Substrate and Target Sequence Length Influence RecTEPsy 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...

Full description

Saved in:
Bibliographic Details
Published inPloS one Vol. 7; no. 11; p. e50617
Main Authors Bao, Zhongmeng, Cartinhour, Sam, Swingle, Bryan
Format Journal Article
LanguageEnglish
Published San Francisco Public Library of Science 30.11.2012
Subjects
Agriculture
Annealing
Bacteria
Biology
Cloning
Deoxyribonucleic acid
DNA
E coli
Efficiency
Engineering
Escherichia coli
Experiments
Genes
Genomes
Homology
Molecular biology
Mutagenesis
Pathogens
Plant pathology
Plasmids
Proteins
Pseudomonas
Pseudomonas syringae
Recombination
Substrates
New York
United States > US
Online AccessGet full text

Cover

More Information
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 RecTEPsy mediated recombination efficiency. These results provide information about the design elements that should be considered when using recombineering.
Bibliography:Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: BS ZB SC. Performed the experiments: ZB. Analyzed the data: BS ZB SC. Contributed reagents/materials/analysis tools: BS ZB. Wrote the paper: BS ZB SC.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0050617