Team:Edinburgh/Experiments
Experiments
Overall experimental plan:
1. Assembly Design
2. Molecular Cloning (JUMP assembly)
3. Enzymes overexpression
4. Explore enzymes to degrade and upcycle recalcitrant waste
a. Chicken feather degradation: Keratinase production and immobilisation
b. Chicken feather upcycle: Glutathione production with ATP-regenerating system
c. Crustaceans wastes upcycle: Chitin extraction
d.Biomass degradation: Cellulase directed evolution, Cellulase immobilisation
e. PET plastic degradation: PETase expression and immobilisation
5. Enzymatic assay
6. Protein immobilisation (sfGFP)
7. Protein stability test after immobilisation
8. Enzyme immobilisation
9. Enzyme stability test and activity assay
Protocols
Cloning and Transformation:
1. Inoculate a single colony of appropriate cells into 10ml LB media and culture overnight at 37°C, 200 rpm
2. Inoculate 1% of the overnight culture into a fresh LB media (100 mL)
3. Incubate at 37°C, 200 rpm until OD600 = 0.3-0.6 (approx. 2 hours)
4. Transfer to 2 x 50 mLtubes and leave on ice for 30 minutes
5. Centrifuge at 4000 x g for 5 minutes at 4°C
6. Gently resuspend pellet in 25 mL ice cold 0.1 M MgCl2 and keep on ice for 30 minutes
7. Centrifuge at 4000 xg, 5 min for 5 minutes at 4°C
8. Gently resuspend pellet in 25 mL ice-cold 0.1 M CaCl2 and incubate on ice for 30 minutes
9. Centrifuge at 4000 xg, 5 min for 5 minutes at 4°C
10. Gently resuspend pellet in 1.25 mL ice cold CaCl2 with 15% Glycerol solution
11. Aliquot 100 uL and flash freeze with liquid nitrogen. Store at – 80°C until required
1. Prepare 50 uL of sterile water in a PCR tubes
2. Pick a single colony and resuspend in 50 uL water, then draw 5 uL out into a new tube
3. Freeze 5 uL cell suspension at -20°C for at least 10 minutes
4. Prepare the master mix and add with 5 uL cell suspension (DNA template) and PCR according to the condition in the table below: following the table below:
Run the PCR product on 1% agarose gel with 100 V for 30 minutes and visualise the result under UV exposure.
1. Defrost chemically competent cells on ice.
2. Add 2ul of plasmid DNA or 10 uL of assembly reaction to 100 uL competent cells
3. Flick the tube to mix and incubate on ice for 30 minutes
4. Heat shock at 42°C for 30 seconds
5. Incubate on ice for 2 minutes
6. Add 1 mL SOC media and recover cellsat 37°C, 200 rpm for 1 hour
7. Spread 100 uL on an appropriate antibiotic LB/agar plate
8. Spin the rest of the cells at 4000 rpm, remove 900 uL of the media, resusupend and spread onto another LB/agar plate
9. Incubate plates at 37°C overnight
1. Determine the concentration of the assembly parts with Nanodrop
2. Use DNA calculator (from Promega) to calculate fmol of each part
3. Prepare the assembly parts in the concentration of 20 fmol/uL
4. Set up 20 uL JUMP assembly reaction using 1 uL of all parts (or 20 fmol)
5. Add 2 uL of 10X T4 ligase reaction buffer, 1 uL of either BsmBI (for Level0 assembly) or BsaI-HF (for Level 1 assembly), and 0.25 uL of T4 ligase
6. Add sterile water to adjust the volume up to 20 uL
7. Incubate the reaction following the tables below
8. Transform the reaction directly to the competent cells
Protein expression and quantification
1. Make BSA protein standard dilutions of 2.0 µg/mL, 1.5 µg/mL, 1.0 µg/mL, 0.5 µg/mL, 0.25 µg/mL and 0.0 µg/mL (dH2O).
2. Prepare an eppendorf tube per BSA standard as well as protein sample and add 1 mL 1x Bradford reagent to the tubes.
3. Transfer 5 µL of each BSA standard as well as protein sample to the respective eppendorf tubes and incubate at room temperature for 10 minutes.
