Molecular identification of Streptomyces isolates and phylogenetic analysis

The genomic DNA was extracted from the cultures of two Streptomyces isolates by using kit (MicroSeq 500 16S rDNA bacterial identification Kits) according to the manufacturer’s instructions. Amplification of 16S rDNA by PCR was done using universal bacterial primer forward F (5′-CGGGCGGTGTGTAC-3′) and reverse R (5′-CAGCCGCGGTAATAC-3′) which amplify a ~ 800 bp. Amplification was carried out in a final volume of 50 μL containing; PCR buffer (1×), Taq DNA polymerase (2.5 U), dNTPs (4 mM), primers (0.4 μM), and template DNA (4 ng) with 100 bp ladder DNA marker. The thermal cycle (PCR) steps were applied as follows: 5 min initial denaturation at 95 ^oC, followed by 30 cycles of 1 min denaturation at 95 ^oC, 1 min primer annealing at 55 ^oC, 1 min extension at 72 ^oC and a final 10 min extension at 72 ^oC. The amplified DNA fragment was separated on 1% (w/v) agarose gel electrophoresis, using TBE buffer containing ethidium bromide (1 μg/mL). A single ~ 800 bp DNA fragment was cut and extracted from the gel, using a Core Bio Gel Extraction Kit. The sequence was determined by the CinnaGen Company. Sequence data of partial 16S rDNA was aligned and analyzed for finding the closest homologous bacteria. The 16S rRNA nucleotide sequence was compared to nucleotide databases using BLASTN program that is available from the National Center for Biotechnology Information (NCBI, 2014) and retrieved ligned using GeneDoc software version 2.6. 002. Phylogenetic trees were built utilizing the neighbor-joining.

Screening for the extracellular proteins attached to AgNPs using SDS-PAGE.

To examine the diversity of extracellular proteins involved in Ag NPs synthesis and secreted from both Streptomyces strains in their numbers and molecular sizes, electrophoretic analysis of concentrated proteins on 12% SDS-PAGE was used. Briefly, after 48 h of adding the extract of the two Streptomyces strains that has been grown for 8 days to silver nitrate, the media of both strains were compiled and concentrated utilizing Amicon Ultra centrifugal filter with cut-off of 10 KDa. The proteins that are larger than 10 KDa was retained and concentrated in the upper part of the filter and their concentration was assessed by using Bradford assay. The extracellular proteins that are associated with Ag NPs was diluted in phosphate buffer saline, pH 7.2 and then 20 μg of the proteins of each strain was added to the wells of 12% polyacrylamide gel containing 10% Sodium dodecyl sulphate (SDS). A low molecular weight marker (Pharmacia Fine Chemicals Company) was used to show the distribution of molecular sizes of extracellular proteins associated with silver nanoparticles. Proteins on the gel were stained with 0.25% Coomassie Brilliant Blue R-250