Substrate and target sequence length influence RecTE(Psy) recombineering efficiency in Pseudomonas syringae

We are developing a new recombineering system to assist experimental manipulation of the Pseudomonas syringae genome. P. syringae is a globally dispersed plant pathogen and an important model species used to study the molecular biology of bacteria-plant interactions. We previously identified ortholo...

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Bibliographic Details
Published inPloS one Vol. 7; no. 11; p. e50617
Main Authors Bao, Zhongmeng, Cartinhour, Sam, Swingle, Bryan
Format Journal Article
LanguageEnglish
Published United States Public Library of Science (PLoS) 2012
Subjects
DNA, Single-Stranded - genetics
DNA, Single-Stranded - metabolism
Genes, Bacterial - genetics
Genetic Engineering - methods
Pseudomonas syringae - genetics
Recombination, Genetic
Sequence Homology, Nucleic Acid
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Summary:We are developing a new recombineering system to assist experimental manipulation of the Pseudomonas syringae genome. P. syringae is a globally dispersed plant pathogen and an important model species used to study the molecular biology of bacteria-plant interactions. We previously identified orthologs of the lambda Red bet/exo and Rac recET genes in P. syringae and confirmed that they function in recombineering using ssDNA and dsDNA substrates. Here we investigate the properties of dsDNA substrates more closely to determine how they influence recombineering efficiency. We find that the length of flanking homologies and length of the sequences being inserted or deleted have a large effect on RecTE(Psy) mediated recombination efficiency. These results provide information about the design elements that should be considered when using recombineering.
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ISSN:1932-6203
DOI:10.1371/journal.pone.0050617