Document Type

Honors Project

First Advisor

Dr. Stephen Baron

Degree Award Date

Spring 2016

Keywords

Polyhydroxybutyrate Depolymerase Gene, Streptomyces sp. 5A, Streptomyces lividans TK24, western blotting, gene cloning, polymerase chain reaction, TOPO-TA cloning vector, electroporation

Disciplines

Biology | Immunology and Infectious Disease | Laboratory and Basic Science Research | Molecular Genetics

Abstract

Polyhydroxybutyrate (PHB) depolymerase is an enzyme produced by the bacterium Streptomyces sp. 5A to degrade the large polymer PHB into monomers that can then be taken into the cell and catabolized. The mechanism for regulating its synthesis is not known at the present time. In order to elucidate the mechanism, western blotting with specific antibodies would allow the monitoring of PHB depolymerase synthesis. In order to obtain antibodies, purified PHB depolymerase must be obtained in milligram quantities from Streptomyces sp. 5A, which is currently not possible. Thus, we attempted to clone and overexpress the gene that corresponds to the PHB depolymerase (phaZ) of this organism, in the heterologous host, Streptomyces lividans TK24. The phaZ gene was amplified by the polymerase chain reaction (PCR) and cloned into Escherichia coli TOP 10 using a TOPO®-TA cloning vector. The phaZ insert in the TOPO vector was transferred to the shuttle plasmid, (pIJ86), producing a pIJ86-phaZ construct. This construct was successfully introduced into E. coli l 2567/pUZ8002 by electroporation. The construct from this strain was successfully cloned into S. lividans TK24 by interspecific conjugation. However, the gene was not expressed in this organism, possibly due to genetic barriers between Streptomyces sp. 5A and S. lividans TK24.

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