A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA.
An improved method for the electrotransformation of wild-type Corynebacterium glutamicum (ATCC 13032) is described. The two crucial alterations to previously developed methods are: cultivation of cells used for electrotransformation at 18 degrees C instead of 30 degrees C, and application of a heat shock immediately following electrotransformation. Cells cultivated at sub optimal temperature have a 100-fold improved transformation efficiency (10(8) cfu micrograms-1) for syngeneic DNA (DNA isolated from the same species). A heat shock applied to these cells following electroporation improved the transformation efficiency for xenogeneic DNA (DNA isolated from a different species). In combination, low cultivation temperature and heat shock act synergistically and increased the transformation efficiency by four orders of magnitude to 2.5 x 10(6) cfu micrograms-1 xenogeneic DNA. The method was used to generate gene disruptions in C. glutamicum.
van der Rest
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ME
Heinrich-Heine-Universität, Düsseldorf, Germany.
Lange
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C
Molenaar
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D
D003352:Corynebacterium / Q000235:genetics* / Q000254:growth & development; D004269:DNA, Bacterial / Q000235:genetics; D018274:Electroporation; D006358:Hot Temperature; D010957:Plasmids / Q000235:genetics; D013696:Temperature; D013997:Time Factors; D014169:Transformation, Bacterial
D004269:DNA, Bacterial