Establishment of gene replacement/disruption system through homologous recombination in Amycolatopsis mediterranei U32.
- Author:
Xiao-Ming DING
1
;
Ni ZHANG
;
Yong-Qiang TIAN
;
Wei-Hong JIANG
;
Guo-Ping ZHAO
;
Rui-Sheng JIAO
Author Information
1. Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
- Publication Type:Journal Article
- MeSH:
Actinomycetales;
drug effects;
genetics;
growth & development;
DNA, Bacterial;
genetics;
Drug Resistance, Microbial;
genetics;
Genes, Bacterial;
genetics;
Mutagenesis;
Nebramycin;
analogs & derivatives;
pharmacology;
Plasmids;
genetics;
Recombination, Genetic
- From:
Chinese Journal of Biotechnology
2002;18(4):431-437
- CountryChina
- Language:Chinese
-
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.
