Nucleic Acids Research Advance Access originally published online on September 26, 2006
Nucleic Acids Research 2006 34(18):5259-5269; doi:10.1093/nar/gkl548
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Nucleic Acids Research, 2006, Vol. 34, No. 18 5259-5269
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites
School of Biological Sciences/IfM 911 Hergot Avenue Louisiana Tech University Ruston, LA 71272, USA 1 Molecular Genetics and Microbiology, University of Texas Austin 1 University Station A5000 Austin, TX 78712-0162, USA 2 University of Arizona Life Sciences North 458, 1501 N. Campbell Avenue, Tucson, AZ 85724, USA
*To whom correspondence should be addressed. Tel: +1 318 257 5141; Fax: +1 318 257 5104; Email: voziyan{at}latech.edu
Received April 21, 2006. Revised June 27, 2006. Accepted July 13, 2006.
As a tool in directed genome manipulations, site-specific recombination is a double-edged sword. Exquisite specificity, while highly desirable, makes it imperative that the target site be first inserted at the desired genomic locale before it can be manipulated. We describe a combination of computational and experimental strategies, based on the tyrosine recombinase Flp and its target site FRT, to overcome this impediment. We document the systematic evolution of Flp variants that can utilize, in a bacterial assay, two sites from the human interleukin 10 gene, IL10, as recombination substrates. Recombination competence on an end target site is acquired via chimeric sites containing mixed sequences from FRT and the genomic locus. This is the first time that a tyrosine site-specific recombinase has been coaxed successfully to perform DNA exchange within naturally occurring sequences derived from a foreign genomic context. We demonstrate the ability of an Flp variant to mediate integration of a reporter cassette in Escherichia coli via recombination at one of the IL10-derived sites.
*Correspondence may also be addressed to Makkuni Jayaram. Tel: +1 512 471 5546; Fax: +1 512 471 5537; Email: jayaram{at}icmb.utexas.edu