Target：Make LB medium

Material：

Procedure：

1. Place the weighing paper on the scale and weigh the net weight.
2. Weigh out 2.5 grams of tryptone on a scale and add it to the flask.
3. Place the weighing paper on the scale and weigh the net weight.
4. Weigh out 1.25 grams of yeast extract on a scale and add it to the flask.
5. Place the weighing paper on the scale and weigh the net weight.
6. Weigh out 2.5 grams of NaCl on a scale and add it to the flask.
7. Measure out 250mL of ddH2O with a graduated cylinder and add it to the flask.
8. Write the name of the medium on the flask and the sealing film, then seal the mouth of the flask with the sealing film.
9. Shake the flask gently to even out the medium.
10. Put it into the high-pressure steam sterilization pot, sterilize for 121 degree Celsius, 20 minutes.

Target：Culture bactreia

Material：

Procedure：

1. The hands were disinfected with ethyl alcohol, and the hands were put into the aseptic console to do the experiment.
2. Lable the cell culture tube (name of the bacteria solution, operator’s name, date).
3. Use a injection syringe to add 3mL LB medium to the cell culture tube.
4. Use a pipette to add 3μL Kan+ to the cell culture tube.
5. Use a pipette to add 5μL IsPETase to the cell culture tube.
6. Gently shake the tube and wrap the sealing film around the side of the lid.
7. Make another tube with the same solution.
8. Repeat the process with dsPETase.
9. Repeat the process with MHET.
10. Repeat the process with PET28a.
11. Bundle four in groups and put them in a constant temperature shaker at 37 degrees Celsius for 12-16 h.

Target：To extract the target plasmid pET28a

Material：

1. Buffer Sp1：to give a suitable environment
2. Buffer Sp2：Lysis of cell membrane
3. Buffer Sp3：stop the reaction
4. adsorption column
5. collecting pipe
6. centrifuge tube
7. Adsorption film：uptake plasmid
8. Wash solution：flushing impurity
9. Elution buffer：flushing target plasmid
10. Nanodrop：test DNA concentration

Procedure：

1. Check that RNase A has been added to Buffer SP1.
2. Check whether anhydrous ethanol has been added to the Wash Solution.
3. Check whether Buffer SP2 and SP3 are precipitated.
4. Adsorption column balance: Add 500μL Buffer S to the adsorption column (the adsorption column is put into the collection tube), centrifuge for 1min at 12,000 Xg, dump the waste liquid in the collection tube, and put the adsorption column back into the three collection tubes.
5. Take 1.5-5mL of overnight cultured bacterial solution, centrifuge 8,000Xg for 2 minutes to collect bacterial bodies, and discard the medium.
6. Add 250μL Buffer SP1 to the precipitate and completely suspend the bacteria.
7. Add 250 μL Buffer SP2 and immediately gently reverse the centrifugal tube for 10 times and mix well. Let stand at room temperature for 2-4 minutes.
8. Add 350 μL Buffer SP3 and immediately gently reverse the centrifuge tube 5-10 times to mix.
9. 12,000Xg centrifuge for 5-10 min. Transfer the supernatant into the adsorption column, centrifuge 8,000Xg for 30 seconds, and drain the liquid in the collection tube.
10. (Optional) Add 500 μL Buffer DW1, centrifuge 9,000Xg for 30 seconds, and drain the liquid in the collection tube.
11. Add 500 μL Wash Solution, centrifuge 9,000Xg for 30 seconds, and drain the liquid in the collection tube.
12. Repeat Step 9 once .
13. The empty adsorption column was centrifuged at 9,000Xg for 1 minute.
14. Put the adsorption column into a clean 1.5mL centrifuge tube, add 50-100 μL Elution Buffer in the center of the adsorption film, stand at room temperature for 1 minute, and centrifuge for 1 minute. Preserve the DNA bath in the tube.

