RESUMO
To clone bifunctional vectors in streptomycetes, it was necessary to define the restriction-modification system of Streptomyces flavopersicus. Plasmid DNA from bifunctional vectors pIJ699 and pXED3-13, isolated from E. coli strains with different methylation systems: E. coli DH5 alpha (dam+ dcm+), E. coli MB5386 (dam dcm), E. coli CB51 (dam dcm+), E. coli NM544 (dam+ dcm), was used for transformation of protoplasts from strain S. flavopersicus. Restriction of dcm-methylated DNA from S. flavopersicus was established. As a host in the intermediate cloning strain E. coli NM544 (dam+ dcm) should be used, as the dcm-transmethylase-dependent strain S. flavopersicus does not process DNA from this strain.
Assuntos
Enzimas de Restrição-Modificação do DNA/metabolismo , Streptomyces/enzimologia , Clonagem Molecular , Metilação de DNA , DNA Recombinante/metabolismo , Escherichia coli/genética , Plasmídeos , Streptomyces/genética , Transformação GenéticaRESUMO
Protoplasts of Streptomyces flavopersicus with the highest regeneration frequency were isolated from late log phase mycelium grown in a two-stage culture system with 2% glycine in the medium. Of the 11 regeneration media tested, R9 was selected as the most efficient with 29 degrees C as the best temperature.
