AL Goldstein

AS Khalil

B Canton

B Deplancke

BA Blount

C Janke

C Mateus

C Tagwerker

CC Guet

CJ Murakami

Claudiu V. Giuraniuc

DG Gibson

E Garí

G Bellí

G Rodrigo

GL Gallia

I Sadowski

J Hasty

JJ Lohmueller

JS Dymond

JS Reece-Hoyes

K Nishimura

Kevin G. Hardwick

M Gossen

MA Sheff

Murray MacPherson

RS Sikorski

S Ausl?ander

S Regot

T Ellis

T Nagai

VV Kushnirov

Y Sasaki

Y Suzuki

Yasushi Saka

PLoS ONE

English

arXiv

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

Claudiu V Giuraniuc

Directory of Open Access Journals

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

Giuraniuc, Claudiu V

MacPherson, Murray Iain

Saka, Yasushi

The Open University

A gateway-compatible yeast one-hybrid system.

A novel highthroughput (htp) cloning strategy for site-directed designed chimeragenesis and mutation using the gateway cloning system.

A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in saccharomyces cerevisiae.

A synthetic biology framework for programming eukaryotic transcription functions.

A tunable zinc finger-based framework for Boolean logic computation in mammalian cells.

A variant of yellow uorescent protein with fast and efficient maturation for cell-biological applications.

A versatile toolbox for pcrbased tagging of yeast genes: new uorescent proteins, more markers and promoter substitution cassettes.

An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast.

An auxin-based degron system for the rapid depletion of proteins in nonplant cells.

Combinatorial synthesis of genetic networks.

Complete chemical synthesis, assembly, and cloning of a mycoplasma genitalium genome.

Composition and acidification of the culture medium inuences chronological aging similarly in vineyard and laboratory yeast.

Destabilized green uorescent protein for monitoring dynamic changes in yeast gene expression with ow cytometry.

Disintegrator vectors for single-copy yeast chromosomal integration.

Distributed biological computation with multicellular engineered networks.

Diversity-based, model-guided construction of synthetic gene networks with predicted functions.

Engineered gene circuits.

Enhanced yeast one-hybrid assays for high-throughput gene-centered regulatory network mapping.

Evaluation of an autoregulatory tetracycline regulated system.

Evidence for high specificity and efficiency of multiple recombination signals in mixed dna cloning by the multisite gateway system.

Methods in yeast genetics:

Optimized cassettes for uorescent protein tagging in saccharomyces cerevisiae.

Programmable single-cell mammalian biocomputers.

Rapid and reliable protein extraction from yeast.

Rational diversification of a promoter providing fine-tuned expression and orthogonal regulation for synthetic biology.

Refinement and standardization of synthetic biological parts and devices.

Sequence analysis of a complete 1.66 mb prochlorococcus marinus med4 genome cloned in yeast.

Synthetic chromosome arms function in yeast and generate phenotypic diversity by design.

The elg1 replication factor c-like complex functions in pcna unloading during dna replication. Mol Cell.

Three new dominant drug resistance cassettes for gene disruption in saccharomyces cerevisiae.

Transcriptional activation by tetracyclines in mammalian cells.

Yeast one-hybrid assays for gene-centered human gene regulatory network mapping.

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

Crossref

Gateway Vectors for Efficient Artificial Gene Assembly In Vitro and Expression in Yeast Saccharomyces cerevisiae

http://dx.doi.org/10.1371/journal.pone.0064419

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

Abstract

Similar works

Full text

Available Versions

Directory of Open Access Journals

The Open University

Crossref