Integrative modules for efficient genome engineering in yeast

Authors:

Triana Amen1 and Daniel Kaganovich1

doi: 10.15698/mic2017.06.576
Volume 4, pp. 182 to 190, published 05/06/2017.

PDF Download PubMed Link Supplemental Information
Affiliations:

1 Department of Cell and Developmental Biology, Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel.

Keywords: 

vector, bidirectional promoter, integrative plasmid, genetic integration, yeast, Saccharomyces cerevisiae.

Corresponding Author(s):

Daniel Kaganovich, dan@cc.huji.ac.il

Conflict of interest statement:

The authors declare no conflicts of interest.

Please cite this article as:

Triana Amen and Daniel Kaganovich (2017). Integrative modules for efficient genome engineering in yeast. Microbial Cell 4(6):182-190. doi: 10.15698/mic2017.06.576

© 2017 Amen and Kaganovich. This is an open-access article released under the terms of the Creative Commons Attribution (CC BY) license, which allows the unrestricted use, distribution, and reproduction in any medium, provided the original author and source are acknowledged.

Abstract:

We present a set of vectors containing integrative modules for efficient genome integration into the commonly used selection marker loci of the yeast Saccharomyces cerevisiae. A fragment for genome integration is generated via PCR with a unique set of short primers and integrated into HIS3, URA3, ADE2, and TRP1 loci. The desired level of expression can be achieved by using constitutive (TEF1p, GPD1p), inducible (CUP1p, GAL1/10p), and daughter-specific (DSE4p) promoters available in the modules. The reduced size of the integrative module compared to conventional integrative plasmids allows efficient integration of multiple fragments. We demonstrate the efficiency of this tool by simultaneously tagging markers of the nucleus, vacuole, actin, and peroxisomes with genomically integrated fluorophores. Improved integration of our new pDK plasmid series allows stable introduction of several genes and can be used for multi-color imaging. New bidirectional promoters (TEF1p-GPD1p, TEF1p-CUP1p, and TEF1p-DSE4p) allow tractable metabolic engineering.