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United States Department of Agriculture

Agricultural Research Service

Research Project: PSEUDOMONAS SYSTEMS BIOLOGY

Location: Plant-Microbe Interactions Research

Title: Substrate and target sequence lengths influence RecTEPsy recombineering efficiency in Pseudomonas syringae

Authors
item Swingle, Bryan
item Bao, Zhongmeng -
item Cartinhour, Samuel

Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 23, 2012
Publication Date: November 30, 2012
Citation: Swingle, B.M., Bao, Z., Cartinhour, S.W. 2012. Substrate and target sequence lengths influence RecTEPsy recombineering efficiency in Pseudomonas syringae. PLoS One. 7(11):e50617. DOI: 10.1371/journal.pone.0050617.

Interpretive Summary: We have developed a technology that aids experimental investigation of Pseudomonas syringae, a plant pathogenic bacterium. This technology can be used to change specific DNA sequences in bacteria, allowing scientists to efficiently remove and add functions to the bacteria. The altered bacteria can then be tested to determine whether that function is important for an aspect of the bacteria’s lifestyle, such as ability to cause disease on plants. This research tool fundamentally changes the way bacteria are studied and greatly expands the range of options available to scientists working with Pseudomonas syringae. In this report, we studied the details of the DNA template that is used to guide the change in the bacteria’s DNA. We determined that the amount of DNA used and the length of the DNA directing the change are very important to the success of the procedure.

Technical 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 have previously identified orthologs of the lambda Red bet/exo and Rac RecTE genes in P. syringae B728a and confirmed that they function in recombineering using ssDNA and dsDNA substrates. Here we investigate the characteristics of dsDNA substrates to determine the features of these substrates that influence recombineering efficiency. We found that the length of flanking homologies and length of the sequences being inserted or deleted have a large effect on RecTEPsy recombination. These results provide information about the design elements that must be considered when using recombineering and suggest that the relationship between the substrate and the region being targeted is sensitive to variations in length.

Last Modified: 9/21/2014
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