A High-Transformation-Efficiency Cloning Vector for Thermus thermophilus

The cloning vector pMK18 was developed through the fusion of the minimal replicative region from an indigenous plasmid of Thermus sp. ATCC27737, a gene cassette encoding a thermostable resistance to kanamycin, and the replicative origin and multiple cloning site of pUC18. Plasmid pMK18 showed transf...

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Bibliographic Details
Published in:Plasmid 1999-11, Vol.42 (3), p.241-245
Main Authors: de Grado, Myriam, Castán, Pablo, Berenguer, José
Format: Article
Language:English
Subjects:
Cloning, Molecular
Genetic Vectors
Models, Genetic
Thermus thermophilus
Thermus thermophilus - genetics
Transformation, Genetic
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Summary:The cloning vector pMK18 was developed through the fusion of the minimal replicative region from an indigenous plasmid of Thermus sp. ATCC27737, a gene cassette encoding a thermostable resistance to kanamycin, and the replicative origin and multiple cloning site of pUC18. Plasmid pMK18 showed transformation efficiencies from 108 to 109 per microgram of plasmid in Thermus thermophilus HB8 and HB27, both by natural competence and by electroporation. We also show that T. thermophilus HB27 can take pMK18 modified by the Escherichia coli methylation system with the same efficiency as its own DNA. To demonstrate its usefulness as a cloning vector, a gene encoding the β-subunit of a thermostable nitrate reductase was directly cloned in T. thermophilus HB27 from a gene library. Its further transfer to E. coli also proved its utility as a shuttle vector.
ISSN:0147-619X
1095-9890
DOI:10.1006/plas.1999.1427