Team:TJUSLS China/Experiments

EXPERIMENTS

Overview

This year,we selected 26 PETase genes and constructed them successfully. For the first time, seven of them expressed successfully and were further purified, but 19 of them formed inclusion bodies , we further optimized the expression conditions of them. After working hard in this summer, finally,we have successfully generated 26 protein mutants and texted their enzymatic properties. Details of experimental process are as follows.

Protocols & Lab journals

Molecular Cloning

Let’s take the protein sup-1 we firstly purified as an example ,and others are very similar to it.

Overlap PCR

The experimental steps are shown as follows

Mix the ingredients according to the following.
Template 50-1000ng
Sense Primer（10μM） 2.5μl
Anti-sense Primer（10μM） 2.5μl
5x SuperStar Omni Buffer 5μl
dNTP（each 2.5mM） 4μl
SuperStar Omni DNA Polymerase 1μl
ddH2O up to 50μl

Set the PCR instrument procedure according to the following table

ENZYME digestion

3mL restriction
endonuclease xhoI
3mL restriction endonuclease nedI
6mL 10´green buffer
3000ng  gene
Add to 60mL ddH2O
Heat in the water bath or heat blockat 37℃ for 1h

Agarose Gel electrophoresis

  1. Use 1×TAE buffer to prepare 1% Agarose mix in a flask, then put it in the microwave and heat it as long as it takes to completely dissolve the Agarose.
  2. Take out the conical flask, cool it in the wash basin to about 50°C. Add EB quickly, and then mix well.
  3. Pour the Agarose gel into gel tray and insert comb into slots. Let the gel solidify for 20min. Meanwhile, dilute the 10x green buffer to 1x and add to the DNA samples.Place the gel onto the electrophoresis apparatus ensuring that it is totally submerged in 1xTAE buffer.
  4. Carefully load each sample into its designated lane and 2μl DNA marker into a separate lane.Run at 120V for 20 min. If the sample have not completely separated, the time may be extended appropriately.
  5. Check the gel using a gel imager or under UV light, then take a photo with the Gel imager.

Gel purification

  1. Column balancing step: add 500μl buffer BL to the adsorption column CA2 (the adsorption column is put into the collection tube), centrifuge at 12,000 RPM (~13,400×g) for 1 min, dump the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
  2. Remove the single target DNA strip from the agarose gel (remove the excess as much as possible) and put it into a clean centrifuge tube, and weigh it.
  3. Add buffer PN to the glue block (if the gel weight is 0.1g, its volume can be considered as 100 μl, then add 100 μl PN), put in 50℃ water bath, during which gently turn up and down the centrifugal tube, to ensure that the glue block is fully dissolved. If there is any undissolved glue, continue to leave for a few minutes or add more PN until the glue is completely dissolved.
  4. Put the tube on ice. Add the solution obtained in the previous step to an adsorption column CA2 (the adsorption column was placed in the collection tube), place at room temperature for 2 min, centrifuge at 12,000 RPM (~13,400×g) for 30-60 SEC, and dump the waste liquid in the collection tube, place the adsorption column CA2 into the collection tube.
  5. Add 600μl bleach buffer PW to the adsorption column CA2 (check whether anhydrous ethanol has been added before use), centrifuge 30-60 SEC at 12,000rpm (~13,400×g), dump the waste liquid from the collection tube, and put the adsorption column CA2 into the collection tube.
  6. Repeat step 5.Put the adsorption column CA2 back into the collection tube and centrifuge at 12,000 rpm (~13,400×g) for 2 min to remove as much bleach as possible.
  7. Place the adsorption column CA2 at room temperature for several minutes to dry thoroughly to prevent the residual rinse fluid from affecting the next experiment.
  8. Put the adsorption column CA2 into a clean centrifugal tube, and drop an appropriate amount of ddH2O (about 30ul) onto the middle position of the adsorption film, and leave it at room temperature for 2 min.
  9. Final DNA solution was collected by centrifugation at 12,000 RPM (~13,400×g) for 2 min.

Preparation of empty vector-Transformation

  1. Take competent cells (E.coli DH5α)out of -80°C and thaw on ice (approximately 20-30 mins).
  2. Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator.
  3. Mix 1μl of DNA (usually 10 pg - 100 ng) into competent cells. Gently mix by flicking the bottom of the tube with your finger a few times.
  4. Incubate the competent cell/DNA mixture on ice for 20-30 mins.Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 90 secs.
  5. Put the tubes back on ice for 2 min.Add 600μl LB media (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min.
  6. The bacterial liquid was centrifuged at 3500rpm for 3 minutes, 400 microliters of supernatant was discarded, and the bacterial liquid was suspended again.
  7. Plate the transformation onto a LB agar plate containing the appropriate antibiotic.Incubate plates at 37°C overnight.

