Protocols

Protocols

General
Plastic degradation
Bioreporter
Biomass production
Expression system
Future steps

1. General protocols

PCR

Amplification of the PCR template should be achieved. The PCR reaction was used to amplify the commercial genes from IDT of the PETase and MHETase enzymes.
Estimated bench time: 30 minutes
Estimated total time: 2 hours
Safety requirements: Prepare the PCR in the laminar flow to protect the agents from potential contaminations with DNases or other DNA fragments. Additionally, wear gloves to protect the reaction from DNases. Lab coat and goggles for personal protection
Equipment:
  • SimpliAmp™ Thermal Cycler
  • PCR tubes and lids
Material:
  • Nuclease-free water
  • Reverse primer
  • Forward primer
Preparation prior protocol: Prepare Master Mix according to the following table and always add one more reaction to the prepared Master Mix to have a buffer when dividing the Master Mix to the individual tubes
Component Concentration Volume for 1 reaction
10X DreamTaq Buffer 5 µl
dNTP Mix, 2 mM 5 µl
Forward primer 0.05 µmol 0.25 µl
Reverse primer 0.05 µmol 0.25 µl
Template DNA 0.01 µg 1 µl
DreamTaq DNA Polymerase 1.25 U 0.25 µl
Nuclease-free water 38.25
Total volume 50 µL
Protocol:
  1. Mix well by pipetting up-and-down
  2. Run the following PCR programme:
    Step Temperature (°C) Time
    Initial denaturation 1 cycle 95 1-3 mins
    25-40 cycles Denaturation 95 30 sec
    Annealing* 58 30 sec
    Extension 72 1 min for PCR products up to 2 kb. For longer products, the extension time should be prolonged by 1 min/kb.
    Final extension 1 cycle 72 5-15 min
    Cool down 4 ---
    *The annealing times and primer amounts were calculated 0.5µM · DNA 0.01 µg

Agarose gel electrophoresis

Separation of the sample by size with 1% agarose gel
Estimated bench time: 15 minutes
Estimated total time: 60 minutes
Safety requirements: Wear thick gloves (neoprene) due to the usage of Ethidium bromide (carcinogen), a lab coat, and goggles for personal protection
Equipment:
  • Gel tray (Bio-Rad)
  • Gel comb (Bio-Rad) (size may vary depending on the sample volume)
  • Gel caster (Bio-Rad)
Material:
  • Agarose
  • 1x TAE buffer
  • MilliQ water
  • Ethidium bromide
  • Erlenmeyer flask
Preparation prior protocol: Calculations for agarose amount:
Final Agarose percentage 1x TAE Total Volume Agarose
1% 200mL 2g
Protocol:
  1. Weight 2 g of agarose on a scale
  2. Add the agarose to 200mL 1x TAE buffer in an Erlenmeyer flask
  3. Microwave 1min-30s and swirl and continue until the liquid is clear. Then, cool down
  4. Add 40 μl of Ethidium Bromide (final concentration of 0.2 μl/ml). Mix well
  5. Place the comb on the casting tray
  6. Pour the gel into the casting tray
  7. Let gel solidify at room temperature for 30 min
Estimated total time: 2 minutes per sample
Safety requirements: Wear gloves, lab coat, and goggles for personal protection
Material:
  • DNA Gel Loading Dye (6X) (Thermo Scientific TM)
Preparation prior protocol: Calculations for DNA Gel Loading Dye amount:
DNA Gel Loading Dye concentration Ratio dye:sample
6X 1:6
Protocol:
  1. Add the 6X loading dye to the samples
  2. Mix by pipetting up-and-down
Estimated total time: 60 minutes
Safety requirements: Wear gloves, lab coat, and goggles for personal protection
Equipment:
  • Gel chamber
  • Power supply
Material:
  • Solidified 1% agarose gel
  • TAE buffer
  • Samples with dye
  • GeneRuler 1kb DNA ladder (Thermo Scientific TM)
Protocol:
  1. Fill the Gel chamber with TAE up to the mark and place an agarose gel inside it.
  2. Load ~6 mL of DNA ladder
  3. Load ~30 mL of the samples
  4. Ensure that the running buffer is high enough
  5. Run the gel for 30-45 minutes at 120V
Estimated total time: 10 minutes
Safety requirements: Wear gloves, lab coat, and goggles for personal protection
Equipment:
  • Gel documentation system (Bio-Rad)
Material:
  • 70% Ethanol
Protocol:
  1. Stop the gel run when the loading dye line is at the bottom of the gel
  2. Take out agarose gel from the chamber and tilt to remove the excess liquid
  3. Place gel in the gel documentation system
  4. Select Bio-rad image lab on the computer
  5. Select Nucleic Acids and click on Ethidium Bromide gel
  6. Select position gel and agree to Filter 1
  7. Position the gel centered
  8. Select Run protocol, print and save the image

