Electroporation and expression of the broad host-range plasmid pRK2501 in Azotobacter vinelandii.
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 microgram-1 DNA were recovered in the A. vinelandii cell suspension electroporated at 1500 V and 25 microF capacitance (time constant 29.0 ms) and recovered on LB agar amended with 0.5 microgram/ml-1 kanamycin (pRK2501 encodes for both kanamycin and tetracycline resistance). Electroporation at 2500 V and capacitance settings of 25 and 3 microF 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 microF; 2500 V, 3 microF; and 1500 V, 25 microF. Electroporation may be a useful method to genetically transform Azotobacter species for use in physiological and/or genetic studies.
Trevors
J T
JT
Department of Environmental Biology, University of Guelph, Ontario, Canada.
Starodub
M E
ME
D001395:Azotobacter / Q000235:genetics*; D002463:Cell Membrane Permeability; D004560:Electricity; D005822:Genetic Vectors; D007613:Kanamycin Resistance / Q000235:genetics; D008722:Methods; D010957:Plasmids; D014162:Transfection