(In vitro amplification: A section or loop of DNA that is duplicated to increase its number)

Target：To amplify the target gene (IsPETase,dsPETase,MHET, PET28a) by PCR

Principle:

1. Denaturation (high temperature) : Splitting DNA into single strands (breaking hydrogen bonds)

2. Annealing: Bind primer

3. Extension: Insert the base with DNA polymerase (starting at the 3 '- end of the primer)

4. Cycle: n= number of cycles, 2^n DNA

Material:

PCR tube,Template(DNA),Primer(dsPETase-F,dsPETase-R)Mix(DNA polymerase,DNTP,MgCl2,buffer)--attenuation for 2x

ddH2O , PCR amplifierMVortexor, Centrifugal machine

Procedure：

PCR system：

PCR procedure：

Note: 30 cycles of 2,3,4

Target: To use agarose gel electrophoresis to analyze the molecular weight of nucleic acids

Material：

Procedure：

1. Gel: agarose 1g+TAE buffer loom, heat, cool, add nucleic acid dye 5μL, add into the mold (against the negative pole po4^2-) to form glue block, add marker
2. Sample addition: 50μL PCR product
3. Running glue: 180v, 20min
4. Photoscope: UV photoscope (Gel imaging system)

Target：To recover the DNA from the target gel.

Material：

Procedure：

1. Preparation：

Check whether anhydrous ethanol has been added to the Wash Solution.

Check Buffer B2 for precipitation.

Set the water bath to 50°C.

2. Cut the glue block containing the target fragment from the agarose gel and weigh it.
3. Add 3-6 times the weight of the glue block Buffer B2, 50°C water bath for 5-10 minutes sol.
4. (Optional) For fragments <500bp, add 1/3 Buffer B2 volume of isopropyl alcohol.
5. Transfer the sol solution into the adsorption column and centrifuge for 30 seconds at 8,000Xg.Drain the liquid from the collection pipe.
6. Add 500μL Wash Solution, centrifuge 9,000Xg for 30 seconds, and drain the liquid in the collection tube.
7. Repeat Step 6.
8. Centrifuge the empty adsorption column at 9,000Xg for 1 min.
9. Put the adsorption column into a clean 1.5mL centrifuge tube, add 15-40μL Elution Buffer in the center of the adsorption film, leave at room temperature for 1 minute, and centrifuge for 1 minute.Preserve the DNA solution in the tube.

Target：To allow DNA molecules containing homologous sequences to recombine between or within them

Material:

Procedure:

1. Sequentially add 2µL of 5xbuffer, 5.7µL of homologous recombinase, 1µL of PETase28a-1, and 1.3µL of IsPETase.
2. Vortex for one minute and centrifuge for 30s.
3. Put the centrifuge tube in 37℃ for 15 minutes.

Procedure：

1. Sequentially add 2µL of 5xbuffer, 2.8µL of homologous recombinase, 1µL of PETase28a-2, and 4.2µL of MHET.
2. Vortex for one minute and centrifuge for 30s.
3. Put the centrifuge tube in 37℃ for 15 minutes.

Procedure:

1. Sequentially add 2µL of 5xbuffer, 6µL of homologous recombinase, 1µL of PETase28a-1, and 1µL of dsPETase into a centrifuge tube.
2. Vortex for one minute and centrifuge for 30s.
3. Put the centrifuge tube in 37℃ for 15 minutes.

Notes：

1. MHET connects PET28a-2; IsPETase, dsPETase connect PET28a-1
2. Formula: 0.02* Length of target gene fragment (ng)/actual DNA concentration (ng/μL) = actual added volume (μL)
3. Why choose PET28a carrier?

--!! Can be used for E.coli, the usual carrier

--Strong promoter -- T7 promoter, efficient expression of protein

4. E.coli ：

-- DH5α : high copy number, short cycle, generally used for plasmid construction

-- BL21 (DE3) : Short cycle, good protein expression effect (high protein content)

Target: Transform using the heat shock method.

Material:

Procedure：Heat shock method

1. The 10μL homologous recombinant product was put into 10μL receptive cells
2. Leave on ice for 30 minutes
3. Water bath 42 degrees Celsius, 45s
4. 3 minutes on ice
5. Add 900μL LB medium, 37 ° C, 1h (resuscitation)
6. Centrifuge 5000xg, 5min, discard part of the supernatant
7. Apply the remaining E.coliDN5α/BL21 (DE3) to a petri dish with antibiotic Kan+ (kill bacteria)
8. Invert the petri dish and put it in the incubator at 37 degrees Celsius, 12-16h

Target：Verify that the transform is successful

Material：

Procedure:

-- PCR system：

-- PCR procedure：

Note: 30 cycles of 2,3,4

-- Agarose Gel Electrophoresis(check whether the expansion is successful)

