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Optimization of ordered plasmid assembly by gap repair in Saccharomyces cerevisiae
Author(s) -
EckertBoulet Nadine,
Pedersen Mette Louise,
Krogh Berit Olsen,
Lisby Michael
Publication year - 2012
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.2912
Subject(s) - homologous recombination , biology , shuttle vector , saccharomyces cerevisiae , plasmid , dna , genetics , transformation (genetics) , in vitro recombination , helicase , computational biology , homology directed repair , flp frt recombination , recombination , genetic recombination , dna repair , recombinant dna , gene , vector (molecular biology) , nucleotide excision repair , molecular cloning , peptide sequence , rna
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.