Figure 1

Design of the Recombinase-Mediated Cassette Exchange. (A) The scheme of RMCE between the recipient plasmid (pRC59) and the donor plasmid (pRC60). pRC59 contains a floxed cassette, consisting of the truncated 3′URA3 gene and the yeast LEU2 marker; and pRC60 contains the 5′URA3 gene, a floxed yeast MET14 ORF, and the Cre recombinase gene under the GAL1 inducible promoter. The gray color indicates the actin intron. The purple bars represent 34 bp hetero-specific loxP mutants where cassette exchange takes place, marked by broken arrows. The cassette exchange was performed by growing the yeast harboring two plasmids in medium containing galactose for 24 hours, followed by the selection of uracil prototrophs on SD-Uracil plates. The cassette exchange would produce two plasmids, pRC59S and pRC60S. The exchange event was evaluated by PCR using primers (swap-F and swap-R) indicated by red arrows. pRC59S allows the amplification of a 1.1 kb product, in contrast to the 3.6 kb product amplified from the parental pRC59. (B) PCR screening for cassette exchange. Cassette exchange was performed in two yeast strains, W303a and VL6-48. Fifteen colonies from each strain were analyzed by PCR. Lanes 1 to 15: W303a strain; and lanes 16 to 30: VL6-48 strain; M: DNA marker.