Team:BGU ISRAEL/Protocols

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Protocols

Our Protocols & Assays

General Protocols

Terephthalic acid detection protocol

In order to assess our different variants' PET degradation activity we developed an assay for the detection of Terephthalic acid, a product of degradation of PET by LC-Cutinase:
Dissolve Terephtalic acid (TPA) in water with a strong base (NaOH)in a 1:2 concentration ratio.
Prepare a stock solution of 50 mL, 20 mM TPA by adding:

  1. 0.166 gr of TPA
  2. 2.0 mL of 1M NaOH solution
  3. 48 mL double distilled water (DDW)

Vortex for five minutes until the TPA powder is completely dissolved. Titrate the solution with 1M HCl solution to achieve a pH of 7.5.
For a calibration curve:
Prepare 10 mL size batches of different TPA concentrations. Dilute the initial TPA stock solution with DDW to the following final concentrations:

15mM, 10mM, 5mM, 1mM, 0.5mM, 0.1mM, 0.05mM, 0.01mM, 0.005mM, 0.001mM.

Pipette 400 µL of each concentration in a UV transparent 96 wells microtiter plate. Make triplicates of each concentration.


For baseline readings, measure fluorescence in a plate reader using the following values:

  • Excitation: 314 nm.
  • Emission: 426 nm.
  • Gain: 100 Hz to detect high concentrations and 200 Hz to detect low concentrations
  • Number of Flashes: 150 to detect high concentrations and 200 to detect small concentrations

Following measurements, irradiate the samples with UV light (365 nm) for one hour using a UV transilluminator.
Repeat the fluorescence measurements using the plate reader. (if needed, prolong the exposure time of the sample to enhance fluorescence).
Create a calibration curve from the fluorescence values that were received for each concentration. Use the curve to extract the concentrations of TPA in a sample of unknown concentration of TPA.

Detection of PET utilization using soft agar plates using modified E. coli

  • Day 1:

    Transformation and growth of PET-degrading bacteria (E. coli).

  • Day 2:
    1. Dilute stationary phase bacteria by transferring 50μL of the grown bacteria to a new 5mL starter medium.
    2. Incubate the bacteria for 2 hours in 37oC, measure O.D every 90 minutes until an O.D value of 0.5-0.7 is reached (if induction of protein expression is required). When the needed value is reached stop the growth by removing the tubes from the incubator.
    3. Preparation of PET powder:
      1. Pour liquid nitrogen on the PET pellets.
      2. After 30 seconds transfer the pellets to a coffee grinding machine.
      3. Grind the pellet to a fine powder. Repeat with liquid nitrogen if needed.
      4. Sterilization - before the preparation of the plates spray 70% ethanol on the PET powder and leave it to dry in an incubator set to 50oC until the powder dries.
    4. Prepare an M9 liquid medium solution, add 0.7% (w/v) of Bacto-Agar and autoclave.
    5. Prepare a 40-45oC liquid bath
    6. Prepare 15mL vials with 250mg of the PET powder.
    7. Transfer 1mL of bacteria from the grown starters to 1.5mL vial and centrifuge for 1 min at 8000g.
    8. Discard the supernatant and resuspend the bacterial pellet.
    9. Place the PET powder vials and the soft M9 agar solution into a heated bath (40oC).
    10. Transfer 5mL of soft agar to each PET powder vial.
    11. Add a bacterial selection marker (Antibiotics) to each vial.
    12. If induction is needed - add an inducer when bacteria reach appropriate OD, to each vial
    13. Transfer the re-suspended bacteria to the appropriate vial (still inside the heated bath).
    14. Pour the prepared solution from the vials to petri dishes.
    15. Incubate at 37oC for growth.

Growth experiments with various carbon sources

We have prepared various M9 liquid media with different carbon sources in order to test P. putida's ability to utilize different source and its ability to transport them into the cells.
Preparation of the carbon sources for the M9 medium:

  1. Carbon source - Terephthalic acid (TPA). For the preparation of 50mL with a concentration of 200mM:
    • 1.66gr TPA
    • 10ml NaOH 1M
    • Add DDW to a final volume of 50mL
    Dilute the prepared medium for lower concentrations.
  2. Carbon source - Protocatechuic acid (PA). For the preparation of 60ml with a concentration of 500mM:
    • 4.623gr PA
    • 15mL Ethanol 25%
    • Add DDW for a final volume of 60mL
    Dilute the prepared medium for lower concentrations.

