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Applied and Environmental Microbiology, July 2007, p. 4286-4293, Vol. 73, No. 13
0099-2240/07/$08.00+0     doi:10.1128/AEM.00119-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

A Rapid and Efficient Method for Cloning Genes of Type II Restriction-Modification Systems by Use of a Killer Plasmid{triangledown}

Iwona Mruk and Tadeusz Kaczorowski*

Department of Microbiology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland

Received 17 January 2007/ Accepted 18 April 2007

We present a method for cloning restriction-modification (R-M) systems that is based on the use of a lethal plasmid (pKILLER). The plasmid carries a functional gene for a restriction endonuclease having the same DNA specificity as the R-M system of interest. The first step is the standard preparation of a representative, plasmid-borne genomic library. Then this library is transformed with the killer plasmid. The only surviving bacteria are those which carry the gene specifying a protective DNA methyltransferase. Conceptually, this in vivo selection approach resembles earlier methods in which a plasmid library was selected in vitro by digestion with a suitable restriction endonuclease, but it is much more efficient than those methods. The new method was successfully used to clone two R-M systems, BstZ1II from Bacillus stearothermophilus 14P and Csp231I from Citrobacter sp. strain RFL231, both isospecific to the prototype HindIII R-M system.

* Corresponding author. Mailing address: Department of Microbiology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland. Phone: (48-58) 305-6242. Fax: (48-58) 320-2031. E-mail: kaczorow{at}biotech.univ.gda.pl

{triangledown} Published ahead of print on 27 April 2007.

Applied and Environmental Microbiology, July 2007, p. 4286-4293, Vol. 73, No. 13
0099-2240/07/$08.00+0     doi:10.1128/AEM.00119-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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