1. Gel: agarose 1g+TAE buffer loom, heat, cool, add nucleic acid dye 5μL, add into the mold (against the negative pole po4^2-) to form glue block, add marker
2. Sample addition: 10μL PCR product
3. Running glue: 180v, 20min
4. Photoscope: UV photoscope (Gel imaging system

Target：To extract the target plasmid dspET28a

Material：

1. Buffer Sp1：to give a suitable environment
2. Buffer Sp2：Lysis of cell membrane
3. Buffer Sp3：stop the reaction
4. adsorption column
5. collecting pipe
6. centrifuge tube
7. Adsorption film：uptake plasmid
8. Wash solution：flushing impurity
9. Elution buffer：flushing target plasmid
10. Nanodrop：test DNA concentration

Procedure：

1. Check that RNase A has been added to Buffer SP1.
2. Check whether anhydrous ethanol has been added to the Wash Solution.
3. Check whether Buffer SP2 and SP3 are precipitated.
4. Adsorption column balance: Add 500μL Buffer S to the adsorption column (the adsorption column is put into the collection tube), centrifuge for 1min at 12,000 Xg, dump the waste liquid in the collection tube, and put the adsorption column back into the three collection tubes.
5. Take 1.5-5mL of overnight cultured bacterial solution, centrifuge 8,000Xg for 2 minutes to collect bacterial bodies, and discard the medium.
6. Add 250μL Buffer SP1 to the precipitate and completely suspend the bacteria.
7. Add 250 μL Buffer SP2 and immediately gently reverse the centrifugal tube for 10 times and mix well. Let stand at room temperature for 2-4 minutes.
8. Add 350 μL Buffer SP3 and immediately gently reverse the centrifuge tube 5-10 times to mix.
9. 12,000Xg centrifuge for 5-10 min. Transfer the supernatant into the adsorption column, centrifuge 8,000Xg for 30 seconds, and drain the liquid in the collection tube.
10. (Optional) Add 500 μL Buffer DW1, centrifuge 9,000Xg for 30 seconds, and drain the liquid in the collection tube.
11. Add 500 μL Wash Solution, centrifuge 9,000Xg for 30 seconds, and drain the liquid in the collection tube.
12. Repeat Step 9 once .
13. The empty adsorption column was centrifuged at 9,000Xg for 1 minute.
14. Put the adsorption column into a clean 1.5mL centrifuge tube, add 50-100 μL Elution Buffer in the center of the adsorption film, stand at room temperature for 1 minute, and centrifuge for 1 minute. Preserve the DNA bath in the tube.

(In vitro amplification: A section or loop of DNA that is duplicated to increase its number)

Target：To amplify the target gene (Gly8-GIn, Gly8-Ser, Gly8-Thr, Gly8-Cys, Gly8-Asn, Gly8-Tyr, Arg47-Lys, and Arg47-His) by PCR

Principle:

1. Denaturation (high temperature) : Splitting DNA into single strands (breaking hydrogen bonds)

2. Annealing: Bind primer

3. Extension: Insert the base with DNA polymerase (starting at the 3 '- end of the primer)

4. Cycle: n= number of cycles, 2^n DNA

Material:

PCR tube,Template(DNA),Primer,Mix(DNA polymerase,DNTP,MgCl2,buffer)--attenuation for 2x

ddH2O , PCR amplifierMVortexor, Centrifugal machine

Procedure：

PCR system：

PCR procedure：

Note: 30 cycles of 2,3,4

Target: To use agarose gel electrophoresis to analyze the molecular weight of nucleic acids

Material：

Procedure：

1. Gel: agarose 1g+TAE buffer loom, heat, cool, add nucleic acid dye 5μL, add into the mold (against the negative pole po4^2-) to form glue block, add marker
2. Sample addition: 50μL PCR product
3. Running glue: 180v, 20min
4. Photoscope: UV photoscope (Gel imaging system)

Target：To recover the DNA from the target gel.

Material：

Procedure：

1. Preparation：

Check whether anhydrous ethanol has been added to the Wash Solution.

Check Buffer B2 for precipitation.

Set the water bath to 50°C.

