[Establishment of gene replacement/disruption system through homologous recombination in Amycolatopsis mediterranei U32].
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.
Ding
Xiao-Ming
XM
Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Zhang
Ni
N
Tian
Yong-Qiang
YQ
Jiang
Wei-Hong
WH
Zhao
Guo-Ping
GP
Jiao
Rui-Sheng
RS
D000192:Actinomycetales / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D004269:DNA, Bacterial / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D016296:Mutagenesis; D009328:Nebramycin / Q000031:analogs & derivatives* / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D009328:Nebramycin; C011666:apramycin