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A low copy number cosmid.

X M Jiang1, H N Brahmbhatt, N B Quigley

  • 1Department of Microbiology, University of Sydney, New South Wales, Australia.

Plasmid
|September 1, 1987
PubMed
Summary
This summary is machine-generated.

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A novel low-copy-number cosmid, pPR691, was developed for constructing genomic libraries. This cosmid enabled the successful isolation of the Salmonella typhimurium rfb gene cluster.

Area of Science:

  • Molecular Biology
  • Microbial Genetics
  • Recombinant DNA Technology

Background:

  • Cosmids are essential vectors for constructing genomic libraries due to their large insert capacity.
  • Efficient cloning strategies are crucial for isolating specific genetic loci from complex genomes.

Purpose of the Study:

  • To construct a low-copy-number cosmid vector suitable for creating genomic libraries.
  • To isolate the rfb gene cluster from Salmonella typhimurium using the developed cosmid.

Main Methods:

  • Subcloning of cos sites and a kanamycin resistance gene into a pGB2 vector to create the pPR691 cosmid.
  • Construction of a Salmonella typhimurium chromosomal DNA library using pPR691.
  • Isolation of the rfb gene cluster via screening of the cosmid library.

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Main Results:

  • The novel cosmid pPR691, based on the pSC101 replicon, confers resistance to kanamycin, spectinomycin, and streptomycin.
  • pPR691 incorporates restriction sites compatible with the Bates and Swift cloning strategy.
  • The rfb gene cluster from Salmonella typhimurium was successfully isolated from the library constructed with pPR691.

Conclusions:

  • The pPR691 cosmid is an effective tool for constructing genomic libraries.
  • This method facilitates the isolation of specific gene clusters, such as the Salmonella typhimurium rfb locus.