Gel extraction

Estimated bench time: 1 hour
Estimated total time: 1 hour
Safety requirements: Wear UV protective goggles and attach a UV shield to the gel doc for the marking of the gel, while the UV is turned on to protect yourself from the radiation. Wear gloves throughout the experiment to protect yourself from residual ethidium bromide and UV radiation during the gel cutting. Lab coat and goggles for personal protection. The binding buffer is harmful if swallowed and to aquatic life with long-lasting effects. It causes severe skin burns and eye damage.
Equipment:
  • Gel documentation system
  • Centrifuge
  • 1.5 mL eppendorf tubes
  • GeneJet Gel Extraction Kit by Thermo Scientific™
  • Disposable scalpel
  • Heat block
  • Vortex
Material:
  • Provided by GeneJet Gel Extraction Kit by Thermo Scientific™
  • 96 - 100 % Ethanol
  • 3 M sodium acetate, pH 5.2
Protocol:
  1. Use a clean scalpel and cut as close to the band as possible
  2. Add 1:1 binding buffer to the gel volume (1 µL:1 mg)
  3. Incubate for 10 min at 60 °C until the gel is dissolved (invert now and then during incubation)
  4. Vortex mixture
  5. The mixture should have a yellow color indicating an optimal pH, if the color is orange or yellow add 10 µL of 3 M sodium acetate -> pH will become 5.2
  6. Transfer up to 700 µL of the solubilized gel solution to the GeneJET purification column
  7. If the volume exceeds 800 µL have several centrifuge steps of 30 - 60 sec at >12000 x g
  8. Centrifuge for 1 min at >12000 x g
  9. Discard the flow-through and place the column back into the same collection tub
  10. Add 700 µL of Wash Buffer (ethanol previously added to WB)
  11. Centrifuge for 1 min at >12000 x g
  12. Discard the flow-through and place the column back into the same collection tube
  13. Centrifuge the empty GeneJET purification column at >12000 x g for an additional 1 min to completely remove residual wash buffer
  14. Transfer the GeneJET purification column into a clean 1.5 mL microcentrifuge tube
  15. Add 50 μL of nuclease-free water to the center of the purification column and centrifuge for 1 min
  16. Discard the purification column and Store purified DNA at - 20 °C

Nanodrop measurement

Estimated total time: 5 minutes
Safety requirements: Wear gloves, lab coat, and goggles for personal protection
Equipment:
  • Nanodrop
Material:
  • Samples
  • Blank
Protocol:
  1. Select concentration measurement for DNA program and dsDNA
  2. A clean device with ddH2O
  3. Measure blank for calibration and wipe away
  4. Repeat blank measurement
  5. Add 1-2 mL of the sample and measure
  6. Write down values including concentrations and measurements at specific wavelengths to assess the purity

