Transformation of alternan-producing strains of Leuconostoc by electroporation.
Alternan-producing Leuconostoc mesenteroides strain NRRL B-1355 and its glucansucrase-negative derivative NRRL B-21414 were transformed by electroporation using four Gram positive-Gram negative shuttle vectors. Optimal conditions were 400 Omega and 10 kV cm(-1), resulting in transformation efficiencies of up to 3.5 x 10(4) per microg DNA. Relatively low copy numbers and native plasmids made it difficult to visualize the introduced plasmids on ethidium bromide-stained gels and, in some cases, on blot hybridizations. However, PCR analysis indicated that 95% of putative transformants carried plasmid sequences. Direct colony PCR was shown to work well for this system and also for transformants of L. mesenteroides subsp. cremoris.
Leathers
Timothy D
TD
Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDA, Agricultural Research Service, Peoria, IL 61604, USA. leathetd@ncaur.usda.gov
Jones
James D
JD
Wyckoff
Herb A
HA
D001709:Biotechnology / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D018274:Electroporation / Q000379:methods*; D004996:Ethidium / Q000494:pharmacology; D005822:Genetic Vectors; D005936:Glucans / Q000096:biosynthesis*; D007934:Leuconostoc / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014169:Transformation, Bacterial
D005936:Glucans; C439358:alternan; D004247:DNA; D004996:Ethidium