Inducible and constitutive expression using new plasmid and integrative expression vectors for Thermus sp.


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Abstract

Aims:To develop molecular tools and examine inducible and constitutive gene expression in Thermus thermophilus.Methods and Results:Two plasmid promoter probe vectors and an integrative promoter probe vector were constructed using a promoterless thermostable kanamycin nucleotidyltransferase (KmR) cassette. Three expression vectors were constructed based on a constitutive promoter J17, that functions in both Thermus and Escherichia coli. An inducible expression vector was constructed using the heat-shock inducible promoter (70 to 85°C) from the dnaK gene of T. flavus, and the malate dehydrogenase gene (mdh) from T. flavus was cloned and expressed in both E. coli and T. thermophilus HB27.Conclusions:This report describes the construction and use of improved promoter probe and expression vectors for use in Thermus species. The mdh gene can be used as a high temperature (85°C) reporter gene for Thermus sp. The dnaK promoter is thermo-inducible.Significance and Impact of the Study:The expression vectors and molecular tools described here are significant improvements over previously reported vectors for Thermus sp. The mdh gene and the thermo-inducible dnaK promoter will facilitate high temperature studies employing Thermus species.

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