2. Plastic degredation protocols

Thermal pre-treatment

Estimated total time: 2 days
Safety requirements: Wear gloves, lab coat, and goggles for personal protection
Equipment:
  • Oven
  • Heat protective gear
Material:
  • Aluminum foil
Protocol:
  1. place PET on aluminum foil on top of a hot plate at 290 °C (about 1 min or when melted)
  2. Once PET is melted, cover it with another aluminum foil
  3. Use a stainless-steel cylinder to press the melted PET into a flat film (1 min)
  4. Heat the PET film at 290 °C for 15 min
  5. Cover with aluminum foil and put into an ice bath (rapid cooling)
  6. After cooling, separate the PET from the aluminum foil

Chemical pre-treatment

Estimated total time: 3 days
Safety requirements: Wear gloves, lab coat, and goggles for personal protection
Equipment:
  • A fume hood
Material:
  • 10M NaOH
Protocol:
  1. Cut 1×1cm2 pieces from the PET bottle from the middle area of the bottle
  2. Wash and let dry completely, preferably 20h at 40°C
  3. Immerse into NaOH (10M) for 24 h at room temperature
  4. Washed with distilled water, and dried for about 20h
  5. Ready for use

PET depolymerization

Estimated total time: 3 days
Safety requirements: Wear gloves, lab coat, and goggles for personal protection
Equipment:
  • Glass tubes
Material:
  • Purified PETase supernatant
  • PET thermal pre-treated
  • PET chemical pre-treated
  • PET film
Protocol:
  1. Measure all three different of PET and put it into glass tube each
  2. Add 5 ml of PETase supernatant
  3. Incubate in the shaker at 37°C at 150 rpm for 3 days
  4. Analyze the PET plastic with an electro microscope

3. Bioreporter protocols

Restriction enzyme digestion

Estimated bench time: 1 hour
Estimated total time: 1 hour
Safety requirements: Wear gloves, lab coat, and goggles for personal protection from lysis buffer that can cause eye and skin irritation and for the protection of the samples from DNases.
Equipment:
  • Centrifuge
  • 1.5 mL eppendorf tubes
Material:
  • Thermo Scientific GeneJET Plasmid Miniprep Kit.
  • All purification steps should be carried out at room temperature.
  • All centrifugations should be carried out in a table-top microcentrifuge at >12000 × g (10,000-14,000 rpm, depending on the rotor type).
Protocol:
  1. Use 1-5 mL of E. coli culture in LB media for purification of high-copy plasmids.
  2. Resuspend the pelleted cells in 250 μL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
  3. Add 250 μL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
  4. Add 350 μL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.
  5. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
  6. Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
  7. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
  8. Wash the GeneJET spin column by adding 500 μL of Wash Solution I (diluted with isopropanol) and centrifuge for 30-60 sec. Discard the flow-through.Note. This step is essential to remove trace nuclease activity.
  9. Add 500 μL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
  10. Repeat the wash procedure (step 8) using 500 μL of the Wash Solution. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
  11. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube. Add 50 μL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.
  12. For elution of plasmids or cosmids >20 kb, prewarm the Elution Buffer to 70°C before applying to the silica membrane.
  13. Discard the column and store the purified plasmid DNA at -20°C.
Estimated bench time:30 minutes
Estimated total time: 65 minutes
Safety requirements: Wear gloves, lab coat, and goggles for personal protection
Equipment:
  • Incubator 37°C
  • Heat block 80°C
  • 1.5mL Eppendorf
Material:
  • Nuclease-free water
  • Restriction enzymes (FastDigest Ndel and Xhol)
  • Plasmid template
  • Buffer (10x Universal FastDigest)
Preparation prior protocol:
Calculations for the restriction reaction
Component Stock Concentration Final Concentration Volume for 1 reaction
Buffer 10x 1x 2 μL
Ndel restriction enzyme 1U/μL 1 μL
Xhol restriction enzyme 1U/μL 1 μL
Cector 1 μg
Nuclease-free water Fill up to 20μL
Protocol:
  1. Add all the reagents in an Eppendorf tube
  2. Incubate the digestion reaction at 37°C for 30 minutes
  3. Incubate the digestion reaction at 80°C for 5 minutes for inactivating the reaction
Estimated bench time:30 minutes
Estimated total time: 65 minutes
Safety requirements: Wear gloves, lab coat, and goggles for personal protection
Equipment:
  • Incubator 37°C
  • Heat block 80°C
  • 1.5mL Eppendorf
Material:
  • Nuclease-free water
  • Restriction enzimes (FastDigest Ndel and Xhol)
  • Synthetic insert (PETase or MHETase)
  • Buffer (10x Universal FastDigest)
Preparation prior protocol:
Calculations for the restriction reaction
Component Stock Concentration Final Concentration Volume for 1 reaction
Buffer 10x 1x 2 μL
Ndel restriction enzyme 1U/μL 1 μL
Xhol restriction enzyme 1U/μL 1 μL
Synthetic insert 0.2 μg
Nuclease-free water Fill up to 20μL
Protocol:
  1. Add all the reagents in an Eppendorf tube, mix gently and spin down
  2. Incubate the digestion reaction at 37°C for 60 minutes
  3. Incubate the digestion reaction at 80°C for 5 minutes for inactivating the reaction (this step can be omitted if immediately followed with a PCR purification kit)

