Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites.
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.
Bolusani
Swetha
S
School of Biological Sciences/IfM, 911 Hergot Avenue, Louisiana Tech University, Ruston, LA 71272, USA.
Ma
Chien-Hui
CH
Paek
Andrew
A
Konieczka
Jay H
JH
Jayaram
Makkuni
M
Voziyanov
Yuri
Y
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D023281:Genomics; D006801:Humans; D016753:Interleukin-10 / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D015003:Yeasts / Q000235:genetics
C508609:IL10 protein, human; D016753:Interleukin-10; D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase