Transformation of Corynebacterium pseudotuberculosis by electroporation

Songer, J.G.; Hilwig, R.W.; Leeming, M.N.; Iandolo, J.J.; Libby, S.J.

Transformation of Corynebacterium pseudotuberculosis by electroporation

1991

Songer, J.G. | Hilwig, R.W. | Leeming, M.N. | Iandolo, J.J. | Libby, S.J.

Corynebacterium pseudotuberculosis was transformed by electroporation, using pNG2, an erythromycin-resistance plasmid from C diphtheriae. Corynebacterium pseudotuberculosis cultivated in brain-heart infusion broth was washed 3 times with water, and resuspended to a final concentration of about 5 X 10(13) colony-forming units/ml. An electroporator constructed in our laboratory incorporated an electrode with 0.8-mm interelectrode gap, using disposable spectrophotometer cuvettes as containers for electroporation. The pNG2 was prepared in Escherichia coli and 4 to 16 microgram of pNG2 DNA was mixed with 400-microliter amounts of cell suspension in prechilled cuvettes. After incubation on ice for 5 to 10 minutes, the mixture was electroporated at field strengths of up to 18 kV/cm, mixed with 1.5 ml of brain-heart infusion broth, and incubated at 37 C for 2 hours with agitation. Aliquots were then plated on brain-heart infusion blood agar with 15 microgram of erythromycin/ml. Corynebacterium pseudotuberculosis was transformed at a maximal efficiency of approximately 4 X 10(4) transformants/microgram of pNG2 DNA. Most total transformants and most transformants per microgram of pNG2 were generated at a field strength of 18 kV/cm. When the concentration of pNG2 DNA was varied, the average total number of transformants increased through a concentration of 30 microgram/ml, but the efficiency of transformation was highest at the lowest DNA concentration. Transformants contained unmodified pNG2.

Afficher plus [+]