Ligation

Estimated bench time: 15 minutes
Estimated total time: 24 hours
Safety requirements: Wear gloves, lab coat, and goggles for personal protection
Equipment:
  • Fridge
Material:
  • Digested and pure vector
  • Digested and pure synthetic inserts
  • 10X T4 DNA ligase buffer
  • T4 DNA ligase
  • Nuclease-free water
  • 1.5mL Eppendorf
Preparation prior protocol:
Use the following equation to calculate the amount of insert needed:
[Vector (ng) * insert size (kb) / vector size (kb)] * 5 (excess)
Protocol:
  1. Add the reagents in an Eppendorf as indicated:
    10X T4 DNA ligase buffer 2 μl
    Digested and pure vector 50 ng
    Digested and pure synthetic inserts 40 ng
    T4 DNA ligase, 5U/μl (Thermo Fischer Sceintific) 1 μl
    Nuclease-free water 1 μl
  2. Incubate at 4°C overnight OR at 16°C for 1-2 hours

Transformation

Estimated total time: 2 days
Safety requirements: Wear gloves, lab coat, and goggles for personal protection
Equipment:
  • Incubation shaker
  • Spectrophotometer UV-Vis
  • Refrigerated centrifuge
  • Cuvette
  • 50 ml Falcon tubes
Material:
  • LB broth medium
  • Sterile 10% glycerol
  • Sterile MQ water
Protocol:
Day 1:
  1. Grow an overnight culture in 50 mL of LB medium 37C, 250 rpm
  2. Prepare 2x 500 ml of LB medium in two 2L flask

