczykowski S. C. , 'Homologous pairing in vitro stimulated by the recombination hotspot, Chi ' (1991 ) 66 Cell : 361 -371 .

    • Search Google Scholar

  • Dujon, B. (1989) Group I introns as mobile genetic elements: facts and mechanistic speculations — review. Gene 82 , 91–114.

    Dujon B. , 'Group I introns as mobile genetic elements: facts and mechanistic speculations — review ' (1989 ) 82 Gene : 91 -114 .

    • Search Google Scholar

  • Friedman-Ohana, R., Karunker, I., Cohen, A. (1998) Chi-dependent intramolecular recombination in Escherichia coli . Genetics 148 , 545–557.

    Cohen A. , 'Chi-dependent intramolecular recombination in Escherichia coli ' (1998 ) 148 Genetics : 545 -557 .

    • Search Google Scholar

  • Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, P., Kushner, S. R. (1989) New method for generating deletions and gene replacements in Escherichia coli . J. Bacteriol. 171 , 4617–4622.

    Kushner S. R. , 'New method for generating deletions and gene replacements in Escherichia coli ' (1989 ) 171 J. Bacteriol. : 4617 -4622 .

    • Search Google Scholar

  • Hayashi, K., Morooka, N., Yamamoto, Y., Fujita, K., Isono, K., Choi, S., Ohtsubo, E., Baba, T., Wanner, B. L., Mori, H., Horiuchi, T. (2006) Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110. Mol. Syst. Biol. 2 , 2006.0007.

  • Kato, C., Ohmiya, R., Mizuno, T. (1998) A rapid method for disrupting genes in the Escherichia coli genome. Biosci. Biotechnol. Biochem. 62 , 1826–1829.

    Mizuno T. , 'A rapid method for disrupting genes in the Escherichia coli genome ' (1998 ) 62 Biosci. Biotechnol. Biochem. : 1826 -1829 .

    • Search Google Scholar

  • Khasanov, F. K., Zvingila, D. J., Zainullin, A. A., Prozorov, A. A., Bashkirov, V. I. (1992) Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology. Mol. Gen. Genet. 234 , 494–497.

    Bashkirov V. I. , 'Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology ' (1992 ) 234 Mol. Gen. Genet. : 494 -497 .

    • Search Google Scholar

  • Kolisnychenko, V., Plunkett, G., III, Herring, C. D., Fehér, T., Pósfai, J., Blattner, F. R., Pósfai, G. (2002) Engineering a reduced Escherichia coli genome. Gen. Res. 12 , 640–647.

    Pósfai G. , 'Engineering a reduced Escherichia coli genome ' (2002 ) 12 Gen. Res. : 640 -647 .

    • Search Google Scholar

  • Link, A. J., Phillips, D., Church, G. M. (1997) Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. J. Bacteriol. 179 , 6228–6237.

    Church G. M. , 'Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization ' (1997 ) 179 J. Bacteriol. : 6228 -6237 .

    • Search Google Scholar

  • Maniatis, T., Fritsch, E. F., Sambook, J. (1982) Molecular Cloning: A Laboratory Manual . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Sambook J. , '', in Molecular Cloning: A Laboratory Manual , (1982 ) -.

  • Metcalf, W. W., Jiang, W., Daniels, L. L., Kim, S.-K., Haldimann, A., Wanner, B. L. (1996) Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria. Plasmid 35 , 1–13.

    Wanner B. L. , 'Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria ' (1996 ) 35 Plasmid : 1 -13 .

    • Search Google Scholar

  • Monteilhet, C., Perrin, A., Thierry, A., Colleaux, L., Dujon, B. (1990) Purification and characterization of the in vitro activity of I-SceI, a novel and highly specific endonuclease encoded by a group I intron. Nucleic Acids Res. 18 , 1407–1413.

    Dujon B. , 'Purification and characterization of the in vitro activity of I-SceI, a novel and highly specific endonuclease encoded by a group I intron ' (1990 ) 18 Nucleic Acids Res. : 1407 -1413 .

    • Search Google Scholar

  • Myers, R. S., Stahl, F. W. (1994) Chi and the RecBC D enzyme of Escherichia coli . Annu. Rev. Genet. 28 , 49–70.