2. Cut the glue block containing the target fragment from the agarose gel and weigh it.
3. Add 3-6 times the weight of the glue block Buffer B2, 50°C water bath for 5-10 minutes sol.
4. (Optional) For fragments <500bp, add 1/3 Buffer B2 volume of isopropyl alcohol.
5. Transfer the sol solution into the adsorption column and centrifuge for 30 seconds at 8,000Xg.Drain the liquid from the collection pipe.
6. Add 500μL Wash Solution, centrifuge 9,000Xg for 30 seconds, and drain the liquid in the collection tube.
7. Repeat Step 6.
8. Centrifuge the empty adsorption column at 9,000Xg for 1 min.
9. Put the adsorption column into a clean 1.5mL centrifuge tube, add 15-40μL Elution Buffer in the center of the adsorption film, leave at room temperature for 1 minute, and centrifuge for 1 minute.Preserve the DNA solution in the tube.

Target：To allow DNA molecules containing homologous sequences to recombine between or within them

Material:

Procedure:

1. Sequentially add 2µL of 5xbuffer, 5.7µL of homologous recombinase, 1µL of PETase28a-1, and 1.3µL of IsPETase.
2. Vortex for one minute and centrifuge for 30s.
3. Put the centrifuge tube in 37℃ for 15 minutes.

Target: Transform using the heat shock method.

Material:

Procedure：Heat shock method

1. The 10μL homologous recombinant product was put into 10μL receptive cells
2. Leave on ice for 30 minutes
3. Water bath 42 degrees Celsius, 45s
4. 3 minutes on ice
5. Add 900μL LB medium, 37 ° C, 1h (resuscitation)
6. Centrifuge 5000xg, 5min, discard part of the supernatant
7. Apply the remaining E.coliDN5α/BL21 (DE3) to a petri dish with antibiotic Kan+ (kill bacteria)
8. Invert the petri dish and put it in the incubator at 37 degrees Celsius, 12-16h

Goal: The goal of this process is to monitor the colony density of the bacteria 

Materials:

Process

1.  Modulate the culture solution(15ml LB-base, 1ml bacteria sample)
2.  Culture the solution in the shaker, wait for 30minutes
3.  After time is over, extract 1ml solution and replace the solution in the shaker
4.  Move the shaker into the UV-spectrophotometer, record the first three data from the output
5.  Repeat the process for a time interval of 1hour, 2 hours, 4 hours, 6 hours, 10 hours,16h,24h until the colony density(CD600 (600 stands for wavelength)) reaches a value between 0.6 to 0.8

Goal: Using the IPTG to trigger bacteria to produce enzyme as protein

Material:

Process:

1. When OD600 are about 0.6, add 1mm of IPTG, the ratio is 1:1000
2. Shake equally when IPTG is added, put them in constant temperature shaker for 12 hours, 16 degree Celsius.

Goal: Break the cell membrane and extract the protein

The goal of this process is to break the cell membrane and extract the protein.

Materials:

 Process:

1.  Centrifuge the bacterial sample solution to precipitate the solid.(7000W power, 30 minutes)
2.  Remove the supernatant, add 20mL of Lysis buffer, and stir up the precipitate to blend with the buffer.
3. Put the solution on ice and operate with an ultrasonic instrument. (300W power, on for 3 seconds, off for 3 seconds, soak for 30 minutes)

Goal: Collect the target protein by taking the advantage of the binding and elution mechanism of nickel column.

Material

Process:

1. Place the solution that has undergone ultrasonic disruption of the cell membrane into a centrifuge for centrifugation.
2. Load the nickel column and place it on ice for preparation.
3. After the protein solution has been centrifuged, take the supernatant and pour it into a clean test tube. Place the precipitate on ice for later use.
4. Pour a certain amount of the supernatant into the container with the nickel column for rinsing, so that the nickel column is dissolved in the supernatant. Then, rotate and refrigerate it at 4 degrees Celsius for two hours.
5. After refrigeration, remove the refrigerated supernatant and first filter it using the nickel column loading tube.
6. After filtration, place the loading tube with the nickel column on top of the small test tube and add 500 microliters of washing solution to it. Wait for it to filter, repeat this step five times.
7. After elution is completed, seal the loading tube and add 500 microliters of elution solution to it. Shake until the solution becomes cloudy and then let it stand for two minutes.
8. After two minutes of standing, place the loading tube on top of the small test tube and wait for the liquid to filter.
9. Repeat steps 7 and 8 five times to obtain five samples.

Goal: Adding protein sample to gel and observe the protein pillar(success if protein pillar on the gel is obvious)

Preparation:Take the protein samples out from ice and pick number 1,2,7,8,9. Transfer 200ul of them to new tube. Then add 20ul of blue coloring to them and put them in centrifuge, to make sure the solution are stable. Then put them in heater for 10 minutes. 