Day 2:
  1. Inoculate each flask with 10 mL and 20 mL overnight cultivation
  2. Grow the cells for approximately 2 hours by measuring OD every 20 mins, until they reach mid-exponential phase (OD600 of 0.4 best, max 0.6.)
  3. Then, rapidly transfer the growth bottle to an ice-water batch for 15-30 min, mixing occasionally
  4. ~30 min before you plan to centrifuge your cells, set the centrifuge to 4C. Place falcon tubes on ice.
  5. Transfer the cells to pre-chilled 50 ml Falcon tubes
  6. Pellet the cells by centrifugation for 15 minutes at 1000 G. Pour off supernatant
  7. Resuspend the cell pellets in a total 500 ml of ice-cold MQ
  8. Pellet cells by centrifuge15 minutes at 1000 G pour supernatant
  9. Wash again by adding 250 ml of ice-cold 10% glycerol, resuspend
  10. Pellet the cells by centrifugation at 1,000g for 20 min at 4ºC
  11. Wash again by adding 250 ml of ice-cold 10% glycerol, resuspend
  12. Centrifugation at 1,000g for 20 min at 4ºC, resuspend in 10 ml of ice-cold 10% glycerol
  13. Centrifuge at 1,000g for 20 min at 4ºC
  14. Resuspend each pellet in approximately 2.5 mL of 10% glycerol
  15. Divide into 30 µl aliquots in 1.5 ml tubes. Freeze with liquid N2 at -80°C.
Estimated total time: 10 minutes
Safety requirements: Wear gloves, lab coat, and goggles for personal protection
Equipment:
  • MicroPulser Electroporator (Bio-Rad)
  • MicroPulser Electroporation cuvettes 0.2 cm
  • 1.5 ml centrifuge tubes
  • 2 ml of centrifuge tubes
Material:
  • SOC medium
  • LB with antibiotic plates
Protocol:
  1. Pre-cooled: two 1.5 ml sterile tubes, 2 ml of sterile tubes, and cuvettes (pre-washed with ethanol and rinsed with MQ).
  2. Place SOC medium on the ice
  3. Add 25 μl of competent cells and 3 μl of ligation reactions to 1.5 ml sterile tubes
  4. Transfer the total liquid (28 μl) from both tubes to two cuvettes
  5. Program the MicroPulser Electroporator according to bacteria setting: Ec2 bacteria (E.coli; 0.2 cm cuvette)
  6. Place the cuvette into MicroPulser Electroporator to reach the electrode
  7. Press the pulse with one hand and check the code on the display (make sure the bacteria are still alive)
  8. Add 1000 μl of SOC medium to the cuvette
  9. Repeat steps 6-8 for another sample
  10. Transfer the cells from both cuvettes to new 2 ml sterile tubes
  11. Add more than 1000 μl of SOC to each cuvette, and transfer to the 2ml tubes
  12. Incubate the competent cells at 37°C for 1-2 h.
  13. Plate the cells to LB with antibiotics plates. Concentration: 20μl, 40μl, 80μl 100μl, and rest of the liquid (after centrifugation).
  14. Incubate overnight at 37°C

PCR purification

Rapid and efficient removal of short primers, dNTPs, and enzymes.
Estimated bench time: 30 minutes
Estimated total time: 30 minutes
Safety requirements: Wear gloves, lab coat, and goggles for personal protection
Equipment:
  • Microcentrifuge
  • Microcentrifuge tubes 2mLli>
  • Heating block
Material:
  • Binding Buffer
  • Wash Buffer
  • Elution Buffer
  • GeneJET Purification Columns
  • 96-100% Ethanol
  • Isopropanol
  • 3M Sodium acetate (pH 5.2)
Preparation prior protocol:
The Wash Buffer in the kit needs to be diluted with ethanol (96-100%)
Protocol:
  1. Add a 1:1 volume of binding buffer to the digested samples and mix thoroughly
  2. Check the color of the mix. A yellow color indicates an optimal pH for DNA binding, if the color is orange or violet, add 10 mL of 3M sodium acetate (pH 5.2) and mix
  3. Transfer 800 mL of the mix to the GeneJET purification column and centrifuge for 1 minute at 12000g
  4. If the total volume exceeds 800 mL, the solution can be added to the column in stages by discarding the flow-through
  5. Add 700 mL of Wash Buffer to the GeneJET purification column and centrifuge for 1 minute at 12000g
  6. Discard flow through and place the column back into the collection tube
  7. Centrifuge the empty column for 1 minute at 12000g
  8. Transfer the column to a clean 1.5mL microcentrifuge tube, add 50 mL of Elution Buffer, and centrifuge for 1 minute at 12000g
  9. Discard the GeneJET purification column and store the purified DNA at -20°C

