pPSY: A vector for the stable cloning and expression of streptomycete single gene phenotypes in Escherichia coli

D S Philip; Derek S Sarovich; J M Pemberton

doi:10.1016/j.plasmid.2008.02.003

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pPSY: A vector for the stable cloning and expression of streptomycete single gene phenotypes in Escherichia coli

Journal article

Peer reviewed

pPSY: A vector for the stable cloning and expression of streptomycete single gene phenotypes in Escherichia coli

D S Philip, Derek S Sarovich and J M Pemberton

Plasmid, Vol.60(1), pp.53-58

2008

DOI: https://doi.org/10.1016/j.plasmid.2008.02.003

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Abstract

streptomyces

E. coli

stable vector

PCR

Gene expression

pPSY

pPSY is a 12 kb cloning vector derived from the IncW plasmid R388, which provides a rapid and easy way to stably clone phenotypes encoded in DNA segments <10 kb. In the present study three different genes were amplified by PCR, cloned into pGEM-T Easy and sub-cloned into the EcoRI site of pPSY. The first gene, vioA, is a FAD-dependent l-tryptophan amino acid oxygenase from the high G+C Gram-negative bacterium Chromobacterium violaceum. VioA is involved in the synthesis of the indolocarbazole antitumour antibiotic violacein. It was found that vioA was strongly expressed in Escherichia coli from its native promoter. Two other genes encoding recombinase A (recA) and an amylase (amyA), derived from the high G+C Gram-positive streptomycete, Streptomyces lividans, were also tested. Despite recA lacking its native promoter sequence, it was strongly expressed in E. coli using the lac promoter of pGEM-T Easy. Similar to vioA, S. lividans amyA was strongly expressed in E. coli from its native promoter. Unlike pGEM-T Easy, pPSY stably maintained all three genes without the requirement for antibiotic selection. These results demonstrate the applicability of pPSY as a stable amplicon cloning vector for the expression of heterologous genes in E. coli. Crown Copyright © 2008.

Details

Title: pPSY: A vector for the stable cloning and expression of streptomycete single gene phenotypes in Escherichia coli
Authors: D S Philip (Author) - University of Queensland
Derek S Sarovich (Author) - University of Queensland
J M Pemberton (Author) - University of Queensland
Publication details: Plasmid, Vol.60(1), pp.53-58
Publisher: Academic Press
DOI: 10.1016/j.plasmid.2008.02.003
ISSN: 0147-619X
Organisation Unit: Centre for Bioinnovation; University of the Sunshine Coast, Queensland
Language: English
Record Identifier: 99450683502621
Output Type: Journal article

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