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Journal of Bacteriology, October 2008, p. 6392-6397, Vol. 190, No. 19
0021-9193/08/$08.00+0     doi:10.1128/JB.00777-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Highly Efficient In Vitro Site-Specific Recombination System Based on Streptomyces Phage {phi}BT1 Integrase{triangledown} ,{dagger}

Lin Zhang,1 Xijun Ou,1 Guoping Zhao,1,2,3* and Xiaoming Ding1*

State Key Laboratory of Genetic Engineering, Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China,1 Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China,2 Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, Shanghai 201203, China3

Received 3 June 2008/ Accepted 28 July 2008

The Streptomyces phage {phi}BT1 encodes a site-specific integrase of the large serine recombinase subfamily. In this report, the enzymatic activity of the {phi}BT1 integrase was characterized in vitro. We showed that this integrase has efficient integration activity with substrate DNAs containing attB and attP sites, independent of DNA supercoiling or cofactors. Both intra- and intermolecular recombinations proceed with rapid kinetics. The recombination is highly specific, and no reactions are observed between pairs of sites including attB and attL, attB and attR, attP and attL, or attP and attR or between two identical att sequences; however, a low but significant frequency of excision recombination between attL and attR is observed in the presence of the {phi}BT1 integrase alone. In addition, for efficient integration, the minimal sizes of attB and attP are 36 bp and 48 bp, respectively. This site-specific recombination system is efficient and simple to use; thus, it could have applications for the manipulation of DNA in vitro.

* Corresponding author. Mailing address: Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China. Phone for Xiaoming Ding: 86 21 65643616. Fax: 86 21 65650149. E-mail: xmding74{at}fudan.edu.cn. Phone for Guoping Zhao: 86 21 50801919. Fax: 86 21 50801922. E-mail: gpzhao{at}sibs.ac.cn

{triangledown} Published ahead of print on 8 August 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, October 2008, p. 6392-6397, Vol. 190, No. 19
0021-9193/08/$08.00+0     doi:10.1128/JB.00777-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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