Tightly regulated, high-level expression from controlled copy number vectors based on the replicon of temperate phage N15.
A new Escherichia coli host/vector system has been developed to allow a dual regulation of both the plasmid copy number and gene expression. The new pN15E vectors are low copy number plasmids based on the replicon of temperate phage N15, comprising the repA replicase gene and cB repressor gene, controlling the plasmid copy number. Regulation of pN15E copy number is achieved through arabinose-inducible expression of phage N15 antirepressor protein, AntA, whose gene was integrated into the chromosome of the host strain under control of the PBAD promoter. The host strain also carried phage N15 partition operon, sop, allowing stable inheritance of pN15E vectors in the absence of selection pressure. In the first vector, pN15E4, the same PBAD promoter controls expression of a cloned gene. The second vector, pN15E6, carries the phage T5 promoter with a double lac operator repression module thus allowing independent regulation of promoter activity and copy number. Using the lacZ gene to monitor expression in these vectors, we show that the ratio of induction/repression can be about 7600-fold for pN15E4 and more than 15,000-fold for pN15E6. The low copy number of these vectors ensures very low basal level of expression allowing cloning genes encoding toxic products that was demonstrated by the stable maintenance of a gene encoding a restriction endonuclease in pN15E4. The tight control of transcription and the potential to regulate gene activities quantitatively over wide ranges will open up new approaches in the study of gene function in vivo and controlled expression of heterologous genes.
Mardanov
Andrey V
AV
Centre Bioengineering, Russian Academy of Sciences, Moscow, Russia.
Strakhova
Taisia S
TS
Smagin
Vladimir A
VA
Ravin
Nikolai V
NV
D001483:Base Sequence; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005822:Genetic Vectors; D042822:Genomic Instability; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012093:Replicon / Q000235:genetics*
D004279:DNA, Viral; C114636:endodeoxyribonuclease Ecl18kI; D015252:Deoxyribonucleases, Type II Site-Specific