Document Type

Honors Project

First Advisor

Dr. Stephen Baron

Degree Award Date

Fall 2012

Keywords

S. coelicolor, Conjugative Host, pRT Flip Reporter Plasmid Constructs, phaZ Promoter Region

Disciplines

Bacteriology | Biology | Laboratory and Basic Science Research | Microbiology

Abstract

The polyhydroxybutyrate (PHB) depolymerase of Streptomyces sp. SA degrades PHB to 3- hydroxybutyrate monomers, and is synthesized during growth on PHB but not glucose. The promoter region of the PHB depolymerase gene (phaZ) contains possible binding sites for regulators involved in transcriptional control of PHB depolymerase synthesis. To study the function of these sites, the entire phaZ promoter region and that containing a deletion within a suspected binding site were ligated together with a reporter plasmid, pRT Flip, yielding constructs 1 and 3, respectively. Plasmid pRT Flip contains genes determining bioluminescence (lux) and allowing for its transfer from an Escherichia coli donor (strain ET12S67/pUZ8002) to Streptomyces via intergeneric conjugation. Constructs 1 and 3 were separately introduced into E. coli ET12S67/pUZ8002 by electroporation. Because previous attempts to transfer non-recombinant pRT Flip into Streptomyces sp. SA were unsuccessful, the alternative host, Streptomyces coelicolor, was used. Although the organism did indeed receive both the constructs, the S. coelicolor strain harboring construct 1 failed to express the reporter genes when grown on media that would typically promote PHB depolymerase production by Streptomyces sp. SA. Thus, from the conditions tested, S. coelicolor was not a suitable host to evaluate reporter gene expression under control of the phaZ promoter.

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