Protocols

List of Protocols

1. LB (nutrient media) preparation
2. LA (solid media) preparation
3. Plasmid isolation (from E. coli )
   • Plasmid isolation solution I preparation
   • Plasmid isolation solution II preparation
   • Plasmid isolation solution III preparation
4. Restriction Enzyme Digestion
5. Agarose Gel Electrophoresis
6. E. coli competent cells preparation
   • CC solution I preparation
   • CC solution II preparation
   • CC solution III preparation
7. Transformation efficiency for competent cells
8. Ligation and Transformation
9. SDS-PAGE
10. PCR
11. IPTG Induction
12. Lead Adsoprtion Analysis
13. Primer Revival
14. Gene Revival

LB (nutrient media) preparation

1. Add 2 g of Luria Broth mixture to 100 mL of distilled water to make 2% w/v solution in an Erlenmeyer flask.
2. Mix well to form a uniform solution.
3. Make an air-tight cotton plug and close the mouth of the flask using the plug.
4. Autoclave the flask at 121℃, 15 psi pressure for 20-25 minutes. (Cover the cotton plug and the mouth of the flask with a piece of Aluminum foil before keeping inside the autoclave).
5. Sterilized liquid media is ready for use.

LA (solid media) preparation

1. Add 2 g of Luria Broth mixture to 100 mL of distilled water to make 2% w/v solution in an Erlenmeyer flask.
2. Mix well to form a uniform solution.
3. Add 1.5 g of Agar Agar powder to the solution and mix well to make a uniform 1.5% w/v solution.
4. Make an air-tight cotton plug and close the mouth of the flask using the plug.
5. Autoclave the flask at 121℃, 15 psi pressure for 20-25 minutes. (Cover the cotton plug and the mouth of the flask with a piece of Aluminum foil before keeping inside the autoclave).
6. While the media is still in liquid form, pour ~25 mL of media in 4 sterile petri plates each inside a laminar hood.
7. Let the media solidify inside the laminar hood.
8. Once solidified, cover the petri plates with their lids and seal them using parafilm.
9. Sterile solid media is ready for use.

Plasmid isolation (from E. coli)

1. Inoculate a single colony of E. coli in 5 mL LB in a test tube with appropriate antibiotics.
2. Incubate overnight at 37℃ and 180 rpm.
3. Harvest the cells by centrifugation at 12000 rpm for 2 minutes (at room temperature).
4. Discard the supernatant and collect the pellet.
5. Add 1 mL milli-Q water and centrifuge at 12000 rpm for 2 minutes at room temperature.
6. Discard the supernatant and collect the pellet.
7. Resuspend the pellet in 100 μL of Solution I (ice cold).
8. Incubate for 5 minutes in ice.
9. Add freshly prepared Solution II (200 μL).
10. Mix gently by inverting the tubes 4-5 times.
11. Add 150 μL of ice cold Solution III and mix by inverting 3-4 times and keep on ice for 5 minutes.
12. Centrifuge at 12000 rpm at 4℃ for 15 minutes.
13. Transfer the supernatant to a fresh tube.
14. Add 0.7 volume isopropanol (0.7 volume of the total volume of supernatant).
15. Mix by inverting or vortexing.
16. Keep at room temperature for 10 minutes.
17. Centrifuge at 12000 rpm at 4℃ for 15 minutes.
18. Discard isopropanol (supernatant) and add 1 mL 70% ethanol to wash the pellet.
19. Centrifuge at 12000 rpm at 4℃ for 2 minutes.
20. Air dry the pellet overnight.
21. Dissolve the pellet in 40 μL of Elution buffer (10mM Tris-Cl, pH 8.5).
22. Add 0.2 μL RNase.
23. Incubate for 1 hour at 37℃.
24. Store the plasmid at 0℃.

Plasmid Isolation solution I preparation

For 100 mL, pH 8,

1. Add the following to an empty Erlenmeyer flask:
   • 0.908 g glucose
   • 0.30283 g Tris
   • 0.40947 g EDTA
2. Add 10 mL distilled water and mix well.
3. Maintain pH at 8 using HCl.
4. Make up the volume to 100 mL.

Plasmid Isolation solution II preparation

For 1 mL,

1. Add the following to an empty falcon:
   • 100 μL 2 N NaOH
   • 100 μL 10% SDS
   • 800 μL Milli-Q
2. Mix well.

Plasmid Isolation solution III preparation

For 100 mL, pH 4.8,

1. Add 29.442 g Potassium acetate to an empty Erlenmeyer flask.
2. Add 10 mL distilled water and mix well.
3. Maintain pH at 4.8 using glacial acetic acid.
4. Make up the volume to 100 mL.