Preparation of empty vector-Extraction of Plasmid

  1. Collect the E. coli solution into the EP tube. Centrifuge at 12,000 rpm in a rotor for 1 minute. Remove the clear supernatant liquid.
  2. Add 250μL P1 (RNase A added, kept at 4 °C) to the EP tube to suspend bacterial precipitation.
  3. Add 250μL P2 to the EP tube, shake slightly up and down 6-8 times to lyse bacteria.
  4. Add 350μL P3 and invert the tube immediately and gently 6-8 times. Then centrifuge it at 12000rpm, 25℃ for 10 minutes.
  5. Regenerate column CP3 while centrifugation. Add 500μl Buffer BL. Centrifuge for 1 min at 12,000 rpm. Discard the flow-through.Move the clear supernatant liquid to CP3, at 12000rpm, 25℃ centrifuge for 30 seconds.
  6. Add 600μL PW to adsorption column CP3, 12000rpm, 25℃ centrifuge for 30 seconds.Repeat step 6.
  7. Move the adsorption column CP3 to new clean centrifuge tubes and then keep them opening for 5 minutes, so that the ethanol in the PW can be sufficiently volatilized.
  8. Drop 50μL 65°C double-distilled water into the middle of the adsorption membrane, static for 2min. Then centrifuge for 2 min at 12,000 rpm to collect DNA solution in EP tube.

Preparation of empty vector-Enzyme Digestion

(Refer to the protocol above)

Preparation of empty vector-Agarose Gel Electrophoresis

(Refer to the protocol above)

Preparation of empty vector-Gel purification

(Refer to the protocol above)

Ligation

Mix together:
  5×T4 DNA Ligase Buffer 2μl
  T4 DNA Ligase 0.5—1μl
  Vector cut by enzyme
  gene cut by enzyme
  ddH2O Add to 10μl
Heat in water bath or heat block at 22℃ for 30min.

Transformation

(Refer to the protocol above, attention to that DH5α strain was changed into BL21(DE3))

Strain Reservation

In the clean bench, using Micro pipette tip pick a single colony and put it into a LB liquid medium. Put into shaking table 10-12 h. In a clean centrifuge tube, add 600μL bacterium liquid and 400μL glycerin, then put into -80℃ refrigerator.

Massive expression

  1. Transform the plasmid into E.coli(BL21 DE3) used to express PETase protein.
  2. Take monoclone in the culture plate into LB tube and cultivate in shaking incubator overnight(10-12h) to activate bacteria.Test the OD600 number of bacteria, then pipe 5-10μl into each new 5 mL LB tube. Don’t forget to add antibiotic into tubes and mark them.Cultivate in shaking incubator for 3-4 hours until the OD600 of bacteria range from 0.6 to 0.8
  3. we transferred them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 1 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.

Add diggerent tags to help dissolve,including GST MBP SUMO

  1. Preparation of empty vector including pGEX-6p-1,pMATS,PET-28a-SUMO(Refer to the protocol above)
  2. Enzyme Digestion(Refer to the protocol above)
  3. Agarose Gel Electrophoresis(Refer to the protocol above)
  4. Gel purification(Refer to the protocol above)
  5. Ligation(Refer to the protocol above)

Protein Expression and Purification

Portein Expression

Set the gradient of condition to explore how to express it best. The conditions are are showen in the table below

  1. Transform the plasmid into E.coli(BL21 DE3) used to express PETase protein.
  2. Take monoclone in the culture plate into LB tube and cultivate in shaking incubator overnight(10-12h) to activate bacteria.
  3. Test the OD600 number of bacteria, then pipe 5-10μl into each new 5 mL LB tube. Don’t forget to add antibiotic into tubes and mark them.Cultivate in shaking incubator for 3-4 hours until the OD600 of bacteria range from 0.6 to 0.8.Pipet 200μl bacterial liquid as uninduced sample, and take another 600μl to mix with 400μl 50% glycerol to store.
  4. Then add inducer IPTG into each tube in different concentration, and incubate at 16°C for 16 hours or at 37°C for 4 hours shaking at 200-300rpm.After cultivating, pipet 200μl for each as induced sample.
    5. The method to make samples:
    1) Centrifuge the taken bacterial liquid at 12,000rpm for 3 minutes
    2) Drop the supernatant and resuspend the precipitate using 100μl ddw.
    3) Pipet 50μl resuspending liquid to mix with 10μl 6XSDS Loading buffer
    4) Boil it in dry bath at 100°C for 10 minutes

Use SDS-PAGE to check whether the target protein express or not and what the most suitable condition for its expression is.

Protein purification of sup-1

Lysis of the bacteria.

