RRC ID 34913
Author Giuraniuc CV, MacPherson M, Saka Y.
Title Gateway vectors for efficient artificial gene assembly in vitro and expression in yeast Saccharomyces cerevisiae.
Journal PLoS ONE
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.
Volume 8(5)
Pages e64419
Published 2013
DOI 10.1371/journal.pone.0064419
PII PONE-D-13-07010
PMID 23675537
PMC PMC3651225
MeSH Cloning, Molecular / methods* Gene Expression Gene Expression Regulation, Fungal / drug effects Gene Order Genes, Reporter Genetic Vectors / genetics* Indoleacetic Acids / pharmacology Promoter Regions, Genetic Saccharomyces cerevisiae / genetics* Saccharomyces cerevisiae / metabolism Trans-Activators / genetics Trans-Activators / metabolism
IF 2.776
Times Cited 7
Yeast BYP6848(pNHK12) BYP6743(pNHK36) BYP6744(pNHK53)