title: New strategy to improve efficiency for gene replacement in Klebsiella pneumoniae.
abstract: We previously reported the method for introducing gene replacement into Klebsiella pneumoniae through Red-assisted homologous recombination; and it demonstrated that a higher transformation efficiency required long flanking arms at both ends of the linear DNA. The assembly job of the linear DNA is usually time-consuming and laborious. We report here an innovative method for DNA exchange in K. pneumoniae based on PCR-mediated Red recombination. The novel procedure enables rapid gene replacement in K. pneumoniae without prior cloning of the gene of interest; the key modification is to perform PCR reaction to generate linear DNA with extra non-homologous fragments on both ends as mercenary sequences which come from a TA-cloning plasmid. We give a demonstration by deleting the gene dhak1 in K. pneumoniae with high efficiency of about 20 CFU/μg DNA using the new technique.
authors.affiliation: Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong, Shanghai 201210, People's Republic of China.
authors.forename: Dong
authors.initials: D
authors.lastname: Wei
authors.forename: Junsong
authors.initials: J
authors.lastname: Sun
authors.forename: Jiping
authors.initials: J
authors.lastname: Shi
authors.forename: Pengfu
authors.initials: P
authors.lastname: Liu
authors.forename: Jian
authors.initials: J
authors.lastname: Hao
mesh_terms: D004247:DNA / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D059765:Homologous Recombination / Q000235:genetics; D007711:Klebsiella pneumoniae / Q000235:genetics*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic
chemical_list: D004247:DNA