pPSY: a vector for the stable cloning and expression of streptomycete single gene phenotypes in Escherichia coli.
pPSY is a 12kb cloning vector derived from the IncW plasmid R388, which provides a rapid and easy way to stably clone phenotypes encoded in DNA segments <10kb. 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. lividansamyA 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.
Philip
Daniel S
DS
Department of Microbiology and Parasitology, University of Queensland, Research Road, St. Lucia, Brisbane, Qld 4072, Australia.
Sarovich
Derek S
DS
Pemberton
John M
JM
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013302:Streptomyces / Q000235:genetics*