Restriction Enzyme Digestion

1. 25 μL Reaction Mixture:
2. Plasmid/genome to be digested (upto 1 μg., volume decided according to concentration).
3. Suitable buffer - 2.5 μL (chosen according to enzymes)
4. Enzyme 1 - 0.5 to 1 μL (volume according to activity)
5. Enzyme 2 - 0.5 to 1 μL (volume according to activity) [added if double digestion needs to be done]
6. milli-Q water - make up volume to 25 μL

Steps:
1. Make the suitable reaction mixture and mix slowly by gentle pipetting.
2. Incubate the mixture at 37℃ for 1-2 hours.
3. Perform agarose gel electrophoresis of 1 kb ladder, undigested plasmid/genome and digested plasmid/genome on 1% agarose gel. (run 2-3 μL of sample at 1 hour to check if digestion is complete, and full load when digestion is complete).
4. Cut the required band and perform gel extraction to obtain the required digest.

Agarose Gel Electrophoresis

Gel preparation (1% w/v):
1. Measure 0.5 g Agarose powder on a weighing balance.
2. Add it to an Erlenmeyer flask.
3. Add 49 mL distilled water and 1 mL TAE buffer (50 volume) to the flask.
4. Mix well, and microwave the mixture for 1-1.5 minutes, till the agarose completely dissolves to form a clear solution.
5. Let the solution cool till the flask is bearable to touch.
6. Add 2 μL EtBr carefully and mix well. (Use gloves while handling EtBr)
7. Pour the mixture in an agarose gel setting tray.
8. Place the comb to form the required number of wells on the setting tray.
9. Let the gel solidify.

Electrophoresis:
1. Place the solidified gel in the gel run apparatus.
2. Pour 1 volume TAE buffer in the gel run apparatus till it completely covers the surface of the gel.
3. Load the gel with the required samples (mix 1x loading dye with each of the samples before loading) and the suitable ladder.
4. Connect the electrodes of the gel run apparatus with the power pack (wells towards the negative electrode).
5. Switch the power pack on and let the gel run till 3/4th length of the gel.
6. Observe the gel under UV transilluminator to identify the required bands.

E. coli competent cells preparation:

1. For primary culture, isolate a single colony of E. coli DH5ɑ in 5 mL LB. Incubate overnight at 37℃ at 180 rpm.
2. Transfer 1% inoculum (1 mL for 100 mL media) in 100 mL LB to prepare secondary culture. Incubate at 37℃ for 2-3 hours till OD reaches 0.4-0.6.
3. Chill on ice for 20 minutes and centrifuge at 3000 rpm for 10 minutes at 4℃ after putting in oakridge tubes.
4. Discard the supernatant and collect the pellet.
5. Resuspend the pellet in ice-cold Solution I by filling oakridge tubes to half of their volume by length.
6. Centrifuge again at 5000 rpm for 10 minutes at 4℃. Discard the supernatant and collect the pellet.
7. Add 2 mL of ice-cold Solution II.
8. Centrifuge again at 5000 rpm for 10 minutes at 4℃. Discard the supernatant and collect the pellet.
9. Resuspend the pellet in 1 mL ice-cold Solution III.
10. Aliquot 100 μL of resuspended pellet in 10 MCTs each.
11. Competent cells preparation is complete. Store at -80℃.

CC solution I preparation

For 100 mL,

1. Add the following to 100 mL milli-Q water:
   • 1.626 g MgCl2
   • 0.294 g CaCl2
2. Mix well.
3. Store at 0℃.

CC solution II preparation

For 50 mL,

1. Add 0.735 g CaCl2 to 50 mL milli-Q water.
2. Mix well.
3. Store at 0℃.

CC solution III preparation

For 20 mL,

1. Add the following to 17 mL milli-Q water:
   • 3 mL Glycerol
   • 0.294 g CaCl2
2. Mix well.
3. Store at 0℃.

Transformation Efficiency for CC

1. Add 2 μL plasmid to 100 μL of competent cells.
2. Keep mixture on ice for 30 minutes.
3. Give a heat shock at 42℃ for 90 seconds.
4. Keep on ice for 10 minutes.
5. Add 1 mL LB to the mixture.
6. Incubate at 37℃ for 1.5 hours at 180 rpm.
7. Spread 100 μL of culture so obtained on an LA plate (having suitable antibiotics).
8. Incubate the plate overnight at 37℃.
9. To calculate transformation efficiency, use the following formula:
10. Transformation efficiency (cfu/μg) =
    

Ligation-Transformation

1. Make the suitable reaction mixture for both test and control. Perform the following steps for both the test and the control.
2. Incubate at 22℃ for 3 hours.
3. Incubate at 16℃ for 2 hours.
4. Transform the entire mix in 100 μL competent cells solution.
5. Keep the mixture on ice for 30 minutes.
6. Give a heat shock at 42℃ for 90 seconds.
7. Immediately transfer to ice for 5 minutes.
8. Add 1 mL LB.
9. Incubate at 37℃ for 1.5 hours at 180 rpm.
10. Centrifuge at 5000 rpm for 5 minutes at room temperature.
11. Remove 900-910 μL supernatant from the top so that ~100 μL supernatant is left.
12. Resuspend the pellet in the remaining 100 μL supernatant.
13. Spread the resuspended mixture on an LA plate (having suitable antibiotics).
14. Incubate the plate overnight at 37℃.

