جدا نمودن ژن تنظیمی استرپتومایسین فاقد پروموتر [StrR2] از استرپتومایسز گریزئوس

Background and purpose: Polymerase chain reaction (PÇR) is a rather quick and accurate method employed for gene detection and isolation. Primer designing is an important issue in this technique and plays a critical role in considering both the genome properties and cloning of the isolated genes. Streptomycin antibiotic is produced by Streptomyces griseus using str gene cluster with more than 25 genes. This gene cluster contains StrR gene encoding a specific protein regulator of this cluster. The pathway specific transcriptional activator then induces transcription of other genes in the str gene cluster. Ïn this study, the researchers aimed at isolating promoterless StrR2 gene and then cloning it. Materials and methods: To isolate promoterless StrR gene, a set of primers (StrR2) was designed. Ône pair of these primers (St Nes) detected the StrR from the genome. Moreover, Nested-PÇR was then used to detect amplified StrR2 gene. Sites for BamHÏ and XbaÏ were designed in other primers (Str nP1and Str nP2). These primers not only amplified the StrR2 gene but also created restriction enzyme sites in the amplified fragments. Results: Üsing PÇR, the promoterless StrR2 gene was amplified and its structure was confirmed. This gene was successfully cloned, too. The correct structure of the recombinant plasmids was confirmed using different techniques such as gel electrophoresis, PÇR and restriction digestion analysis. Çonclusion: Üsing this vector, one can subclone the promoterless StrR2 gene in the Streptomyces expression vectors containing inducible promoters.