RRC ID |
34913
|
著者 |
Giuraniuc CV, MacPherson M, Saka Y.
|
タイトル |
Gateway vectors for efficient artificial gene assembly in vitro and expression in yeast Saccharomyces cerevisiae.
|
ジャーナル |
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.
|
巻・号 |
8(5)
|
ページ |
e64419
|
公開日 |
2013-1-1
|
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 |
2.74
|
引用数 |
10
|
WOS 分野
|
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
リソース情報 |
酵母 |
BYP6848(pNHK12)
BYP6743(pNHK36)
BYP6744(pNHK53) |