Adaptation of the yeast URA3 selection system to gram-negative bacteria and generation of a {delta}betCDE Pseudomonas putida strain.

ABSTRACT: A general procedure for efficient generation of gene knockouts in gram-negative bacteria by the adaptation of the Saccharomyces cerevisiae URA3 selection system is described. A Pseudomonas putida strain lacking the URA3 homolog pyrF (encoding orotidine-5'-phosphate decarboxylase) was constructed, allowing the use of a plasmid-borne copy of the gene as the target of selection. The delivery vector pTEC contains the pyrF gene and promoter, a conditional origin of replication (oriR6K), an origin of transfer (mobRK2), and an antibiotic selection marker flanked by multiple sites for cloning appropriate DNA segments. The versatility of pyrF as a selection system, allowing both positive and negative selection of the marker, and the robustness of the selection, where pyrF is associated with uracil

SUBMITTER: Galvao TC 

PROVIDER: S-EPMC546819 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature