Somayeh Panahi Moghadam; Zohreh Hojati; Majid Motovali_Bashi
Abstract
Objectives: StrR is a putative pathway specific regulator of streptomycin production in Streptomyces griseus. Because of finding new spo0j domain in strR by bioinformatics methods, the purpose of this study was to suggest another role for strR gene. This domain can be seen in proteins that are involved ...
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Objectives: StrR is a putative pathway specific regulator of streptomycin production in Streptomyces griseus. Because of finding new spo0j domain in strR by bioinformatics methods, the purpose of this study was to suggest another role for strR gene. This domain can be seen in proteins that are involved in initiation of sporulation and normal chromosome partitioning. So, 51 bps of strR in accordance to spo0j domain was deleted to investigate effects of deletion mutation in StrR functions. A unique specific procedure, including three consecutive PCRs known as SOEing PCR has been correctly used for site directed mutagenesis. Application and feasibility of this PCR was studied here.
Materials and Methods: Bioinformatics studies were carried out for comparison of the sequences similarities between StrR and Spo0J proteins. A unique specific procedure, including three consecutive PCRs, was design here in order to delete a 51 bp from the native strR. Other PCRs such as Semi-Nested PCR and RFLP PCR were used for strR isolation and structural confirmation of the isolated strR and deleted strR genes. Routine genetic engineering procedures were conducted in order to clone the native and deleted strR genes into E. coli.
Results: Obtained sequence information, from Conserved Domains Database (CDD) and Clustal W program, has revealed that the StrR is similar to members of ParB family. Here, the strR was initially isolated from Iranian strain of S. griseus (PTCC1127). It was then confirmed as StrR by Semi-Nested PCR and RFLP-PCR. A 51 base pair region of strR gene was deleted by specifically designed overlapped primers. A ten nucleotide overlap region was considered for a set of these primers. The recombinant cassette pSPMstrRΔ17 was constructed and cloned in E. coli.
Conclusion: The sequencing results showed that a specific deletion is produced in the desired site and region in the strR gene. Therefore the designed three steps PCRs (known as SOing PCR) is a very rapid, cheap and precise method for introducing such a deletion in any preferred gene.
