Gateway vectors for efficient artificial gene assembly in vitro and expression in yeast Saccharomyces cerevisiae

TL;DR

This paper introduces Gateway vectors for rapid in vitro assembly and yeast expression of synthetic genes, including engineered transactivators, streamlining synthetic biology workflows in Saccharomyces cerevisiae.

Contribution

Development of versatile Gateway vectors enabling one-step in vitro gene assembly and expression testing in yeast, including novel regulated transactivator constructs.

Findings

Rapid gene assembly from promoter and ORF cassettes

Engineered transactivators can be regulated by doxycycline and auxin

Vectors facilitate synthetic gene construction in yeast

Abstract

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.