Team:Exeter/Experiments

Protocols

Protocols

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This page details the protocols followed in the biological laboratory to develop our final PET degrading enzyme solution. To see our results, check out our Results page.



Transformation of E.coli

  1. Defrost one 100 μL aliquot of E.coli competent cells per transformation, plus one extra as a control (use whole tube and do not re-freeze.)
  2. Add 1-5μL of plasmid DNA (depending on concentration) to selected competent cells, mix by rolling Eppendorf between fingers.
  3. Incubate on ice for 40 mins (allowing the DNA to bind to the cell walls.)
  4. Heat shock in Eppendorf at 42°C for 2 minutes, initiating the movement of DNA into the cells.
  5. Incubate on ice for 5 minutes.
  6. Add 0.7mL of pre-warmed LB medium, incubate at 37°C, 200-220rpm for 60 minutes.
  7. Spin down cells at 8000rpm for 5 minutes.
  8. Remove 0.5mL of the supernatant.
  9. Re-suspend cells in remaining supernatant.
  10. Plate out 200μL on agar laced with 100μg/mL of ampicillin for IDT DNA and 50μg/mL of kanamycin for TWIST DNA.
  11. Incubate at 37°C overnight.

Type IIS Cloning

The one pot cloning strategy utilises the ability of a type II restriction enzyme (Bsal) to cut outside its recognition sequence thereby allowing the formation of a variety off different 4 bp overhangs. Two Bsal are placed at the 5' and 3' end of each DNA segment in reverse orientation allowing two DNA fragments to be ligated seamlessly with the removal of the Bsal recognition sequence. To assemble a complete gene within a plasmid each DNA part(promoter, RBS, CDS, and terminator) are provided as uncut plasmids (entry vectors) and mixed with the destination vector in 'one-pot.' The addition of both Bsal and T4 DNA ligase allows for repeat digestion-ligations cycles to 'amplify' the desired product due to the removal of the Bsal sites in the final product.

For one pot cloning add all the regants together in their allocated volumes, then follow the reaction conditions.

  1. Reagents
    • 20 fmol/μL Destination vector
    • 10x Fast Digest Buffer
    • 10 mM ATP
    • 10U/μL Fast Digest BSal (Thermo)
    • 5U/μL T4 DNA ligase (Thermo)

  2. Final Reaction Volumes
    • 1μL of each entry vector
    • 1μL destination vector
    • 2μL buffer
    • 1μL ATP
    • 1μL Bsal
    • 1μL T4 ligase
    • Then make up with 20μL of water

  3. Reaction conditions
    • 37°C for 2 minutes
    • 22°C for 3 minutes
    • Repeat above steps for 25-50 cycles
    • 37°C for 5 minutes
    • 22°C for 5 minutes
    • 65°C for 10 minutes
    • 10°C hold

Preparation of Competent E.coli cells

  1. Scrape a few cells from an E.coli glycerol stock (e.gDH5α), straight from the -80°C freezer onto a marked space on an LB-agar plate (no antibiotic!) Streak out three times with a wire and grow overnight at 37°C
  2. Innoculate 5ml of LB, in a 50ml Falcon tube, with one colony and incubate overnight at 37°C,200rpm.
  3. Innoculate 40ml of LB in a 350mL conical flask with 0.4mL of the overnight culture.
  4. Grow at 37°C and 200rpm until the OD600 = 0.4-0.5
  5. Transfer the culture to a 50mL Falcon tube and gravest by centrifugation at 5000rpm for 10 minutes at 4°C
  6. Drain the pellet and re-suspend cells in 8mL of Transformation buffer, TF-1.
  7. Place on ice for 15 minutes and then spin as above.
  8. Thoroughly drain the pellet and re-suspend in 4mL of TF-2.
  9. Create 100μL aliquots in 2mL Eppendorfs and immediately freeze in liquid nitrogen.
  10. Store aliquots in a labelled box in -80° freezer.

