Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation

Journal Article · Wed Mar 01 00:00:00 EST 1989 · Applied and Environmental Microbiology; (USA)

OSTI ID:5098256

McIntyre, D A; Harlander, S K ^[1]

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1. Univ. of Minnesota, St. Paul (USA)

The objective of this study was to develop a system for electroporating intact cells of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) with a commercially available electroporation unit. Parameters which influenced the efficiency of transformation included growth phase and final concentration of cells, ionic strength of the suspending medium, concentration of plasmid DNA, and the amplitude and duration of the pulse. Washed suspensions of intact cells suspended in deionized distilled water were subjected to one high-voltage electric pulse varying in voltage (300 to 900 V corresponding to field strengths of 5 to 17 kV/cm) and duration (100 {mu}s to 1 s). Transformation efficiencies of 10{sup 3} transformants per {mu}g of DNA were obtained when dense suspensions (final concentration, 5 {times} 10{sup 10} CFU/ml) of stationary-phase cells were subjected to one pulse with a peak voltage of 900 V (field strength, 17 kV/cm) and a pulse duration of 5 ms in the presence of plasmid DNA. Dilution of porated cells in broth medium followed by an expression period of 2 h at 30{degree}C was beneficial in enhancing transformation efficiencies. Plasmids ranging in size from 9.8 to 30.0 kilobase pairs could be transformed by this procedure.

• OSTI ID: 5098256
• Journal Information: Applied and Environmental Microbiology; (USA), Vol. 55:3; ISSN 0099-2240
• Country of Publication: United States
• Language: English

Related Subjects

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
59 BASIC BIOLOGICAL SCIENCES
DNA
UPTAKE
ELECTRIC FIELDS
BIOLOGICAL EFFECTS
BACTERIA
CELL MEMBRANES
PERMEABILITY
PLASMIDS
RECOMBINANT DNA
USES
CELL CONSTITUENTS
MEMBRANES
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
560400* - Other Environmental Pollutant Effects
550200 - Biochemistry