4. Biomass production

Bioconversion

Estimated bench time: 48 hours
Estimated total time: 6 days
Safety requirements: Wear gloves, lab coat, and goggles for personal protection.
Equipment:
  • Refrigerated centrifuge
  • NanoQuant Plate TECAN
Material:
  • LB broth
  • Terephthalic acid (TPA)
  • Ethylene glycol (EG)
Protocol:
  1. Culture the strains into 5 ml of M9 broth medium containing monomers (50 mM (10 g) ethylene glycol or 20 mM Terephthalic acid). Incubate at 28°C and 150 rpm overnight
  2. Transfer 1 ml of precultures to 1-liter Erlenmeyer flasks containing 250 ml of M9+monomers broth medium, and cells were grown to stationary phase in a shaker at 30°C and 150 rpm
  3. Harvest cultures with centrifugation at 4°C and 13,700 × g for 20 min
  4. Wash with 40 ml of M9 medium without a carbon source. Centrifuge again at 4°C and 13,700 × g for 20 min
  5. Resuspended in 50 ml of M9 medium containing monomers, and the OD600 was adjusted to 7.5. In order to provide a stable pH of 6.7 during the entire experiment, sodium phosphate buffer was used at 0.1 M.
  6. 50 ml of this cell suspension is transferred to 250 ml Erlenmeyer flasks and incubated in a rotary shaker at 28°C and 150 rpm for 120 h. Samples (1.5 ml) are taken at regular time intervals, and cells are removed by centrifugation at 20,000 × g for 1 min (centrifuge 5417 C; Eppendorf) for subsequent high-performance liquid chromatographic (HPLC) analysis of the supernatant.

Monomer uptake analysis using HPLC

Estimated bench time: 2 hours
Estimated total time: 1 day
Safety requirements: Wear gloves, lab coat, and goggles for personal protection.
Equipment:
  • HPLC
  • HPLC vials
  • 0.22 µm Vent Syringe Filter
  • 2 ml eppondorf tubes
Material:
  • Biomass samples
  • Methanol
  • M9 with TPA
Protocol:
  1. Thawing the biomass samples and vortex it gently
  2. Mix 500 µl of methanol to the eppendorf tubes with 1000 µl of biomass samples
  3. Filter with Vent Syringe Filter and store it onto the HPLC vials
  4. For HPLC analysis, samples were mixed with the internal standard xylitol at a final concentration of 10 mM and were filtered through a 0.2-μm-pore-size filter.
  5. HPLC analysis can be carried out on an Agilent system (1200 series) using an Aminex HPX-87H cation-exchange resin column (300 by 7.8 mm; Bio-Rad) at 60°C, with 5 mM H2SO4 as the mobile phase and a flow rate of 0.5 ml/min.
  6. The substrates and products can be quantified using the corresponding standards and a refractive index detector (Agilent 1200 series, G1262A). The detector temperature is set to 35°C.

Protein Bradford Assay

Estimated bench time: 1 hour
Estimated total time: 3 hours
Safety requirements: Wear gloves, lab coat, and goggles for personal protection.
Equipment:
  • UV/VIs spectrophotometer
  • Cuvettes
Material:
  • Biomass samples
  • BSA (bovine serum albumin)
  • Bio-Rad protein assay Dye Reagent (500-0006)
Protocol:
  1. Prepare a 1 mg/ml solution of BSA in water or buffer (weigh accurately) From there prepare dilutions: 0, 5, 10, 25, 50, 75, 100 µg/ml dilutions (some of them are enough)..for a standard line.
  2. Dilute reagent 1:1 in water
  3. Prepare 3 dilutions of your protein sample that will be approximately in the standard range (in same solvent like BSA).
  4. Use cuvettes: 100 µl standard or protein/ cuvette + 900 µl reagent
  5. Let stand for at least 5 but less than 30 min
  6. Measure absorption at 595 nm
  7. Create standard line for BSA (A595 vs concentration) and calculate the concentration of protein from the A595 values and the standard line

