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.
Article
in Chinese
| WPRIM
| ID: wpr-256190
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Pharmacology
/
Plasmids
/
Recombination, Genetic
/
Actinomycetales
/
DNA, Bacterial
/
Drug Resistance, Microbial
/
Mutagenesis
/
Genes, Bacterial
/
Genetics
/
Nebramycin
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2002
Type:
Article
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