A second generation snp-derived Escherichia coli-Streptomyces shuttle expression vector that is generally transferable by conjugation.
An Escherichia coli-Streptomyces shuttle vector (pJN100) was constructed, by inserting an origin of transfer (oriT), derived from the E. coli broad host range plasmid RK2, into pANT1202, a high-copy-number vector for gene expression in Streptomyces. The resulting conjugably transferable vector contains the pANT1202-derived SnpR (LysR-like protein) activated snpA promoter that drives strong heterologous expression of proteins. We initially demonstrated that plasmid pJN100 was transferred with high frequency (10(-5-7) exconjugants per recipient) into several Streptomyces strains that were refractory to transformation by other means. Plasmid pJN100 was also shown to be stable in E. coli and Streptomyces. We confirmed functional protein expression by using a pJN100 derivative to complement a mutant of Streptomyces griseus with a disrupted chromosomal copy of the gene nonM, a gene encoding an essential reductase in the nonactin biosynthesis gene cluster. High levels of protein expression were confirmed using Western blotting to assess the production of the serine esterase NonR, an enzyme responsible for nonactin resistance in the nonactin producer S. griseus.
Nikodinovic
Jasmina
J
Department of Chemistry, University of Montana, Missoula, MT 59812-1656, USA.
Priestley
Nigel D
ND
D001483:Base Sequence; D015153:Blotting, Western; D002874:Chromosome Mapping; D003227:Conjugation, Genetic / Q000235:genetics; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D018014:Gene Transfer Techniques; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000235:genetics*
D017931:DNA Primers