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Transformation of Corynebacterium pseudotuberculosis by electroporation
J G Songer1, R W Hilwig, M N Leeming
1Department of Veterinary Sciences, University of Arizona, Tucson 85721.
American Journal of Veterinary Research
|August 1, 1991
Summary
This study optimized electroporation for transforming Corynebacterium pseudotuberculosis using the pNG2 plasmid. High transformation efficiency was achieved at 18 kV/cm, yielding up to 4 x 10^4 transformants/micrograms of DNA.
Area of Science:
- Microbiology
- Molecular Biology
- Genetic Engineering
Background:
- Corynebacterium pseudotuberculosis is a significant animal pathogen.
- Efficient genetic manipulation tools are crucial for studying C. pseudotuberculosis.
- Electroporation is a common method for bacterial transformation.
Purpose of the Study:
- To optimize the electroporation protocol for transforming Corynebacterium pseudotuberculosis.
- To determine the optimal conditions for high-efficiency transformation using the pNG2 plasmid.
- To characterize the resulting transformants.
Main Methods:
- Corynebacterium pseudotuberculosis cells were prepared and concentrated.
- Electroporation was performed using a laboratory-built electroporator and disposable cuvettes.
- Cells were mixed with pNG2 DNA (erythromycin-resistance plasmid) and subjected to varying field strengths.
- Transformants were selected on erythromycin-containing agar plates.
Main Results:
- Maximal transformation efficiency of approximately 4 x 10^4 transformants/micrograms of pNG2 DNA was achieved.
- Optimal field strength for maximum transformants and efficiency was 18 kV/cm.
- Transformation efficiency was highest at the lowest pNG2 DNA concentration tested.
- Transformants contained unmodified pNG2 plasmid.
Conclusions:
- Electroporation is an effective method for transforming Corynebacterium pseudotuberculosis.
- The optimized protocol yields high transformation efficiency.
- This method facilitates genetic studies of Corynebacterium pseudotuberculosis.