  1. Resuspend the frozen cell paste as best you can in the Lysis Buffer using a 10 mL pipet or whatever means necessary. Let this suspension incubate for 20 minutes at room temperature, or until the suspension becomes turbid and viscous due to release of the bacteria's genomic DNA.
  2. Smash the bacteria.
  3. Centrifuge at 18,000 rpm in a big rotor for 40 minutes at 4°C. Save the pellet and the supernatant.

Affinity chromatography of glutathione transferase (take GST tag fused with MUT-1 protein as an example）

  1. Remove the GST column from the 4℃ refrigerator. Wash the column with GST-binding buffer for 10 minutes to balance the GST column.
  2. Add the protein solution to the column, let it flow naturally and bind to the column.
  3. Add GST-Washing buffer several times and let it flow. Take 5μl of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
  4. We do enzyme cutting in the column. Add 1mg PPase to the column, overnight restriction enzyme digestion under 4℃.
  5. Add GST-Washing buffer several times. Check as above.
  6. Collect the eluted proteins for further operation. Recycle columns: Wash with 2×GSH buffer until Coomassie Brilliant Blue doesn’t turn blue when tested. Wash with 6M Guanidine hydrochloride , let stand for 10 min then drain. Wash with ddH2O for three times.

Immobilized metal ion affinity chromatography

  1. Remove the Ni column from the 4℃ refrigerator, which contains 20% alcohol. Wash the column with water for one time. Change to Ni-binding buffer for another time and balance the Ni column.
  2. Add the protein solution to the column, let it flow naturally and bind to the column. Repeat until the medium turns gray ( usually twice ).
  3. Add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
  4. We do enzyme cutting in the column. Add 1mg ULPase to the column, overnight restriction enzyme digestion under 4℃.
  5. Add Ni-Washing buffer several times. Check as above.
  6. Collect the eluted proteins for further operation. Recycle columns: Wash with 0.2mM EDTA , let stand for 10 min then drain. Wash with 6M Guanidine hydrochloride, let stand for 10 min then drain. Wash with ddH2O for three times. Fill up with NiSO4, place on a shaker overnight at 4℃.

Gel filtration of SUP-1

  1. According to the size of the protein PETase, firstly select the superdex75 gel column to adapt to the size of protein.Connect the proper column which adapts to target protein to AKTA high pressure tomographic system, use buffer of gel filtration chromatography (buffer: 25mMTris， 150mMNaCl，pH7.5) to prebalance the column of gel filtration chromatography of about 1.5 columns.
  2. Concentrate the protein to less than 1mL by centrifuging at 4℃, 3400rpm for 10 minutes in a high-speed centrifuge to remove insoluble substances and bubbles.Add the protein to the column of gel filtration chromatography through a 1mL sample collecting loop. The protein is eluted at a flow rate of 0.5mL/min. The eluent was collected according to the peak position.
  3. Use SDS-PAGE to check the result

Thermostability assay of Protein

Treatment of PET films

  1. Punch PET films with a hole puncher with a diameter of 0.6mm.
  2. The PET films was respectively soaked in 1mL 0.5 % Triton X-100, 1mL 10 mM Na2CO3, and 1mL distilled water, each remained 50°C for 30 min.
  3. The PET film was then air-dried at 37°C for the reaction.

Degradation of PET film

  1. The PET film was soaked in 300 μl of glycine/NaOH (pH 9.0) with 500 nM of the PETase Mutant enzyme(Good Fellow).
  2. The reaction mixture was incubated at 40 °C to 80 °C for 18 hours.

Termination Reaction

  1. After PET film was removed from reaction mixture, the enzyme reaction was terminated by heating at 85 °C for 10 min.
  2. The samples were centrifuged at 13,500 x g for 10 min, and the supernatant was analyzed by HPLC and UV-Vis.
  3. Reactions were performed in 1.5 mL microfuge tubes.

HPLC analysis of degradation product MHET

  1. The MHET peak area is used to calculate the amount of MHET produced in each PET hydrolysis reaction. Standard MHET was obtained from MHET which was bought from Sigma.
  2. Sampled 50μL for HPLC measurement，which was performed on a Waters e2695 equipped with a HyPURITY C18 column (4.6 × 250 mm).
  3. The mobile phase was methanol/18 mM phosphate buffer (pH 2.5) at a flow rate of 0.5 mL/min
  4. The effluent was monitored at a wavelength of 240 nm.
  5. The typical elution condition was as follows: 0 to 30 min, 25 % (v/v) methanol; 30 to 50 min, 25-100 % methanol linear gradient.

"Bulk" UV spectrophotometry to estimate PET degradation products

  1. Sampled 3μL for HPLC measurement
  2. Each time taked 1μL for measurement. Using a NanoDrop 1000 (TermoFisher Scientifc) to estimate PET degradation products, which acted a spectrophotometer where all degradation products contribute to absorbance.

Journals