Sample collection and mer operon library construction
Panipat industrial area was selected for soil sample collection because it is reported to have much higher concentration of mercury than the permissible limit. The soil was taken 10 cm under the ground level by digging it and was kept in sterile container at 4^oC for further investigation.

In order to determine the concentration of mercury present in the soil sample, 10 gm of sample was weighed to check Hg concentration present in the soil using CV-AAS.

MOBIO kit was used to isolate metagenomic DNA from the soil sample following the protocol provided by the manufacturer. Before constructing the library the DNA was quantified using UV-absorption at 260nm at dilution fold of 500. After the quantification the partial restriction digestion was performed to get the desired fragment size of 5 to 10Kb in order to make clones.

For restriction digestion reaction, sterile micro-centrifuge tube was used to set up the reaction of 150µl which consist of 100ml of DNA, 15µl of 10X enzyme buffer, 1.5ml of Sau 3A1 and 33.5ml autoclave distilled water. The reaction was setup in the ice bucket and  immediately transferred to 16^oC for 3h. The reaction was terminated by heating the mixture at 70^oC in water bath. The digested metagenomic DNA was run on 1% Agarose using gel electrophoresis assembly (BIORAD) and the fragments of the DNA in range of 5-10Kb were gel eluted using HiYield Gel/PCR DNA Mini Kit (Real Biotech Corporation, Taiwan). Ligation reaction was setup using digested DNA and dephosphorylated pUC 19 vector digested with Bam HI along with T4 DNA ligase enzyme. The mixture was incubated at 4^oC for 16-18h and reaction was terminated by heat inactivation of enzyme at 70^oC. Electroporation at 200W, 25mF and 2.5kV was done by introducing recombinant vector into host bacteria (E.coli DH10B) with aid of Micropulser II (BIO RAD). Immediately, the tube was poured with 1ml of Luria Broth (LB) and  kept in shaker incubator at 180rpm for 1h. Transformed cells (100µl) were spread on the Luria Bertani Agar (LBA) plates supplemented with ampicillin, X-gal and IPTG and incubated at 37^oC for 16h.

Screening and selection of mercury resistant clones
The white colonies were picked and streaked on the LBA plates which were supplemented with ampicillin and 5ppm of Mercury Chloride (HgCl₂), incubated at 37^oC for 24-48h. The clones which were able to grow in the presence of 5ppm of HgCl₂ were transferred to fresh LB supplemented with 10ppm of HgCl_2. The clones showing visible growth at 10ppm were further transferred to 20ppm, 30ppm and so on up to 100pm for acclimatization of clones and examined by taking OD at 600nm. The clone which showed maximum growth after acclimatization at highest HgCl₂ concentration was selected for further studies. To check the presence of merB gene, the clone was grown in LB with 5ppm and 10ppm of Methyl Mercury Chloride (CH₃HgCl₂), incubated at 37^oC for 18h and OD was taken at 600nm.

Antibiotics resistance assay
Antibiotic resistance of the selected clone was checked through disk diffusion method against various antibiotic listed in Table 1. Freshly cultured clone was spread on the LBA plates and antibiotic discs were placed onto it, incubated at 37^oC for 18h. Later, zone of inhibition was measured.

Table (1):
Antibiotics used for antibiotic resistance activity.

Investigation of Hg volatilization by CV-AAS
The clone was inoculated in LB media containing 10 to 100ppm of HgCl₂ and incubated at 37^oC;180rpm. The culture was drawn periodically on 2^nd, 4^th and 6^th day under sterile conditions in tubes and centrifuged at 8000rpm for 8 mins. The supernatant was used for CV-AAS analysis (Analytical Jena make (Vario-6) cold vapor Hg-AAS).

In-vivo efficacy of the clone for Hg Volatilization
The Panipat soil (non-sterile) was taken and mixed with the sterile soil to make 5 kg of mixture of around 10ppm Hg concentration (3:2) wt/wt). Overnight grown culture (500ml);(1OD) was sprayed onto the soil mixture. The soil sample was periodically taken out on 2^nd, 4^th and 6^th day. The samples were used for CV-AAS in order to check the ability of clone to transform the toxic Hg to non-toxic form on the contaminated site.

Entrapment of clone to check its repeatability
Immobilization was done to check the efficiency of the clone to convert the Hg from toxic form to non-toxic form of mercury by repeated use. Two methods were used for immobilization listed below (3 cycles).

Sodium alginate Entrapment method
Pre-grown culture (16h) was mixed with sodium alginate (3%w/v) in the ratio of 1:4 and mixed well to make slurry under sterile conditions. The slurry was added to 0.2M of Calcium Chloride (CaCl₂) drop-wise using sterile tips at room temperature in laminar air flow. Then the beads were stored at 4^oC for 1h. After cooling the beads were washed 2-3 times with autoclaved distilled water and inoculated in conical flask containing 30ml of LB supplemented with 10, 20 and 30ppm of HgCl₂ respectively. The flasks were incubated at 37^oC; 180rpm and beads were drawn at the time interval of 24h (for 3 days). The beads were washed and re-inoculated to LB having same concentration of HgCl₂. This procedure was repeated thrice to check reusability of entrapped clone. CV-AAS of drawn samples at different time intervals were done.

Polyacrylamide gel entrapment method
Cell suspension of pre-grown culture (16h) was prepared by adding 2ml of culture into 10ml of chilled autoclaved distilled water and kept aside. 10 ml of the Potassium Phosphate Buffer (pH 7.0, 0.2M) was prepared, Acrylamide (2.85g), Bis-acrylamide (0.15g), Ammonium persulphate (10mg) and TEMED (1ml) were added to the buffer. The chilled cell suspension was mixed with polyacrylamide gel and poured into a sterile petri plate for solidification. After solidification, the cubes (5 mm x 5mm x 5mm) were cut using sterile scalpel and kept in Sodium Phosphate Buffer (pH 7.0, 0.2M) for 1h at 4^oC. The cubes were washed 3-4 times and put into conical flask containing LB (30ml) supplemented with 10, 20 and 30ppm of HgCl₂. The flasks were incubated at 180rpm, 37^oC and cubes were drawn each day after 24h (for 3 days). The cubes were washed and re-inoculated to LB having same concentration of HgCl₂. The drawn samples were used for CV-AAS analysis.