Add prepared solutions as a carbon source for the preparation of the M9 medium as needed.
After preparation of the media we have prepared a Corning 24 wells plate for continuous measurement of growth in a plate reader.
The design for the partitioning of the plate is as follows:

1 2 3 4 5 6
A Water Water Water Water Water Water
B Water Carbon Source X mM Carbon Source Y mM Carbon Source Z mM Carbon Source X mM No Bacteria Water
C Water Carbon Source X mM Carbon Source Y mM Carbon Source Z mM Glucose Water
D Water Water Water Water Water Water

Preparation Notes

  1. Water surrounding the plate is important as it prevents the evaporation of the media. Add water if needed throughout the experiment.
  2. Wells B5 and C5 are negative and positive controls respectively.
  3. X,Y and Z are annotations for different concentrations of the carbon source tested for growth.
  4. Every well should contain 2.0mL of growth medium.

The experiment:

  1. Prepare starters from the tested bactria for over-night incubation in a 5.0mL liquid M9 medium with glucose as a carbon source.
  2. After sufficient growth is achieved centrifuge the bacteria for 10 min at 4000 rpm (repeat if needed). Discard the supernatant and add 500μL of the lowest concentration of tested growth medium to the vial and re-suspended the bacteria.
  3. After resuspension transfer 100μL of bacteria to the 24 well plate according to the above design and add the growth medium of appropriate concentration up to a volume of 2.0mL.
  4. Take an initial measurement in the plate reader with a wavelength of 610nm for a base O.D value.
  5. Incubate the plate in the appropriate temperature and measure O.D at 610nm twice a day for a week.
  6. Make sure that the water has not evaporated from the plate. Add if necessary.
  7. After a week analyze results.
*Measurements can be preformed in a 96 wells plate with smaller volumes of growth media.
  • Remember to fill the outer wells with water to prevent evaporation of the growth media.
  • Each well should be filled with 200μL of growth medium and 20μL of bacteria from the starters.
  • The rest of the experiment is as with a 24 wells plate.

Preparation of PET pellets for SEM imaging

We incubated PET pellets with LC-Cutinase expressing bacteria and viewed the samples under a scanning electron microscope (SEM).
Preparation of the samples in LB liquid growth medium:
  1. Grow and induce LC-Cutinase expressing bacteria as in the pNP-Butyrate assay.
  2. Prepare a 500mL LB liquid growth medium in a 2L Erlenmeyer flask for each of the tested variants.
  3. Add 5.0mL of the appropriate bacteria to each flask. (Control - Medium with PET and no bacteria and a medium with PET and bacteria transformed with the pACYC vector with no insert)
  4. Add 3 PET pellets to the flask after sterilization with ethanol and drying.
  5. Add the appropriate antibiotics. (we used 0.5mL of Chloroamphenicol (CMP))
  6. Grow the bacteria in the appropriate temperature and when an O.D value of 0.5-0.7 is reached add an inducer for protein expression (IPTG for the pACYC vector).
  7. The next day remove 250mL out of each flask and replace with fresh 250mL of growth medium. Add more inducer and antibiotics (we added 0.25mL IPTG and 0.25mL CMP).
  8. On the following day remove the PET pellets. Dry 2 of them with paper towels, the remaining one wash with 70% ethanol and then dry.
  9. Examine pellets under SEM.

E. coli growth inside a dialysis bag with PET as a carbon source

In this protocol we describe how to grow E. coli expressing a PET degrading enzyme (LC-Cutinase for example) inside a dialysis bag with PET as a carbon source. This method is used so to allow other microorganisms to grow outside the bag and utilize the PET degradation products as nutrients.

Reagents and lab tools:
  1. 1L Beaker.
  2. 1 Dialysis Bag (with a cut-off of lower of ca. 10KDa according to the PET degrading enzyme's molecular weight) treated with 70% ethanol.
  3. Two clamps and a floater.
  4. 1L of Sterile M9 medium supplemented with Shredded PET and antibiotics.
  5. E. coli BL21 strain (or any strain fitted for protein expression) transformed with LC-Cutinase (or other PET degrading enzyme) on a low-copy-number plasmid.
  6. 1 Magnetic stirrer.
  7. 1 Magnet
  8. 1.0mL of IPTG 1M (or other inducer, according to the expression vector used).

