New method for generating deletions and gene replacements in Escherichia coli

C. M. Hamilton, M. Aldea, B. K. Washburn, Paul Lee Babitzke, S. R. Kushner

Research output: Contribution to journalArticle

564 Citations (Scopus)

Abstract

We describe a method for generating gene replacements and deletions in Escherichia coli. The technique is simple and rapid and can be applied to most genes, even those that are essential. What makes this method unique and particularly effective is the use of a temperature-sensitive pSC101 replicon to facilitate the gene replacement. The method proceeds by homologous recombination between a gene on the chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication. Thus, after transformation of the plasmid into an appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44°C. Subsequent growth of these cointegrates at 30°C leads to a second recombination event, resulting in their resolution. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the original copy of the gene. The procedure can also be used to effect the transfer of an allele from a plasmid to the chromosome or to rescue a chromosomal allele onto a plasmid. Since the resolved plasmid can be maintained by selection, this technique can be used to generate deletions of essential genes.

Original languageEnglish (US)
Pages (from-to)4617-4622
Number of pages6
JournalJournal of Bacteriology
Volume171
Issue number9
DOIs
StatePublished - Jan 1 1989

Fingerprint

Gene Deletion
Plasmids
Escherichia coli
Chromosomes
Genes
Genetic Recombination
Alleles
Replicon
Temperature
Homologous Recombination
Essential Genes
Sequence Homology
DNA Replication
Growth

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

Cite this

Hamilton, C. M. ; Aldea, M. ; Washburn, B. K. ; Babitzke, Paul Lee ; Kushner, S. R. / New method for generating deletions and gene replacements in Escherichia coli. In: Journal of Bacteriology. 1989 ; Vol. 171, No. 9. pp. 4617-4622.
@article{d69ac3895c2b4ae99d0a9caa8b6cb730,
title = "New method for generating deletions and gene replacements in Escherichia coli",
abstract = "We describe a method for generating gene replacements and deletions in Escherichia coli. The technique is simple and rapid and can be applied to most genes, even those that are essential. What makes this method unique and particularly effective is the use of a temperature-sensitive pSC101 replicon to facilitate the gene replacement. The method proceeds by homologous recombination between a gene on the chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication. Thus, after transformation of the plasmid into an appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44°C. Subsequent growth of these cointegrates at 30°C leads to a second recombination event, resulting in their resolution. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the original copy of the gene. The procedure can also be used to effect the transfer of an allele from a plasmid to the chromosome or to rescue a chromosomal allele onto a plasmid. Since the resolved plasmid can be maintained by selection, this technique can be used to generate deletions of essential genes.",
author = "Hamilton, {C. M.} and M. Aldea and Washburn, {B. K.} and Babitzke, {Paul Lee} and Kushner, {S. R.}",
year = "1989",
month = "1",
day = "1",
doi = "10.1128/jb.171.9.4617-4622.1989",
language = "English (US)",
volume = "171",
pages = "4617--4622",
journal = "Journal of Bacteriology",
issn = "0021-9193",
publisher = "American Society for Microbiology",
number = "9",

}

New method for generating deletions and gene replacements in Escherichia coli. / Hamilton, C. M.; Aldea, M.; Washburn, B. K.; Babitzke, Paul Lee; Kushner, S. R.

In: Journal of Bacteriology, Vol. 171, No. 9, 01.01.1989, p. 4617-4622.

Research output: Contribution to journalArticle

TY - JOUR

T1 - New method for generating deletions and gene replacements in Escherichia coli

AU - Hamilton, C. M.

AU - Aldea, M.

AU - Washburn, B. K.

AU - Babitzke, Paul Lee

AU - Kushner, S. R.

PY - 1989/1/1

Y1 - 1989/1/1

N2 - We describe a method for generating gene replacements and deletions in Escherichia coli. The technique is simple and rapid and can be applied to most genes, even those that are essential. What makes this method unique and particularly effective is the use of a temperature-sensitive pSC101 replicon to facilitate the gene replacement. The method proceeds by homologous recombination between a gene on the chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication. Thus, after transformation of the plasmid into an appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44°C. Subsequent growth of these cointegrates at 30°C leads to a second recombination event, resulting in their resolution. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the original copy of the gene. The procedure can also be used to effect the transfer of an allele from a plasmid to the chromosome or to rescue a chromosomal allele onto a plasmid. Since the resolved plasmid can be maintained by selection, this technique can be used to generate deletions of essential genes.

AB - We describe a method for generating gene replacements and deletions in Escherichia coli. The technique is simple and rapid and can be applied to most genes, even those that are essential. What makes this method unique and particularly effective is the use of a temperature-sensitive pSC101 replicon to facilitate the gene replacement. The method proceeds by homologous recombination between a gene on the chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication. Thus, after transformation of the plasmid into an appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44°C. Subsequent growth of these cointegrates at 30°C leads to a second recombination event, resulting in their resolution. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the original copy of the gene. The procedure can also be used to effect the transfer of an allele from a plasmid to the chromosome or to rescue a chromosomal allele onto a plasmid. Since the resolved plasmid can be maintained by selection, this technique can be used to generate deletions of essential genes.

UR - http://www.scopus.com/inward/record.url?scp=0024340114&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024340114&partnerID=8YFLogxK

U2 - 10.1128/jb.171.9.4617-4622.1989

DO - 10.1128/jb.171.9.4617-4622.1989

M3 - Article

VL - 171

SP - 4617

EP - 4622

JO - Journal of Bacteriology

JF - Journal of Bacteriology

SN - 0021-9193

IS - 9

ER -