Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Yeast. 2012 Aug;29(8):323-34. doi: 10.1002/yea.2912. Epub 2012 Jul 17.

Optimization of ordered plasmid assembly by gap repair in Saccharomyces cerevisiae.

Author information

  • 1Department of Biology, University of Copenhagen, Ole Maaloees Vej 5, DK-2200, Copenhagen N, Denmark.

Abstract

Combinatorial genetic libraries are powerful tools for diversifying and optimizing biomolecules. The process of library assembly is a major limiting factor for library complexity and quality. Gap repair by homologous recombination in Saccharomyces cerevisiae can facilitate in vivo assembly of DNA fragments sharing short patches of sequence homology, thereby supporting generation of high-complexity libraries without compromising fidelity. In this study, we have optimized the ordered assembly of three DNA fragments into a gapped vector by in vivo homologous recombination. Assembly is achieved by co-transformation of the DNA fragments and the gapped vector, using a modified lithium acetate protocol. The optimal gap-repair efficiency is found at a 1:80 molar ratio of gapped vector to each of the three fragments. We measured gap-repair efficiency in different genetic backgrounds and observed increased efficiency in mutants carrying a deletion of the SGS1 helicase-encoding gene. Using our experimental conditions, a gap-repair efficiency of > 10(6) plasmid-harbouring colonies/µg gapped vector DNA is obtained in a single transformation, with a recombination fidelity > 90%.

Copyright © 2012 John Wiley & Sons, Ltd.

PMID:
22806834
[PubMed - indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for John Wiley & Sons, Inc.
    Loading ...
    Write to the Help Desk