Transformation of Corynebacterium pseudotuberculosis by electroporation

J. Glenn Songer From the Department of Veterinary Science, University of Arizona, Tucson, AZ 85721 (Songer, Hilwig, Leeming), Division of Biology, Kansas State University, Manhattan, KS 66506 (landolo), and Department of Molecular Biology, Scripps Clinic and Research Foundation, La Jolla, CA 92137 (Libby).

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Ronald W. Hilwig From the Department of Veterinary Science, University of Arizona, Tucson, AZ 85721 (Songer, Hilwig, Leeming), Division of Biology, Kansas State University, Manhattan, KS 66506 (landolo), and Department of Molecular Biology, Scripps Clinic and Research Foundation, La Jolla, CA 92137 (Libby).

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Michael N. Leeming From the Department of Veterinary Science, University of Arizona, Tucson, AZ 85721 (Songer, Hilwig, Leeming), Division of Biology, Kansas State University, Manhattan, KS 66506 (landolo), and Department of Molecular Biology, Scripps Clinic and Research Foundation, La Jolla, CA 92137 (Libby).

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John J. landolo From the Department of Veterinary Science, University of Arizona, Tucson, AZ 85721 (Songer, Hilwig, Leeming), Division of Biology, Kansas State University, Manhattan, KS 66506 (landolo), and Department of Molecular Biology, Scripps Clinic and Research Foundation, La Jolla, CA 92137 (Libby).

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Stephen J. Libby From the Department of Veterinary Science, University of Arizona, Tucson, AZ 85721 (Songer, Hilwig, Leeming), Division of Biology, Kansas State University, Manhattan, KS 66506 (landolo), and Department of Molecular Biology, Scripps Clinic and Research Foundation, La Jolla, CA 92137 (Libby).

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Summary

Corynebacterium pseudotuberculosis was transformed by electroporation, using pNG2, an erythromycin-resistaiice 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 × 1013 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 μg of pNG2 dna was mixed with 400-μl 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 μg of erythromycin/ml. Corynebacterium pseudotuberculosis was transformed at a maximal efficiency of ≈ 4 × 104 transformants/μg 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 μg/ml, but the efficiency of transformation was highest at the lowest dna concentration. Transformants contained unmodified pNG2.

Summary

Corynebacterium pseudotuberculosis was transformed by electroporation, using pNG2, an erythromycin-resistaiice 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 × 1013 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 μg of pNG2 dna was mixed with 400-μl 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 μg of erythromycin/ml. Corynebacterium pseudotuberculosis was transformed at a maximal efficiency of ≈ 4 × 104 transformants/μg 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 μg/ml, but the efficiency of transformation was highest at the lowest dna concentration. Transformants contained unmodified pNG2.

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