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Protocol & Safety

Protocol & Safety

Agarose Gel Preparation
Protocol

  1. Add 50 mL of TAE buffer to a 100 mL buffer.
  2. Next measure 0.5 g of agarose powder and add it to the TAE buffer in the beaker.
  3. Heat the solution in the microwave until it starts bubbling (around about 45 seconds on high). Check if the solution is clear if not repeat step 3.
  4. Pour the agarose gel solution into a gel casting chamber with the comb specific to the number of wells wanted.
  5. Add 3 μL of thiazole orange to the gel (BEFORE gel begins to solidify) in the chamber and mix it until it cannot be seen.
  6. Allow the gel to cool, once done it is ready for adding samples.

Safety
Component Risk Risk Prevention
Heated Agarose gel Burns and scalds Wear protective clothing, such as heat-proof gloves, when handling the heated container.
Ampicillin Stock Preparation
Protocol

  1. Weigh 1 g of Ampicillin trihydrate powder using an analytical balance.
  2. Add 10 mL of water.
  3. Gradually add NaOH until the solution becomes clear. When the solution is completely clear, stop adding NaOH. DO NOT overshoot as the addition of too much NaOH will result in a yellow solution which will no longer be usable.
  4. Take a syringe and attach a filter to it.
  5. Add the solution to the syringe 1 mL at a time and Transfer the solution from the syringe to an Eppendorf tube.
  6. Store the ampicillin stock in the fridge (-20°C) for future use.

Safety
Component Risk Risk Prevention
Ampicillin trihydrate powder (ThermoFisher SCIENTIFIC, 2018). Acute toxicity, Repeated Dose Toxicity, Reproduction & Development Toxicity, Genetic Toxicity, Carcinogenicity (Carcinogen status: Group 3). Causes skin irritation, Causes serious eye irritation, May cause an allergic skin reaction, May cause allergy or asthma symptoms or breathing difficulties if inhaled. Wash face, hands and any exposed skin thoroughly after handling. Wear protective gloves/protective clothing/eye protection/face protection. Avoid breathing dust/fume/gas/mist/vapors/spray. In case of inadequate ventilation wear respiratory protection. Contaminated work clothing should not be allowed out of the workplace. Use only outdoors or in a well-ventilated area.
5 M NaOH (Merck, 2017). May be corrosive to metals. Causes severe skin burns and eye damage. Wear protective gloves/ protective clothing/ eye protection/ face protection. Response: IF SWALLOWED: Rinse mouth. Do NOT induce vomiting. IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. IF exposed or concerned: immediately call a POISON CENTER or doctor/ physician. When not using the solution, close the lid to avoid any spillage.
Shaking the microfuge tube. Make sure the lid is tightly closed before shaking. Wear gloves and protective goggles at all times.
Electrocution/ burns from centrifuge or vortex. Make sure that no liquids are around the devices. Make sure that the appliances are tagged before use. If unsure on how to operate them, ask the supervisors. Be careful when using the devices.
Overnight Culture/Flask
Protocol
Seed Culture

  1. Place 5 mL of LB broth into a labelled falcon tube.
  2. Place 2.5 μL of (200 mg/μL) Ampicillin stock solution into the same falcon tube.
  3. Inoculate a single colony of bacteria from an agar plate into the tube.
  4. Incubate at 37°C overnight in a shaking incubator.

Flask Culture (IPTG induced)

  1. Place 100 mL of LB broth into a labelled 500 mL flask.
  2. Place 100 μL of (200 mg/μL) ampicillin stock solution into the flask.
  3. Inoculate a previously prepared seed culture.
  4. Inoculate an IPTG at 1:500 w/v to flask. If control skip this step.
  5. Incubate at 18°C overnight in a shaking incubator.

Safety
Component Risk Risk Prevention
Ampicillin trihydrate powder (ThermoFisher SCIENTIFIC, 2018). Acute toxicity, Repeated Dose Toxicity, Reproduction & Development Toxicity, Genetic Toxicity, Carcinogenicity (Carcinogen status: Group 3). Causes skin irritation, Causes serious eye irritation, May cause an allergic skin reaction, May cause allergy or asthma symptoms or breathing difficulties if inhaled. Wash face, hands and any exposed skin thoroughly after handling. Wear protective gloves/protective clothing/eye protection/face protection. Avoid breathing dust/fume/gas/mist/vapors/spray. In case of inadequate ventilation wear respiratory protection. Contaminated work clothing should not be allowed out of the workplace. Use only outdoors or in a well-ventilated area.
LB broth (SIGMA-ALDRICH, 2014). May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed.

Personal precautions: Use personal protective equipment. Avoid dust formation. Avoid breathing vapours, mist or gas. Ensure adequate ventilation. Avoid breathing dust.

Environmental precautions: Do not let product enter drains. Methods and materials for containment and cleaning.

Streaking Single Colonies
Protocol

  1. Prepare LB-AMP agar plate for antibiotic selection and label the plates.
  2. Collect samples of interest.
  3. Sterilise a pipette using 70% ethanol and a paper towel.
  4. Set up a Bunsen burner for sterile inoculation.
  5. Using a pipette, scrape some cells from the sample.
  6. Inoculate bacterial cells from the sample of interest onto the agar plate.
  7. Streak out the culture, swapping pipette tips between streaks to maintain sterility.
  8. Incubate at 37°C overnight.