5. Expression system

Protein Expression

Estimated bench time: 2 hours
Estimated total time: 6 days
Safety requirements: Wear gloves, lab coat, and goggles for personal protection.
Equipment:
  • Erlenmeyer flasks
Material:
  • LB-kanamycin agar plates
  • LB-kanamycin broth
  • IPTG
Protocol:
Selection and Overnight Culture:
  1. Spread transformed cells onto LB agar plates supplemented with 100 μg/mL kanamycin.
  2. Incubate the plates overnight at 30°C
  3. Select a single colony from the agar plate and use it to inoculate 15 mL of LB medium containing 100 μg/mL kanamycin in a suitable culture tube.
  4. Grow the culture overnight at 37°C and 130 rpm in a shaking incubator
Expression Culture:
  1. Prepare 1L baffled Erlenmeyer flasks, each containing 200 mL of LB medium supplemented with 100 μg/mL kanamycin.
  2. Inoculate each flask with 2 mL of the overnight culture
  3. Incubate the flasks in a shaking incubator at 33°C and 130 rpm until the culture reaches an optical density at 600 nm (OD600) of 1.
  4. Induce protein expression by adding 1 mM IPTG to the flasks when the culture reaches an OD600 of 1.
  5. Lower the temperature to 16°C when the culture reaches an OD600 of 2.5
  6. Continue incubating the flasks at 16°C and 130 rpm for a total expression time of typically 18 hours. (24-48h)

SDS-PAGE

For the preparation of an SDS-gel to separate protein samples according to size
Estimated bench time: 30 minutes
Estimated total time: 2 hours
Safety requirements: Wear gloves, lab coat and goggles for personal protection and due to working with SDS, which is a flammable solid and combustible dust with acute inhalation toxicity targeting the respiratory system, forming dusts and mists, causing skin corrosion/ irritation, serious eye damage/ eye irritation. β-mercaptoethanol, which causes eye, skin, and respiratory tract irritation and is harmful if swallowed.
Equipment:
  • Heat Block
Material:
  • Sample buffer
  • Colour protein standard
Protocol:
  1. Dilute the pellet samples 5-10x with dH2O.
  2. Mix 75 µl sample (diluted pellet, supernatant, and concentrated supernatant) with 25 µl of Laemmli SDS sample buffer containing β-ME
  3. Boil the samples at 94°C for 5 min in a heat block.
  4. Pipette the 25 µl of each sample into the wells
For running the SDS gel
Estimated bench time: 5 min
Estimated total time: 5 min
Safety Requirements:
Wear gloves, lab coat, and goggles for personal protection from the ethanol while cleaning the gel chamber. The actual gel chamber set-up takes place on the bench top.
Equipment:
  • SDS-gel chamber by Thermo Scientific™
Material:
  • 70% ethanol
  • Water
Protocol:
  1. Clean 1 glass plate with 0.75 mm spacers with ethanol and leave to dry
  2. Assemble glass slide and pre-made gel on tabletop of bench and insert into the chamber (important white part goes into the centre)
  3. Insert glass slides into the chamber turning the short glass to the inside
  4. Clip the side pieces to ensure that the glass-slides are securely fastened
  5. Check that the glass slides are levelled at the bottom
For the separation of an SDS-gel to separate protein samples according to size.
Safety Requirements: Wear gloves, lab coat, and goggles for personal protection.
Note: Instead of making the SDS-gel ourselves, we used Mini-PROTEAN® Precast Gel from Bio-Rad.
Separating proteins according to their molecular mass to confirm successful protein expression
Estimated bench time: 30 minutes
Estimated total time: 2 hours
Safety requirements: Wear thicker neoprene gloves, lab coat and goggles for personal protection.
Equipment:
  • Power supply
  • SDS-gel chamber by Thermo Scientific™
Material:
  • SDS gel
  • 1x running buffer
Protocol:
  1. Set up the running device with two gels on each side
  2. Fill the inner chamber with 1x running buffer until it covers the glass plates. Avoid air bubbles. If any leaking occurs, reassemble the unit again
  3. Fill the lower buffer chamber with 1x running buffer until the sandwiches are fully immersed
  4. Carefully remove the comb, if air bubbles or residual acrylamide in the pockets remain, wash the pockets with 5x running buffer twice at the sink
  5. Load samples
  6. Run the gel at 120 V for 1 hour
Separating proteins according to their molecular mass to confirm successful protein expression and to visualise the run gel
Estimated bench time: 30 minutes
Estimated total time: 2 days
Safety requirements: Wear thicker neoprene gloves, lab coat and goggles for personal protection.
Equipment:
  • Plastic box
Material:
  • MilliQ water
  • Coomassie Brilliant solution
Protocol:
  1. Take the gel sandwich out of the electrophoresis chamber
  2. Separate the glass plates from the gel
  3. Loosen the gel from the plate by rinsing it with water and let the gel slip into the plastic box..
  4. Remove the water
  5. Add Coomassie Brilliant blue solution to cover the gel and incubate overnight while gently shaking.
  6. Remove excess rinsing it with a small amount of MQ.
  7. Destain with a destaining solution with gentle shaking overnight
  8. Scan the gel