    Stahl F. W. , 'Chi and the RecBC D enzyme of Escherichia coli ' (1994 ) 28 Annu. Rev. Genet. : 49 -70 .

    • Search Google Scholar

  • Pósfai, G., Koob, M. D., Kirkpatrick, H. A., Blattner, F. R. (1997) Versatile insertion plasmids for targeted genome manipulations in bacteria: isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli 0157:H7 genome. J. Bacteriol. 179 , 4426–4428.

    Blattner F. R. , 'Versatile insertion plasmids for targeted genome manipulations in bacteria: isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli 0157:H7 genome ' (1997 ) 179 J. Bacteriol. : 4426 -4428 .

    • Search Google Scholar

  • Pósfai, G., Kolisnychenko, V., Bereczki, Z., Blattner, F. R. (1999) Markerless gene replacement in Escherichia coli stimulated by a double-stand break in the chromosome. Nucleic Acids Res. 27 , 4409–4415.

    Blattner F. R. , 'Markerless gene replacement in Escherichia coli stimulated by a double-stand break in the chromosome ' (1999 ) 27 Nucleic Acids Res. : 4409 -4415 .

    • Search Google Scholar

  • Pósfai, G., Plunkett, G., III, Fehér, T., Frisch, D., Keil, G. M., Umenhoffer K., Kolisnychenko, V., Stahl, B., Sharma, S. S., de Arruda, M., Burland, V., Harcum, S. W., Blattner, F. R. (2006) Emergent properties of reduced-genome Escherichia coli . Science 312(5776) , 1044–1046.

    Blattner F. R. , 'Emergent properties of reduced-genome Escherichia coli ' (2006 ) 312 Science : 1044 -1046 .

    • Search Google Scholar

  • Riley, M., Abe, T., Arnaud, M. B., Berlyn, M. K. B., Blattner, F. R., Chaudhuri, R. R., Glasner, J. D., Horiuchi, T., Keseler, I. M., Kosuge, T., Mori, H., Perna, N. T., Plunkett, G., III, Rudd, K. E., Serres, M. H., Thomas, G. H., Thomson, N. R., Wishart, D., Wanner, B. L. (2006) Escherichia coli K-12: a cooperatively developed annotation snapshot-2005. Nucleic Acids Res. 34 , 1–9.

    Wanner B. L. , 'Escherichia coli K-12: a cooperatively developed annotation snapshot-2005 ' (2006 ) 34 Nucleic Acids Res. : 1 -9 .

    • Search Google Scholar

  • Rinken, R., Thomas, B., Wackernagel, W. (1992) Evidence that recBC -dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding. J. Bacteriol. 174 , 5424–5429.

    Wackernagel W. , 'Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding ' (1992 ) 174 J. Bacteriol. : 5424 -5429 .

    • Search Google Scholar

  • Russell, C. B., Thaler, D. S., Dahlquist, F. W. (1989) Chromosomal transformation of Escherichia coli recD strains with linearized plasmids. J. Bacteriol. 171 , 2609–2613.

    Dahlquist F. W. , 'Chromosomal transformation of Escherichia coli recD strains with linearized plasmids ' (1989 ) 171 J. Bacteriol. : 2609 -2613 .

    • Search Google Scholar

  • Sambrook, J., Fritsch, E. F., Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual . pp. 1.82–1.84, A.1–A.2, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Maniatis T. , '', in Molecular Cloning: A Laboratory Manual , (1989 ) -.

  • Serres, M. H., Goswami, S., Riley, M. (2004) GenProtEC: an updated and improved analysis of functions of Escherichia coli K-12 proteins. Nucleic Acids Res. 32 , D300.

    Riley M. , 'GenProtEC: an updated and improved analysis of functions of Escherichia coli K-12 proteins ' (2004 ) 32 Nucleic Acids Res. : D300 -.

    • Search Google Scholar

  • Stahl, F. W. (2005) Chi: a little sequence controls a big enzyme. Genetics 170 , 487–493.

    Stahl F. W. , 'Chi: a little sequence controls a big enzyme ' (2005 ) 170 Genetics : 487 -493 .

