Novel method for genomic promoter shuffling by using recyclable cassettes.

Genetic elements of interest can be introduced into the Saccharomyces cerevisiae genome via homologous recombination. The current method is to link such an element to a selectable marker gene to be integrated into the target locus. However, the marker gene in this method cannot be reused, which limits repeated manipulation of the yeast genome. An alternative method is to utilize a counterselectable gene, such as URA3, with flanking tandem repeats. After integration, URA3 along with one copy of the repeat can be popped out via internal recombination, leaving behind one copy of the unwanted repeat. Here we describe a novel concept of genetic element shuffling in which the tandem repeats are made of the desired genetic element, so that after integration and popping out, only one copy of the element remains at the desired locus to function. As a proof of principle, we constructed three recyclable cassettes (PPGK1-URA3-PPGK1, PGAL1-URA3-PGAL1, and PtetO7-URA3-PtetO7) and integrated them upstream of an engineered chromosomal PHIS3-mCherry-Myc locus. After promoter shuffling, the mCherry-Myc gene was regulated precisely as anticipated.