Safety
Component Risk Risk Prevention
Bunsen burner. Fire hazard, burn risk. Keep flammable materials away from the flame. Secure all loose hair and clothing. Ensure the gas line is secure.
Carbonic Anhydrase Assay
Equipments

  1. PPE
  2. Micropipettes and tips
  3. Milli Q water
  4. Tris buffer
  5. NaCl
  6. ZnSO4
  7. Ni-NTA resin

Protocol

  1. Prepare following solutions:
    • Binding buffer (0.5 M NaCl, 5 mM imidazole, 0.1 mM ZnSO4, 100 mM tris sulfate buffer),
    • Wash buffers (same as above except with 40 mM, 60 mM and 100 mM of Imidazole),
    • Elutation buffer (1 M imidazole and the rest the same),
    • Desalting buffer (no imidazole and the rest the same).
  2. After Growing Overnight with Flask, add solution for each treatment into Falcon tubes and centrifuge.
  3. Remove media from pelletised cells of each treatment.
  4. Add binding buffer and lyse cells for each treatment.
  5. Spin to pelltise cells (store supernatant and lysis) for each treatment.
  6. Pass supernatant through syringe for condition of further study or if you wanna study more thoroughly both.
  7. Add supernatant to gravity column with Ni-NTA resin and collect first pass through (store first pass through).
  8. Your Ni-NTA now has your protein of interest.
  9. Add in succession and different wash buffer in increasing concentration collection 3 faction of 2 mL each for each wash buffer.
  10. Add eluion buffer and collect 9 fractions of 2 mL each (with each three being E1, E2, E3 to show all protein has been eluted).
  11. Perform bradford assay to determine initial protein qualitatively. If protein concentration is low across the board add 15 μL to each well of SDS-PAGE. If protein concentration is build standard curve to determine.

Carbonic Anhydrase Assay
Equipments

  1. PPE
  2. Stirile work space
  3. Micropipettes and tips
  4. Milli Q water
  5. Tris buffer
  6. NaCl
  7. ZnSO4
  8. Cell lysing reagent (Bugbuster)
  9. CA inhibitor (acetazolamide or furosemide)
  10. p-nitrophenyl acetate (p-NPA) in ethanol anhydrous at 200 mM
  11. p-nitrophenol (p-NP) 1 mM
  12. 96 well plate
  13. Plate reader

Protocol

  1. Make the assay buffer by adding Tris, NaCl and ZnSO4 in the ratio where in solution, the concentration of the substances are 25 mM Tris, 75 mM NaCl, 0.02 mM ZnSO4. 3 mL of this should be sufficient.
  2. Lyse the bacterial cells with lysing reagent.
  3. Centrifuge for 20 minutes at 13000 rpm.
  4. Set up a Bunsen burner for sterile inoculation.
  5. Collect Supernatant and dilute 10 folded with the assay buffer.
  6. Estimate the concentration of hpCA by running the solution on SDS gel.
  7. Dilute the solution further to 0.2 μM (this can be slightly lower as hpCA is more active than human CA).
  8. Dilute p-NPA to 0.06 mM in Assay buffer (Only do this right before measurement as water also lyse it).
  9. Half the solution and inhibitor to one, inhibitor concentration should be 40 μM.
  10. Make the standards by adding p-NP, in well A1 add 2 μL, A2 add 4 μL, A3 add 8 μL, A4 add 12 μL, A5 add 16 μL, A6 add 20 μL, then fill the well to 100 μL with the assay buffer.
  11. Add 50 μL of the solution without inhibitor to well B1 to B8.
  12. Add 50 μL of the solution with the inhibitor to well C1 to C8.
  13. Add 50 μL of the lysed bacteria solution to wells B2 to B8 and C2 to C8, skipping 1 as a blank.
  14. Measure the wells at 405 nM for 1 hour with 1 min interval.

Urease Assay
Equipments

  1. PPE
  2. Fume hood
  3. Stirile work space
  4. Micropipettes and tips
  5. Milli Q water
  6. Cell lysing reagent (Bugbuster)
  7. Phenol red sodium salt
  8. Urea salt
  9. Ammonia
  10. 96 well plate
  11. Plate reader

Protocol

  1. Prepare phenol red indicator by dissolving phenol red sodium salt in water at a concentration of 0.012 g/L.
  2. Prepare urea stock solution by dissolving urea in water at a concentration of 200 mM, dilute to 1 in 50 prior to use.
  3. Prepare ammonia standard by taking 10 μL of 27% ammonia solution and dilute in 50 mL of water. (This process must be carried out in fume hood).
  4. Prepare positive control by hydrating previously characterised urease in water at concentration of 1 mM (CU).
  5. Collect and concentrate urease enzymes from cell lysate and measure concentration, dilute urease enzyme to a concentration of 1 mM (EU).
  6. Make the standards by adding dissolved ammonia in wells:
    • A2 & B2 add 2 μL,
    • A3 & B3 add 4 μL,
    • A4 & B4 add 8 μL,
    • A5 & B5 add 12 μL,
    • A6 & B6 add 16 μL,
    • A7 & B7 add 20 μL,
    then fill all wells including A1 and B1 to 100 μL with water.
  7. Load negative controls by loading 50 μL of diluted ammonia solution into wells D1 to D4 then load 50 μL of water to fill to 100 μL.
  8. Load positive controls by adding 50 μL of dilute ammonia solution into wells D5 to D8 then load 50 μL of CU into the wells.
  9. Load samples by adding EU in wells:
    • E1 & F1 add 10 μL,
    • E2 & F2 add 20 μL,
    • E3 & F3 add 25 μL,
    • E4 & F4 add 30 μL,
    • E5 & F5 add 35 μL,
    • E6 & F6 add 40 μL,
    • E7 & F7 add 45 μL,
    • E8 & F8 add 50 μL,
    then add 50 μL of dilute ammonia to each well, finally fill all wells to 100 μL with water.
  10. Add 30 μL of phenol red indicator to all wells.
  11. Incubate for 30 to 60 minutes then measure the results in the plate reader at 560 nm absorbance or start measuring after 30 minutes at 1 minute interval with 30 repeats for kinetics.