    • Search Google Scholar

  • Woodcock, D. M., Crowther, P. J., Doherty, J., Jefferson, S., DeCruz, E., Noyer-Weidner, M., Smith, S. S., Michael, M. Z., Graham, M. W. (1989) Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res. 17 , 3469–3478.

    Graham M. W. , 'Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants ' (1989 ) 17 Nucleic Acids Res. : 3469 -3478 .

    • Search Google Scholar

  • Zhang, Y., Buchholz, F., Muyrers, J. P., Stewart, A. F. (1998) A new logic for DNA engineering using recombination in Escherichia coli . Nat. Genet. 20 , 123–128.

    Stewart A. F. , 'A new logic for DNA engineering using recombination in Escherichia coli ' (1998 ) 20 Nat. Genet. : 123 -128 .

    • Search Google Scholar
  • Collapse
  • Expand
Cover Acta Biologica Hungarica
Acta Biologica Hungarica
Print ISSN:
0236-5383
Online ISSN:
1588-256X

Senior editors

Editor(s)-in-Chief: Elekes, Károly

Editorial Board

    1. Csányi, Vilmos (Göd)
    1. Dudits, Dénes (Szeged)
    1. Falus, András (Budapest)
    1. Fischer, Ernő (Pécs)
    1. Gábriel, Róbert (Pécs)
    1. Gulya, Károly (Szeged)
    1. Gulyás, Balázs (Stockholm)
    1. Hajós, Ferenc (Budapest)
    1. Hámori, József (Budapest)
    1. Heszky, László (Gödöllő)
    1. Hideg, Éva (Szeged)
    1. E. Ito (Sanuki)
    1. Janda, Tibor (Martonvásár)
    1. Kavanaugh, Michael P. (Missoula)
    1. Kása, Péter (Szeged)
    1. Klein, Éva (Stockholm)
    1. Kovács, János (Budapest)
    1. Brigitte Mauch-Mani (Neuchâtel)
    1. Nässel, Dick R. (Stockholm)
    1. Nemcsók, János (Szeged)
    1. Péczely, Péter (Gödöllő)
    1. Roberts, D. F. (Newcastle-upon-Tyne)
    1. Sakharov, Dimitri A. (Moscow)
    1. Singh, Meharvan (Fort Worth)
    1. Sipiczky, Mátyás (Debrecen)
    1. Szeberényi, József (Pécs)
    1. Székely, György (Debrecen)
    1. Tari, Irma (Szeged)
    1. Vágvölgyi, Csaba (Szeged),
    1. L. Zaborszky (Newark)

 

Acta Biologica Hungarica
P.O. Box 35
H-8237 Tihany, Hungary
Phone: (36 87) 448 244 ext. 103
Fax: (36 87) 448 006
E-mail: elekes@tres.blki.hu

Indexing and Abstracting Services:

  • Biological Abstracts
  • BIOSIS Previews
  • CAB Abstracts
  • Chemical Abstracts
  • Current Contents: Agriculture
  • Biology and Environmental Sciences
  • Elsevier BIOBASE
  • Global Health
  • Index Medicus
  • Index Veterinarius
  • Medline
  • Referativnyi Zhurnal
  • Science Citation Index
  • Science Citation Index Expanded (SciSearch)
  • SCOPUS
  • The ISI Alerting Services
  • Zoological Abstracts

 

Acta Biologica Hungarica
Language English
Size  
Year of
Foundation		 1950
Publication
Programme		 changed title
Volumes
per Year		  
Issues
per Year		  
Founder Magyar Tudományos Akadémia
Founder's
Address		 H-1051 Budapest, Hungary, Széchenyi István tér 9.
Publisher Akadémiai Kiadó
Publisher's
Address		 H-1117 Budapest, Hungary 1516 Budapest, PO Box 245.
Responsible
Publisher		 Chief Executive Officer, Akadémiai Kiadó
ISSN 0236-5383 (Print)
ISSN 1588-256X (Online)

Monthly Content Usage

Abstract Views Full Text Views PDF Downloads
Dec 2024 20 1 1
Jan 2025 32 0 0
Feb 2025 35 0 0
Mar 2025 45 0 0
Apr 2025 18 4 5
May 2025 9 1 0
Jun 2025 0 0 0