4. Calibrate a spectrophotometer with a blank of distilled water
5. Measure A595
6. Make a standard curve using standards and use this to determine protein concentration of samples.
1. Inoculate 1% of overnight cultures of E. coli BL21(DE3) with level 1 constructs encoding gene with Si-tag and untagged level 1 constructs (as a negative control) in 200ml of fresh LB media containing 50μg/ml of Kanamycin and incubate it at 37°C in a shaking incubator until the OD reaches to 0.5 at 600 nm measured by a spectrophotometer.
2. Then, add IPTG to a final concentration of 1mM and incubate overnight at 18°C.
3. After overnight incubation, centrifuge the cultures in a centrifuge at 4000rpm for 10 min at 4°C, weigh the cell pellets and store them at -20°C.
4. Resuspend the cell pellets to 0.1 g/mL in the lysis buffer (100mM Tris-HCl pH 8.0 or 100 mM Sodium Phosphate buffer pH 6.5, with 0.5 mM EDTA and 100 uM PMSF)
5. Lyse cell by sonication on ice in 5s pulses at 55% amplitude for 10 minutes.
6. Centrifuge the lysates at 4500 rpm for 20 min and at 4°C
7. Prepare 50 mg/mL of the Celite beads in the buffer (100 mM Tris-HCl pH 8.0 or 100 mM Sodium phosphate buffer pH 6.5).
8. Add samples to the beads solution in the amount that dilute sample 10 times
9. Incubate all samples overnight at 4°C on the rotating mixer.
10. After overnight incubation, centrifuge the tubes at 4000 rpm for 5 minutes to settle the beads.
11. Resuspend the beads in 1 mLl of the buffer, spin the tubes at 3500g for 3 minutes and discard the wash buffer.
12. Perform two more washes following the same protocol.
13. After removing the third wash buffer, resuspend the beads with the starting volume of the buffer.
14. Use 100 uL of the bead suspension to measure fluorescence or enzymatic activity.
15. For the stability test, incubate the immobilised and free enzyme at 37°C on the rotating mixer, then draw sample out at various time point to measure the fluorescence or assay activity
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Enzymatic Assay
1. Methylumbelliferyl-cellobioside
- Take 20μl of the 200μl samples from the immobilisation experiment
- Add methylumbelliferyl-cellobioside (MUC) to a final concentration of 0.2mM into the 20μl samples.
- Take 1ml of phosphate-buffered saline in a UV cuvette and measure the absorbance by Jenway 7305 spectrophotometer at 348nm and then blank the spectrophotometer with the same buffer.
- Incubate the samples at 37°C for an hour.
- Place the samples on ice to stop the reaction and measure the absorbance at 348nm.
- To determine the enzymatic activity of Cex in the sample, take the difference between the absorbance before and after incubation and relate to a standard curve of 4-methylumbelliferone.
2. O-nitrophenol-xylopyranoside
- Add 60μl the sample to 300μl of 0.5% w/v O-nitrophenyl-xylopyranoside (ONPX), 150μl sodium acetate (pH 5.0) and 90μl.
- Add 200μl of this reaction to the 800μl sodium phosphate buffer (pH 12.0) and transfer to a UV cuvette.
- Measure the absorbance at 405 nm.
- Place the remaining reaction in a thermomixer set at 300rpm for 2 hours at room temperature.
- After 2 hours, take 200μl from the reaction and add to a UV cuvette containing 800μl sodium phosphate buffer (pH 12.0) and measure the absorbance at 405 nm.
- To determine the enzymatic activity of Cex in the sample, relate the difference in absorbance before and after incubation to a standard curve of 4-nitrophenol.
