Plasmid extraction kit

The expression vector (pET102/D-TOPO), Ni-NTA purification system, anti-His Tag antibody, and HRP-conjugated anti human against IgG were purchased (Invitrogen, Frankfurt, Germany). Primers were synthesized by Metabion (Frankfurt, Germany). Micro bicinchoninic acid (BCA) protein assay Kit (Pierce, Rockford, United States) and Pfu DNA polymerase (Stratagene, La Jolla, United States) were provided. Viral Nucleic acid extraction Kit, PCR product purification Kit, and Plasmid extraction Kit all were obtained from Roche (Frankfurt, Germany). The Escherichia coli BL21 (DE3) strain was provided by virology laboratory of Iranian Blood Transfusion Organization. Polyvinylidene difluoride (PVDF) membrane and horseradish peroxidase (HRP) substrate for western blotting were obtained from Amersham (London, United Kingdom).

Pure metagenoms were PCR amplified for bacterial and archaeal 16S rRNA genes using the universal primers (Table 1, Supplementary data). One hundred Nano gram of DNA was used in a PCR reaction mixture (final volume of 50 µL)
containing 1.5 mM MgCl₂, 1X Reaction buffer, 0.2 mM dNTP, 5 pmoL of each primer and 2.5 U Taq DNA polymerase. PCR was performed with an initial denaturation at 95 °C for 5 min, followed by 30 cycles of 95 °C for 60 s (denaturation), 50-54 °C for 60 s (annealing), 72 °C for 1.5 min (extension) and 72 °C for 10 min (final extension). The PCR products were visualized on 1% agarose gel in TAE buffer and then purified using the Roche High pure PCR purification kit.
The amplicons were ligated into pTz57 R/T vector, according to the Fermentas’s protocol. Ligation products were transformed into E. coli DH5α cells by heat shock transformation method ( 16
) and screened on LB/Ampicillin/IPTG/X-Gal plates in 37 °C for 16 h. The positive clones were selected based on the blue-white screening method; accordingly, white colonies were considered as recombinant clones and confirmed by PCR using vector specific primers M13F (5ˊ-GTAAAACGACGGCCAG-3ˊ) and M13R (5ˊ- CAGGAAACAGCTATGAC-3ˊ) ( 17 (link)
). Then, plasmids were extracted from positive clones by plasmid extraction Kit (Roche, Germany) for sequencing (Sanger method, Macrogen, South Korea).