New strategy to improve efficiency for gene replacement in Klebsiella pneumoniae.
J Ind Microbiol Biotechnol
; 40(5): 523-7, 2013 May.
Article
em En
| MEDLINE
| ID: mdl-23478882
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Marcação de Genes
/
Klebsiella pneumoniae
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article