1. DNS activity assay
- Evenly mix 400µl crude enzyme lysate of recombinant endoglucanase with 1.6ml CMC-PBS solution (2%CMC with PBS buffer).
- Prepare the blank control with 400µl crude enzyme and 1.6ml PBS buffer without the substrate.
- Incubate the samples in a water bath at 37°C for 4h.
- Add 70µl of the sample to a new 1.5ml microcentrifuge tube containing 70µl of DNS solution.
- Heat the tube for 12min at 90°C and then put it on ice for 30 min to stop the reaction.
- Measure the absorbance by spectrophotometer (Genova Nano, JENWAY) at 540 nm, with three parallel samples in each group.
- Later, plot the glucose standard curve by measuring the absorbance of different glucose concentrations at 540 nm.
- Firstly, prepare 1mg/ml glucose stock and dilute it to 0, 0.2, 0.4, 0.6, 0.8, 1mg/ml, respectively.
- Mix all of them with equivalent DNS solution and heat for 12 min and then measure the absorbance at 540 nm.
2.4-MUC activity assay
- Take 20 µl crude lysate into a 1.5ml microcentrifuge tube.
- Add 20 µl of 10mM 4-MUC and 960 µl PBS buffer (pH=7.0) and then incubate at 37°C for 1h.
- Prepare the sample in triplicate.
- Prepare the blank control the same as each sample except for replacing crude enzyme extract lysate with 20 µl bugbuster master mix solution.
- Measure the absorbance by spectrophotometer at OD348.
- Prepare the standard curve of 4-MU with different concentrations.
1. Milk plate screening
- Autoclave 10% milk solution and LB agar separately, and then cool and mix to a final concentration of 1% milk.
- Add an appropriate antibiotic before pouring. Plate the transformants onto milk plates and incubate for 48-72 h at 30°C. Halos of clearance around colonies were analysed.
2. Ninhydrin assay
- Add clarified cell lysate or commercial enzyme solution to the substrate (BSA, or chopped feathers) and incubate (37°C, 100 mM potassium phosphate buffer pH=8, 1000 rpm).
- The reaction stops at the specified timepoint by addition of equal volume of 10% trichloroacetic acid (TCA).
- Remove the precipitated protein by centrifugation (10000g, 10 min).
- Add the supernatant to ninhydrin reagent (Sigma-Aldrich) in a 2:1 ratio and boil for 10 min.
- Measure the absorbance at 570 nm. Colour generation will be proportional to free amino acids released into the solution.
- Compare reaction samples to a standard curve prepared with varying concentrations of amino acid (Glycine, L-lysine dihydrochloride, L-cysteine).
3. Keratin azure assay
- Add 1 mL total volume of clarified cell lysate, supernatant, or commercial enzyme in 50 mM Tris-HCl (pH=8) to 20 mg keratin azure, pre-equilibrated in buffer.
- Incubate for 60-90 min at 37°C with constant agitation.
- Centrifuge the samples at 13,500g for 10 min and measure the absorbance of supernatant at 595nm.
1. para-Nitrophenol butyrate (pNPB)
- Add 100µg of 2 mM para-Nitrophenol butyrate (pNPB) substrate per mL of the soluble portion of the cell lysates.
- Add 45 mM Na2HPO4-HCl (pH 7.0), 90 mM NaCl, and 10% (v/v) DMSO and incubate the reaction mixture for 30 minutes at 250 rpm and at 30°C for all PETase-containing constructs and at 72°C for all Leaf-Branch Compost Cutinase-containing constructs
- Measure the absorbance at 415 nm for the production of para-Nitrophenol (pNP).
2. Absorbance of PET film)
- Treat the PET film with 70% ethanol and distilled water and air-dried.
