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Electroporation and expression of the broad host-range plasmid pRK2501 in Azotobacter vinelandii

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Abstract

Azotobacter vinelandii cells were transformed via high-voltage electroporation, with the broad host-range plasmid pRK2501. The number of transformants was dependent on the applied voltage, capacitance, and recovery procedure after electroporation. For example, Log. 4.44 transformants μg−1 DNA were recovered in the A. vinelandii cell suspension electroporated at 1500 V and 25 μF capacitance (time constant 29.0 ms) and recovered on LB agar amended with 0.5 μg/ml−1 kanamycin (pRK2501 encodes for both kanamycin and tetracycline resistance). Electroporation at 2500 V and capacitance settings of 25 and 3 μF did not produce any transformants. Cell survival was also poor at high voltages. A. vinelandii transformants were not recovered on N-free agar medium. In addition, no viable cells were recovered on N-free agar after electroporation at 2500 V, 25 μF; 2500 V, 3 μF; and 1500 V, 25 μF. Electroporation may be a useful method to genetically transform Azotobacter species for use in physiological and/or genetic studies.

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