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  1. May 2019
    1. 2 mL of an overnight culture of E. coli cells was inoculated into 100 mL LB medium and incubated with vigorous shaking at 30 °C until A600 of 0.8 was reached. •Cells were collected in 50 mL plastic (Falcon) tubes, cooled for 15 min on ice and centrifuged in a pre-cooled centrifuge (4,000 rpm for 10 min at 4 °C). •The pellet was suspended in 20 mL of ice-cold 50 mM CaCl2-15% glycerol solution, maintained on ice for 15 min and centrifuged again at 4,000 rpm for 10 min at 4 °C. •Pellet was resuspended in 2 mL of ice-cold 50 mM CaCl2-15 % glycerol solution, kept on ice for 30 min and aliquoted in 400 μL in microcentrifuge tubes. These were stored at -80 °C until required.
    2. as well as commercial methods (MN kit, Germany; Mo-Bio kit, CA, USA; Zymo soil DNA kit, CA, USA) according to the manufacturer’s protocols and compared in terms of DNA yield and purity.
    3. The soil DNA from Pantnagar and Lonar soil samples were also extracted by various manual (Desai and Madamwar, 2007; Agarwal et al., 2001; Yamamoto et al., 1998
    4. Comparison of yield and purity of crude DNA
    5. Various strains of Escherchia coli (DH5α, XL1Blue, DH10B) were used as hosts for the propagation of recombinant vectors. In addition, Bacillus subtilis was used as a host for the expression of xylanase gene from the recombinant vector pWHMxyl. Different vectors used in this investigation are listed in
    1. Human 0+ or AB+ RBC was obtained from a donor and mixed with heparin (50 units/ml of blood) and centrifuged at 500 g for 10 min with minimu1p. de-acceleration. The supernatant was removed carefully and the pelleted RBCS were washed 3 times with RPMI 1640 to remove serum and buffy coat. Equal amount of RPMI 1640 media was added to packed RBC volume to achieve 50% hem~tocrit and stored at 4°C till: further use
    2. Preparation of RBCsfor culture
    1. Cancer cell lines of human origin, HUT102, T-cell leukemia; K562, erythroleukemia; COL0205; colon adenocarcinoma; MCF7, breast adenocarcinoma; A431, epidermoid carcinoma; A549, lung carcinoma and HeLa, cervical carcinoma and J774A.I, mouse monocyte-macrophage; and L929, mouse fibroblast were obtained from ATCC. All the cell Jines were maintained in RPMI 1640 supplemented with antibiotic antimycotic solution, 2 mM glutamine and I 0% heat inactivated foetal calf serum (Life Technologies, Maryland, USA). E. coli strain DH5a was used for DNA manipulation, cloning and mutagenesis. Strains CJ236 and DH5aF' were used. for oligonucleotide mediated site directed mutagenesis. BL21 (A.DE3) strain containing T7 RNA polymerase gene under the control of lac promoter, was used for expression of the recombinant proteins.
    2. Cell Lines and Bacterial Strains
    1. Bacto -tryptone, Bacto -agar and Bacto -yeast extract were from Difco Laboratories, Detroit, USA. Fetal calf I serum, Ham s F-12 medium ( DMEM ) , I Iscove s Laboratories, USA.
    2. Media.
    1. was used to amplify the oligonucleotides (Promega Biotech, Madison, USA). pGEMT-Easy and p-TARGET cloning vectors were also obtained from Promega. In vitro transcription was carried out using Riboprobe transcription system (Promega Biotech, Madison, U.S.A.). BCA protein assay kit was obtained from Pierce Biotechnology (Rockford, IL, U.S.A.). Reverse transcription was carried out using lmProm-TI™ Reverse Transcriptase kit from Promega. Luciferase activity in the cell extracts was measured using Luciferase assay System (Promega Biotech., U.S.A.).
    2. Qiaprep spin mini kit and Qiagen plasmid midi kit (West Sussex, U.K.) were used for isolation of DNA. Isolation of DNA fragments from gel was carried out using QiaGel extraction kits or PCR products were purified using nucleotide removal kit from Qiagen (West Sussex, U.K.). PCR core system I
    3. Kits
    1. For bacterial isolates, a single colony from a nutrient agar slant was inoculated into 50 ml of nutrient broth in a 250 ml Erlenmeyer flask. These flasks were incubated at 37±1°C in a incubator shaker till an optical density of 0.6 at 660nm. Now these cultures were used to inoculate 50 ml of the tannase production medium in 250 ml Erlenmeyer flasks using 2% v/v inoculum. These flasks were incubated at 37±1°C in an incubator shaker (Multitron AG-27; Switzerland) at 200 rpm for 72h. The experiments were carried out in triplicates. Samples (2.0 ml for bacteria and same for fungi) were withdrawn at regular intervals of 12h upto 72 h. The samples thus obtained were centrifuged at 10,000 rpm in a refrigerated centrifuge (SIGMA 4K15 Germany) for 10 min at 4°C. The supernatant/s were analyzed for tannase activity
    2. 1N Hydrochloric acid (HCl)* One normal hydrochloric acid was prepared by adding 1.0 ml concentrated HCl to 10.0 ml of double distilled water. •1N Sodium hydroxide (NaOH)* One normal sodium hydroxide was prepared by dissolving 4.0 g of NaOH in 100 ml of double distilled water. *: These were used for adjusting the pH of the medium. •Tween-80Tween-80 used as surfactant was prepared by adding 100 μl of concentrated Tween-80 to 100 ml of double distilled water and autoclaved.
    3. tock Solution