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- Title
Transformation of Saccharopolyspora erythraea by electroporation of germinating spores: construction of propionyl Co-A carboxylase mutants.
- Authors
English, R S; Lampel, J S; Boom, TJ Vanden
- Abstract
The introduction of plasmid DNA into germinating spores of an industrially improved strain of Saccharopolyspora erythraea was accomplished by electroporation. Various parameters affecting the efficiency of electroporation were examined. The most critical factor was the extent of spore germination. Electrocompetence was limited to a 4-h period following the initial emergence of the germ tube. Electroporation efficiencies as high as 2 × 10[sup 5] CFU μg[sup -1] of plasmid DNA were obtained using electrocompetent germlings. The optimal field strength was 12-14 kV cm[sup -1] with a pulse duration of 15-20 ms. Electrocompetent germlings were stored at -80°C without a significant decrease in transformation efficiency. The utility of this protocol was demonstrated by isolating a propionyI-CoA carboxylase mutant through targeted gene disruption and replacement.
- Subjects
PLASMIDS; ELECTROPORATION; PLANT spores; GERMINATION
- Publication
Journal of Industrial Microbiology & Biotechnology, 1998, Vol 21, Issue 4/5, p219
- ISSN
1367-5435
- Publication type
Article
- DOI
10.1038/sj.jim.2900576