- Incubate 100 µg/mL of the soluble fraction of cell lysates with biaxially 31 orientated two PET films (1 cm × 1 cm) supplied by DuPont Fujifilm Technologies for 3 hours in the buffer containing 50 mM Na2HPO4- HCl (pH 7.0), 50 mM glycine-NaOH (pH 10.0) at 30°C for all PETase-containing enzymes and at 72°C for all LCC-containing enzymes at 200 rpm.
- Terminate the reaction by heat treatment (85°C for 10 minutes).
- Collect the supernatant of the reaction mixtures and measure the absorbance at 260 nm for all the degradation products of PET.
- Due to commercial unavailability of MHET, establish its calibration curve using BHET (Sigma-Aldrich) at varying concentrations (0.01-0.1 mM).
3. Fluorescence of PET film)
- Adjust the fluorescence filter for excitation at 355 nm and emission at 460 nm by using a microplate reader (GREINER 96 F-BOTTOM).
- Place TPA (Merck Millipore) solution aliquots (dissolved in 0.1 M NaOH) at the various concentration (0.05-1.00 mM) in a 96-well microplate and add to a final volume of 150 µL with 0.1 M phosphate buffer (pH 8.5).
- Add 25 µL of 5 mM EDTA (Ethylenediamine tetraacetic acid) from Sigma-Aldrich in each well.
- Add 25 µL of freshly prepared 10 mM FeSO4 (Sigma-Aldrich) to initiate the hydroxylation of TPA molecule and incubating at room temperature for 20 minutes prior to analysis.
- To determine the amount of TPA produced by the reaction, add 100 µL of the supernatant from the reaction mixture along with the other chemicals as mentioned (Wei et al., 2012).
1. GshF can be assayed by the endpoint measurement of the glutathione produced.
2. Take 10 mM MgCl2, 20 mM glycine, 20 mM L-glutamic acid, 20 mM L-cysteine, 10 mM ATP, and 5 mg/mL wet weight cell crude extract in 100 mM Tris-HCl pH 8.0, with a total volume of 1 mL.
3. Take the crude lysate of E. coli Rosetta (DE3) pLysS as the negative control.
4. Add crude extract and incubate at room temperature for 10 minutes to initialise the reaction
5. Draw 25 uL of sample out to determine the GSH.
6. Detect the luminescence signal using Greiner, 96-well, F-bottom microtiter plate with the microplate reader.
7. Determine the amount of GSH following the protocol provided with the detection kit.
*KatG was assay based on the peroxidase activity
1. The reaction will take place in two different volumes: 50 uL and 1 mL.
2. Make a 1-mL reaction in UV/VIS cuvette composed of 4 mM 2,2'-Azino-bis (3-Ethylbenzthiazoline-6- Sulfonic Acid) (ABTS), 0.01% w/w H2O2, 0.04 mg/mL BSA, 0.008% v/v TritonX100 and 1 mg/mL wet weight cell crude extract in Sodium acetate buffer pH 5.0.
3. Measure the increment of absorbance at 405 nm with UV/VIS spectrophotometer every 5 or 10 seconds for 4.5 minutes.
4. Set the reaction in a 96-well F-bottom microtiter plate and measure oxidised ABTS produced from an elevated A405 with POLARstar Omega plate reader.
5. Take crude lysate of E. coli BL21 (DE3) as the negative control.
1. Perform the reaction in 1 mL and 50 uL; consist of 50 uM sodium pyruvate, 2 mM ABTS, 0.2 mM Thiamine pyrophosphate (TPP), 0.01 mM flavin adenine dinucleotide (FAD), 1 mM EDTA, 10 mM MgCl2, 0.04 mg/mL BSA, 0.008% v/v TritonX-100 and 20 ug/mL of protein in KatG crude extract in 20 mM 24 potassium phosphate buffer pH 5.0.
2. Add 10 uL L. plantarum crude extract for 1 mL-reaction and 10 uL Pox5 from cell-free protein synthesis in 50 uL-reaction.
3. Take crude extract of E. coli BL21 (DE3) and/or as the negative CFPS reaction.