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

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Claudiu V. Giuraniuc, Yasushi Saka

United Kingdom

Synthetic biology is rapidly expanding thanks to the rational design and construction of artificial gene regulatory networks. It allows for the analysis of isolated networks in simple model organisms including the budding yeast Saccharomyces cerevisiae while eliminating variables such as unknown endogenous interactions of the network components. However these gene networks require the assembly of DNA fragments encoding for functional biological parts in a defined order and can prove time costly. To address this time-constraint we have created a series of Gateway vectors that facilitates the construction of an artificial gene from a promoter and open reading frame cassette by one-step recombination reaction in vitro. Depending on the vector backbone used the resulting expression vector can then be introduced as a plasmid into S. cerevisiae or integrated into the genome. This approach allows rapid assembly of the synthetic gene and testing of its function in yeast. 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 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 allowing for a finer degree of temporal control. Taken together, the method for combinatorial gene assembly described here is versatile and would be a valuable tool for yeast synthetic biology.