Buffers and LB

Buffers

  • Buffer A: 50 mM Tris, 0.5 mM NaCl, 20 mM imidazole, pH 7.5
  • Buffer B: 50 mM Tris, 0.5 M NaCl, 0.5 M imidazole, pH 7.5
  • GF Buffer: 100 mM NaPhosphate Buffer, 100 mM NaCl, pH 7.5

Luria Bertani media (LB)

LB is a non-specific growth medium for bacteria and contains a complex extract of biological material. Tryptone is the peptide formed by pancreatic digestion, and is an organic source of nitrogen. Should be autoclaved in flasks that are 1/4 full. This allows the air to circulate during propagation i.e. 5ml volumes in 50ml Falcon tubes, 50ml volumes in 250ml flasks and 250ml in 1 litre flasks.

  1. Tryptone - 10g/L
  2. Yeast Extract - 5g/L
  3. NaCl - 10g/L
  4. Water - No. of Litres required

Expression of Cytosolic PETase and BHETase

Expression of Cytosolic PETase

  1. Set overnights of transformed cells with the genes encoding for the PETase variants and incubate at 37°C, 220rpm.
  2. Transfer 1 ml of the overnights into flasks containing 50 ml of LB media and the appropriate antibiotic and incubate at 37°C, 220rpm until OD 0.6-0.8.
  3. Induce expression by adding 1µl IPTG / 1ml media and incubate overnight at 18o C, 160 rpm.
  4. Centrifuge at 5000rpm for 10 minutes at 20°C.
  5. Remove supernatant.
  6. Resuspend pellets in 10ml buffer A and disrupt cells by sonication on ice.
  7. Centrifuge at 12,000rpms for 20 minutes at 4°C.
  8. Collect the supernatant.
  9. Store in the cold room.

Expression of Cytosolic BHETase

  1. Set overnights of transformed cells with the genes encoding for the BHETase variants and incubate at 37°C, 220rpm.
  2. Transfer 1 ml of the overnights into flasks containing 50ml of LB media and the appropriate antibiotic and incubate at 37°C, 220rpm until OD 0.6-0.8.
  3. Induce expression by adding 1µl IPTG / 1ml media and incubate overnight at 18°C, 160rpm.
  4. Centrifuge at 10,000g for 20 minutes at 4°C.
  5. Remove supernatant.
  6. Resuspend pellets in 10ml buffer A and disrupt cells by sonication on ice.
  7. Centrifuge at 12,000rpm for 20 minutes at 4°C.
  8. Collect the supernatant.
  9. Store in the cold room.

Protein Purification

  1. The protein was purified using a 1 ml HisTrap FF crude column (GE Healthcare, Little Chalfont, England) using a gradient from 20 to 500 mM imidazole in 50 mM Tris-HCl pH 7.5, 0.5 mM NaCl.
  2. The enzyme was then applied to a calibrated Superdex 75 HiLoad 16/60 gel filtration (GF) or Superdex 200 HiLoad 16/60 column (GE Healthcare, Little Chalfont, England) and eluted with one column volume of 50 mM NaPhosphate Buffer, 0.1 M NaCl, pH 7.5 at 1.0 ml min-1.

Esterase Assay

Activity was measured by spectrophotometrically flowing the hydrolysis of p-nitrophenyl acetate (pNPA) into acetate and p-nitrophenol. This was performed at room temperature in buffer containing 50 mM NaPhosphate buffer pH7.5, 100 mM NaCl. A range of substrate concentrations were tested and a blank used to subtract the auto-hydrolysis of the pNPA. The production of p-nitrophenol was measured at 405 nm.

Thermal Stability Assay

The thermostability of the enzyme was investigated incubating enzyme samples at a range of temperatures (20 °C - 90 °C) for one hour using the gradient function in a SensOQuest LabCycler (Geneflow) before samples are cooled to 4 °C and assayed for activity using the esterase assay method described previously.

PETase Assay with Fibres

The PETase assay was carried out using fibres obtained from the washing machine and filter as part of this project. 35 µg of fibres was incubated in 500 µl of enzyme at a range of concentrations (50 to 2000 µM) in 50 mM Na Phosphate buffer pH 7.5 with 50 mM NaCl. The fibres were incubated with the enzyme solution for 76 hours before the reaction was terminated by heating at 80 °C for 15 mins. The reaction was then analysed by HPLC.