SDS-PAGE

1. Take approximately 1 ml of bacterial culture from each of the samples and centrifuge at 12,000 rpm for 2 min.
2. Discard the supernatant and wash the pellet with MilliQ.
3. Centrifuge the suspension and discard the supernatant.
4. Add 50μl of SDS-lysis buffer to the pellet and resuspend the pellet gently in the lysis buffer.
5. Boil the sample at 95°C for 10 min.
6. Centrifuge the suspension at 12,000 rpm for 5 min.
7. Load 15 μl of supernatant on an SDS-PAGE gel.
8. Run the gel until the dye reaches 3/4th the length of the gel at 80V.
9. Stain the gel with CBB R-250 solution for 15 min.
10. De-stain the gel with a destaining solution.

Polymerase Chain Reaction

1. Mix the following in a single PCR tube:
   • Standard Taq buffer - 2.0 μL
   • dNTP - 1.5 μL
   • Forward Primer - 1.0 μL
   • Reverse Primer - 1.0 μL
   • Taq Polymerase - 0.2 μL
   • Template - volume decided according to concentration (1μg needed in the mixture)
   • MilliQ - make up volume to 20 μL
2. Keep the PCR vial in the thermal cycler and set the following cycle conditions:
   • Initial denaturation - 95°C - 3 mins (for standard PCR) / 10 mins (for colony PCR)
   • Denaturation - 95°C - 1 minute
   • Annealing - Annealing temperature (based on primers) - 1 minute
   • Extension - 72°C - Duration dependent on size of template (extension speed of Taq polymerase - 1 kb/minute)
   • Final extension - 72°C - 10 minutes
   • Hold - 4°C
3. Analyze the products using gel electrophoresis.

IPTG Induction

1. Incubate a culture of plysS + plasmid with the required insert in 5 mL of LB with suitable antibiotics, overnight at 37℃ at 180 rpm (primary inoculation).
2. Use 50 μL of the above culture as inoculum and inoculate another 5 mL of LB having suitable antibiotics and incubate at 37℃ at 180 rpm till OD reaches 0.3-0.4 (secondary inoculation).
3. Once OD reaches 0.3-0.4, collect and store (at 4℃) a 1 mL sample in a sterile MCT before inducing the culture with 5 μL of IPTG and further incubating it at 37℃ at 180 rpm.
4. Collect and store (at 4℃) a 1 mL sample after 3 hours, 5 hours and overnight of incubation after induction each.
5. For each of the collected samples, pellet down the cells by centrifugation.
6. Collect the supernatant in a separate tube and save the pellet.
7. Suspend the pellet in 80μL dye and 80μL of the supernatant in 20μL dye.
8. Heat both of them at 95℃ for 10 minutes.
9. Centrifuge both of them at 12000 rpm for 10 minutes.
10. Perform Agarose gel electrophoresis of 16-17μL of each of the two samples.

Lead Adsorption Analysis

1. Grow overnight cultures of MBD-plysS and PbrR-plysS in 5 mL LB + Kan.
2. Use primary cultures as inoculum for secondary inoculation in 2 flasks each.
3. At 0.3 OD, induce one flask with IPTG.
4. After induction, collect 40 ml samples from both flasks at 6 hours and 12 hours after incubation.
5. Pellet down the cells from the collected samples and discard the supernatant.
6. Resuspend the cells in 40 mL Basal Salt Media.
7. To this 40mL, add 400ppb of lead nitrate solution.
8. Incubate the flasks at 37℃ at 180 rpm.
9. Collect 10 ml samples from the flasks at 3 hours, 6 hours, 9 hours and 12 hours of incubation each.
10. Centrifuge the collected samples at 5000 rpm for 10 minutes.
11. Collect 5 mL of supernatant and filter sterilize using a 0.2u filter.
12. To this 5 mL supernatant, add 5 mL of 5% conc. Nitric acid.
13. Mix the tubes gently and submit for ICP-MS.

Minimal Media Composition:

1. Na2HPO4 - 2 g/L
2. KH2PO4 - 1 g/L
3. NH4Cl - 3.59 g/L
4. MgCl2.7H2O - 0.4 g/L

Primer Revival

For 20 mL,

1. Centrifuge the lyophilized primer at 12000 rpm for 10 minutes at room temperature.
2. Add elution buffer (specific volumes for specific primers) to get to a concentration of 100 μg/μL.
3. Vortex thoroughly.
4. Centrifuge at 12000 rpm for 5 minutes at room temperature.
5. To make the working solution, mix 5 μL of stock primer along with 95 μL of elution buffer.

Gene Revival

1. Centrifuge the tube at 3000 rpm for 1 minute at room temperature to ensure that the material is at the bottom of the tube.
2. Add elution buffer to get to a final concentration of 10 ng/μL.
3. Vortex briefly.
4. Incubate at 50℃ for 20 minutes.
5. Briefly vortex and centrifuge.