  Construction of synthetic genetic networks requires the assembly of DNA
fragments encoding functional biological parts in a defined order. Yet this may
become a time-consuming procedure. To address this technical bottleneck, we
have created a series of Gateway shuttle vectors and an integration vector,
which facilitate the assembly of artificial genes and their expression in the
budding yeast Saccharomyces cerevisiae. Our method enables the rapid
construction of an artificial gene from a promoter and an open reading frame
(ORF) cassette by one-step recombination reaction in vitro. Furthermore, the
plasmid thus created can readily be introduced into yeast cells to test the
assembled gene's functionality. As flexible regulatory components of a
synthetic genetic network, we also created new versions of the
tetracycline-regulated transactivators tTA and rtTA by fusing them to the
auxin-inducible degron (AID). Using our gene assembly approach, we made yeast
expression vectors of these engineered transactivators, AIDtTA and AIDrtTA and
then tested their functions in yeast. We showed that these factors can be
regulated by doxycycline and degraded rapidly after addition of auxin to the
medium. Taken together, the method for combinatorial gene assembly described
here is versatile and would be a valuable tool for yeast synthetic biology.