6. Future steps

Estimated bench time: 20 min
Estimated total time: 1.5 h
Safety requirements:
Wear gloves, lab coat, and goggles for personal protection and protection from the imidazole contained in the lysis buffer, which is harmful if swallowed, causes severe skin burns and eye damage. It also may damage fertility and may damage an unborn child.
Equipment:
  • Sonicator
  • Rotator
Material:
  • Lysis buffer
  • Ice
Preparation prior protocol:
  1. Prepare a lysis buffer according to the buffer protocols.
Protocol:
  1. Lyse bacterial pellet in 10 ml of the lysis buffer
  2. Add suspension to 50 ml falcons and incubate for 20 minutes at RT in a rotator
  3. Sonicate on ice. Use six 10 second bursts at 200-300 W (16-20 amp) with a 10 second cooling period between each burst. Use a sonicator equipped with a microtip
  4. Centrifuge for 30 minutes at 12,000 rpm at 4°C
  5. Remove supernatant to a fresh tube and take a 5 μl sample and store at -20°C for SDS
  6. Take sample from pellet and store at -20°C for SDS
Estimated bench time: 1 h
Estimated total time: 1 h
Safety requirements:
Wear gloves, lab coat and goggles for personal protection from imidazole, PBS buffer and HisPurTM Ni-NTA Resin. Imidazole is harmful if swallowed, causes severe skin burns and eye damage. May damage fertility and may damage an unborn child. PBS buffer can cause serious eye irritation and HisPurTM Ni-NTA Resin may cause allergy, asthma symptoms, or breathing difficulties if inhaled, an allergic skin reaction, cancer, damage to an unborn child, damage to organs through prolonged or repeated exposure. Harmful to aquatic life with long-lasting effects.
Equipment:
  • Centrifuge
  • 2 mL eppendorf tubes
  • 1.5 mL eppendorf tubes
Material:
  • Ni-NTA spin column (His-tag) purification from Qiagen
  • Lysed bacterial cells
Preparation prior protocol:
  1. Prepare buffer A and B according to the buffer protocols
Protocol:
  1. One column can contain around 750 μl of the lysed solution
  2. Take 350 μl of sample supernatant and 350 μl of buffer A into an eppendorf tube and mix by inverting the tube a few times. If you have a lot of samples you can take a bigger falcon tube and place 5 ml of sample and 5 ml of buffer A into the tube.
  3. Place the 700 μl of mixture into the Ni-NTA column
  4. Incubate in RT for 5 min
  5. Centrifuge the column for 1 min at 3600 x g
  6. Discard the flowthrough
  7. If you have more sample, repeat step 3 to 5 until all sample has been used
  8. Add 500 μL of wash buffer
  9. Centrifuge 1min 3600 x g
  10. Discard flowthrough
  11. Repeat step 8 - 11
  12. Remove the collection tube and replace it with a new 1.5 mL eppendorf tube
  13. Add 100 μL of buffer B
  14. Incubate for 5min in RT
  15. Centrifuge for 1 min 3600 x g
  16. Save and remove the eppendorf tube