Establishment of gene replacement/disruption system through homologous recombination in Amycolatopsis mediterranei U32 / 生物工程学报
Chinese Journal of Biotechnology
; (12): 431-437, 2002.
Artigo
em Chinês
| WPRIM (Pacífico Ocidental)
| ID: wpr-256190
Biblioteca responsável:
WPRO
ABSTRACT
A gene replacement/disruption system of Amycolatopsis mediterranei U32 was developed based on the established electroporation conditions as well as appropriate selective markers. Through two-step selection, ahbas gene in U32 was replaced by a promoterless alpha-amylase gene constructed on the plasmid pDK110 of E. coli. The first single-crossover and the second double-crossover frequencies were approximately 0.5%-0.7% and 2%, respectively. Denaturation of the plasmid pDK110 increased the integration frequency about 7-10 folds, while electric shock treatment of the single-crossover recombinants increased the frequency of second crossover recombination about 5 folds. Employing denatured DNA fragments containing an apramycin-resistance gene flanked with regions of the respective genes, One-step disruption of rifO and amrA genes of U32 was also achieved with an efficiency of 30-50 transformants per microgram of DNA.
Texto completo:
Disponível
Base de dados:
WPRIM (Pacífico Ocidental)
Assunto principal:
Farmacologia
/
Plasmídeos
/
Recombinação Genética
/
Actinomycetales
/
DNA Bacteriano
/
Resistência Microbiana a Medicamentos
/
Mutagênese
/
Genes Bacterianos
/
Genética
/
Nebramicina
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2002
Tipo de documento:
Artigo