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Broad-host range expression vectors containing manipulated meta-cleavage pathway regulatory elements of the TOL
J L Ramos1, M Gonzalez-Carrero, K N Timmis
1Department of Medical Biochemistry, University Medical Centre, University of Geneva, Switzerland.
Abstract:
The construction of pERD20 and pERD21, two broad-host range expression vectors, is described. The vectors contain the Pm promoter of the meta-cleavage pathway operon of the TOL plasmid pWWO; this promoter is present within a polylinker which provides a number of downstream cloning sites close to the transcription initiation site. Transcription from the Pm promoter in these vectors is controlled not by the natural positive regulator of Pm, the Xy1S protein, but by an Xy1S mutant analogue, Xy1S2tr6, which inhibits an altered effector specificity and can mediate a 3-8-fold higher level of transcription than can Xy1S in a wide range of temperatures. Controlled expression of cloned genes can be achieved in a broad spectrum of Gram negative bacteria grown at a wide range of temperatures.