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

PLoS One. 2012;7(11):e50617. doi: 10.1371/journal.pone.0050617. Epub 2012 Nov 30.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism
  • Genes, Bacterial / genetics*
  • Genetic Engineering / methods*
  • Pseudomonas syringae / genetics*
  • Recombination, Genetic*
  • Sequence Homology, Nucleic Acid

Substances

  • DNA, Single-Stranded

Grants and funding

This research was supported by the U. S. Department of Agriculture, Agricultural Research Service. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.