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Applied and Environmental Microbiology, December 2004, p. 7156-7160, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7156-7160.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Restoration of Gene Function by Homologous Recombination: from PCR to Gene Expression in One Step

Ido Yosef,1,{dagger} Noga Bloushtain,1 Michal Shapira,2 and Udi Qimron1*

Department of Microbiology and Immunology, Faculty of Health Sciences,1 Department of Life Sciences, Faculty of Natural Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel2

Received 15 June 2004/ Accepted 31 July 2004

We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete {lambda}PR or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage {lambda} Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding ß-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer-Sheva 84105, Israel. Phone: 972 8 6477282. Fax: 972 8 6477626. E-mail: ehudq{at}bgu.ac.il.

{dagger} Present address: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

Applied and Environmental Microbiology, December 2004, p. 7156-7160, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7156-7160.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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