Preparation steps:
  1. Pour the M9 medium into the Beaker with 6.5gr of PET.
  2. Keep 1.0 mL M9+PET as a blank measurement.
  3. Insert the M9 medium+PET and E. coli (after reaching an O.D value of 0.1 at 610nm) into the dialysis bag.
  4. Insert the dialysis bag into the beaker.
  5. Stir, add IPTG (or other inducer) to the dialysis bag every day according to the amount of M9 medium used.
  6. Measure O.D value at 610nm twice a day, for E. coli growth curve.
  7. Measure terephthalic acid content to evaluate degradation efficiency using "Terephtalic acid detection assay".

pNP-Butyrate degradation protocol

Day 1:

  1. Transform the chosen genes into E.Coli BL21 competent cells
  2. From transformation volume, take 100uL and grow in 5.0mL starters with appropriate antibiotics
  3. Centrifuge the rest of the transformation volume and sow on LB agar plates with appropriate antibiotics, to check transformation efficiency

Day 2:

  1. Dilute the starters (1:100)- take 50uL to a new 5.0mL starter- grow in 50mL test tubes
  2. Grow bacteria until O.D is between 0.5 to 0.7.
    Measuring O.D:
    1. Take 100uL of starter and add 900uL of LB
    2. Check O.D at 600nm
    3. Don’t forget to multiply the result by x10
    (O.D measurements should be about every 1.5h; usually it takes the bacteria 3-4h to reach correct O.D)
  3. Meanwhile, thaw the IPTG (concentration: 1M)
  4. INDUCTION: Add 5uL of IPTG to the 5.0mL starters (to get a conc. of 1mM)
  5. Leave tubes in shaker incubator in 37°C overnight

Day 3:

Preparations of pNP-B: (needs to be done only on day III, because pNPB degrades spontaneously)
  1. Prepare a solution of 200mM pNPB in ethanol or acetonitrile
  2. Prepare 9.9mL of 25 mM Tris-HCl (pH 8.0)
  3. Add 100uL of the pNPB solution to the Tris buffer, to get a 2mM conc. of pNPB
Preparations of protein supernatant:
  1. Centrifuge the 5.0mL E. coli starters at 8000xg for 30 minutes at 4°C to separate the supernatant and cells.
  2. Collect supernatant into a new test tube
pNPB degradation assay:
  1. Mark the appropriate wells in a 200uL 96-well plate (clear, not white!)
  2. Add 100uL of protein sup to each well
  3. Add 100uL of the pNPB solution, to get a 1mM final conc.
  4. Check O.D in a plate reader at 405nm

Preparation of SM liquid growth medium (for R. ruber)

SM is a liquid minimal medium used in assays of bacterial strains that degrade PE (polyethylene).
Add to one liter of distilled water:
  1. 1.0 gr NH4NO3
  2. 0.2 gr MgSO4*7H2O
  3. 1.0 gr K2HPO4
  4. 0.1 gr CaCl2*2H2O
  5. 0.15 gr KCl
  6. 0.1 Yeast extract
  7. 1.0 mg of the following - FeSO4*6H2O, ZnSO4*7H2O and MnSO4

Protein purification (for LC-Cutinase)

E. coli BL21-Rosetta (DE3)-pLys transformants with pACYC-LCC (LC-Cutinase) were cultivated at 37°C. When the absorbance of the culture at 600 nm reached ≈1.0, 1mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to the culture medium, and cultivation was continued overnight. The culture medium was centrifuged at 8,000 × g for 30 min to separate the supernatant and cells. The protein in the supernatant was precipitated by the addition of ammonium sulfate to 70% of the saturated concentration and then dissolved in 10 mM Tris-HCl (pH 7.0) containing 1 mM EDTA and 1 mM dithiothreitol (DTT). The solution was dialyzed against the same buffer overnight and applied to a column (1.0 mL) of SP-Sepharose (GE Healthcare) equilibrated with the same buffer. The protein was eluted from the column by linearly increasing the NaCl concentration from 0 to 1.0 M at 0.3 M NaCl. The production level of the protein in E. coli cells and the purity of the protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) (Laemmli, 1970) using a 12% polyacrylamide gel, followed by staining with coomassie brilliant blue (CBB).

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Ben-Gurion University of the Negev
Ben Gurion 1, Beer Sheva 8410501, Israel

Mail: igembgu2016@gmail.com

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