BHET Assay

The assay enzyme activity reactions (100 µl) with 100 µg/ml substrate in 40 mM Na Phosphate buffer pH 7.5 with 50 mM NaCl and 20 % (v/v) DMSO were incubated at 25 °C with the enzyme at 3 different concentrations (250, 500 and 1000 µM) and the reaction incubated for 24 hours at 30 °C. The reaction is terminated by heating at 80 °C for 15 mins. BHET was purchased pure form Sigma Aldrich. The reaction was then analysed by HPLC.

HPLC Assay

Samples (10 µl) was analysed using a high-performance liquid chromatography system (HPLC, Agilent 1200) using an Eclipse Plus C18 column (Agilent, UK). The mobile phase was 99.9 % Water with 0.1 % Formic Acid at a flow rate of 0.8 ml min-1 and the effluent monitored at 240 nm. The typical elution condition was 10 min with 20% - 80% acetonitrile. The amounts of products (BHET, MHET and TPA) were calculated by comparison to a standard curve. All samples were analysed in triplicate and the data averaged and standard errors calculated.

SDS-Page

Sample Preparation

  1. Add 10 ul of sample into an Eppendorf tube.
  2. Transfer 1 ml of the overnights into flasks containing 50ml of LB media and the appropriate antibiotic and incubate at 37°C, 220rpm until OD 0.6-0.8.
  3. Add 10 ul Bolt LDS Sample Buffer (4X).
  4. Add 4 ul Bolt/NuPAGE Reducing Agent (10X).
  5. Make up to 40 ul using deionized water.
  6. Heat samples at 70 degrees C for 10 min.

Gel Run

  1. Mix 20 mL of 20X Bolt MOPS SDS Running Buffer with 380 mL of deionized water.
  2. Fill the chamber of the gel tank with the buffer.
  3. Load 10 ul of the ladder into one of the gel’s wells.
  4. Load 40 ul of the sample into each of the wells.
  5. Run the gel for 32 min at 200 V.

Gel Staining

  • Generon Quick Comassie Stain
  • 25 ml of stain and incubate with gel for 1 hour
  • Destain gel in 100 ml of distilled water for 1 hour

Western Blot

Transfer

  1. Run SDS PAGE according the aforementioned method.
  2. Soak PVDF membrane in methanol.
  3. Add “1-step transfer buffer” in plastic gel box.
  4. Place 2 filter papers in plastic gel box.
  5. Place PVDF membrane in the box.
  6. Place 2 more filter papers on top.
  7. Make sure everything is soaked in transfer buffer.
  8. Leave minimum of 5 minutes.

Gel Sandwich

  1. Make a gel sandwich on the fast transfer adding in this order:
    • Filter paper
    • Filter paper
    • PVDF membrane
    • Gel
    • Filter paper
    • Filter paper
  2. Run program Mirella custom (25 V; 29 min; 1.3A)

Make Antibody Solutions

  1. Prepare 30 ml iBind mix by adding:
    • 6 ml iBind buffer
    • 300 ul
    • 23.7 ml H2O
  2. Prepare 2 ml of primary antibody by adding:
    • 2 ml iBind mix
    • 2 ul anti his-tag raised in mouse antibody
  3. Prepare 2 ml of secondary antibody by adding:
    • 2 ml iBind mix
    • 2 ul anti mouse raised in goat-alkaline phosphatase conjugated antibody

Prepare iBind

  1. Soak membrane in methanol.
  2. Add 6 ml iBind mix to a plastic gel box.
  3. Add the membrane to the box.
  4. Put iBind card into iBind.
  5. Add 5 ml of iBind mix all over the card.
  6. Add 1ml of iBind to center of the card.
  7. Place membrane upside down into the center of the card.
  8. Close iBind.
  9. Pipette into top of iBind:
    • 2 ml primary antibody mix
    • 2 ml iBind mix
    • 2 ml secondary antibody mix
    • 6 ml iBind mix
  10. Leave for 3 hours.

Visualisation

  1. Dissolve one tablet of SigmaFast BCIP/NBT in 10 ml ddH2O.
  2. Visualise in lid until ladder develops.
  3. Store in ddH2O.