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Team:BIOTEC Dresden/Modeling v2
From 2009.igem.org
In genetics, FLP-FRT recombination is a site-directed recombination technology used to manipulate an organism's DNA under controlled conditions in vivo. It is analogous to Cre-Lox recombination. It involves the recombination of sequences between short Flippase Recognition Target (FRT) sites by the Flippase recombination enzyme (FLP or Flp) derived from the 2µ plasmid of the baker's yeast Saccharomyces cerevisiae. The 34bp long FRT site sequence is : 5'-GAAGTTCCTATTCtctagaaaGTATAGGAACTTC-3'. Flippase (flp) binds to the 13-bp 5'-GAAGTTCCTATTC-3' and to the reverse complement of 5'-GTATAGGAACTTC-3' (5'-GAAGTTCCTATAC-3'). The FRT site is cleaved just before 5'-tctagaaa-3', the 8bp asymmetric core region, on the top strand and behind this sequence on the bottom strand.[1] Several variant FRT sites exist. Recombination can occur between two identical FRT sites but generally not between non-identical FRT sites Many available constructs include the sequence 5'-GAAGTTCCTATTCC-3' immediately upstream the FRT site (resulting in 5'-GAAGTTCCTATTCCGAAGTTCCTATTCtctagaaaGTATAGGAACTTC-3') but this sequence is dispensable for recombination. Because the recombination activity can be targeted to only one target organ, or a low level of recombination activity can be used to consistently alter the DNA of only a subset of cells, FLP-FRT can be used to construct genetic mosaics in multicellular organisms. Using this technology, the loss or alteration of a gene can be studied in one target organ of interest, even if experimental animals could not survive the loss of the gene in other organs. The effect of altering a gene can also be studied over time, by using an inducible promoter to trigger the recombination activity late in development - this prevents the alteration from affecting overall development of an organ, and allows single cells lacking the gene to be compared to normal neighboring cells in the same environment.
A very similar study using eukaryotic DNA:
http://www.ncbi.nlm.nih.gov/pubmed/10581237
kinetic analysis of Flp activity - DNA binding and recombination models: http://www.ncbi.nlm.nih.gov/pubmed/9813124
Thermostability of Flp recombinase (We are using the F70L variant because it is sufficiently slow to give a time course): http://www.ncbi.nlm.nih.gov/pubmed/8932381
