|BAO, ZHONGMENG - Cornell University - New York|
Submitted to: Molecular Genetics of Bacteria and Phage
Publication Type: Abstract Only
Publication Acceptance Date: 6/1/2010
Publication Date: 8/24/2010
Citation: Swingle, B.M., Bao, Z., Markel, E.J., Cartinhour, S.W. 2010. Recombineering using RecET from Pseudomonas syringae. Molecular Genetics of Bacteria and Phage. 61/34.
Technical Abstract: Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecET proteins encoded by E. coli bacteriophage. The ability of the pseudomonad encoded proteins to promote recombination was tested in P. syringae pv. tomato DC3000 using a quantitative assay based on recombination frequency. The results show that the Pseudomonas RecT homolog is sufficient to promote recombination of single stranded (ss)DNA oligos and that efficient recombination of double stranded (ds)DNA requires the expression of both the RecT and RecE homologs. Additionally we illustrate the utility of this recombineering system to make targeted gene disruptions in the P. syringae chromosome.