Materials:

1. Organize the gel model first, assemble the two layers of glass together, remain 1.5mm space between them. 
2. Put all devices in the model together, then verify whether it’s sealed by adding purifies water between the glass carefully, them observe it and check whether there is water in bottom. 
3. If it’s sealed, pour the water out then make the lower gel. 
4. Add 4ml of lower layer glue buffer solution and adhesive solution to the flask by using pipette, then add 80ul coagulant accelerator, mix them together. 
5. When it’s mixed, transfer the solution to the model using pipette, take 1ml a time and slowly drip them in the gap, make sure to drip equally in both side of the glass.
6. Left it still about 15 minutes, and see if it becomes solid, then add some ethanol on the top of gel to make the surface of the gel flat. After that, make the upper layer.
7. Add 1ml of upper layer glue buffer solution with pink color and adhesive solution to another flask, then add 20ul coagulant accelerator to it and mix them together. Using pipette to transmit the mixture to the model, the process is similar to lower layer one. Then add a comb like devices to make spaces for protein substance to intake. 
8. Left it still for 15 minutes until it become solid. 
9. Take the protein sample from previous experiment out of the heater with blue coloring and use pipette to transfer these substance to the gel, drip them vertically and make sure it do not spill to other gap. 
10. Wait for about 2 hours until the marking are appeared in the gel, them analyze the results. 

Goal:Using HPLC method to test the degrading rate of MHET

Materials:

Sample process:

1. Write the name of protein name on new microtude.
2. Weight 0.5g PETase particles and transfer them to new microtube
3. Add 200ul protein samples(such as IsPETase,dsPETase, His, Gln, Asn) to that microtude and put them on scroll machine for about 10 seconds, until the mixture are will mixed.

MHET HPLC process:

a. Experimental Instrument Parameters

Detector: DAD

Column: Thermo Scientific C18, 250 mm * 4.6 mm i.d., 5 μm particle size

Column Temperature: 30 °C

Flow Rate: 1 mL/min

Wavelength: 242 nm

Injection Volume: 20 μL

Mobile Phase: Phosphate buffer solution (containing 5 mmol/L potassium dihydrogen phosphate, 0.04% phosphoric acid) : Methanol = 70 : 30 (v/v)

b. Standard Solution Preparation

An accurate amount of ethylene phthalate (PET) standard was weighed into a 5 mL brown volumetric flask. An appropriate amount of 0.1 mol/L NaOH solution was added, and the mixture was sonicated to dissolve the standard. The solution was then diluted to the mark with ultrapure water, mixed thoroughly to prepare a 1 mg/mL single-standard stock solution, and stored at -20 °C.

The standard working curve was prepared using ultrapure water for dilution.

c. Sample Extraction Process

The sample was homogenized, filtered through a 0.45 μm membrane filter, and then subjected to HPLC analysis.

Goal: Calculating the speed of plastic degrading and determain the most efficient one. 

Material:

Sample Process:

1. Weigh 1g of plastic particles in each test tube and write the tag, write the name that is going to add.
2. Add the according enzyme to the test tube about 400ul for each, then add 400ul of MHETase to each test tube about 400ul.
3. Left it in the refrigerator for 48h and 72h, add additional enzyme if it’s not enough.
4. Take some sample to test the degrading rate after 72 hours.

TPA and EG HPLC process:

1. Experimental Instrument Parameters

Detector: DAD

Column: Thermo Scientific C18, 250 × 4.6 mm, 5 μm

Column Temperature: 30 °C

Flow Rate: 1 mL/min

Wavelength: 242 nm

Injection Volume: 20 μL

Mobile Phase: Phosphate buffer solution (containing 5 mmol/L potassium dihydrogen phosphate and 0.04% phosphoric acid) : Methanol = 70 : 30

2. Standard Solution Preparation

Accurately weigh an appropriate amount of terephthalic acid standard into a 5 mL brown volumetric flask. Add a suitable volume of 0.1 mol/L NaOH solution, and dissolve by sonication. Dilute to the mark with ultrapure water, mix well to prepare a 1 mg/mL single-standard stock solution, and store at -20 °C.

Prepare the standard working curve using ultrapure water.

3. Sample Treatment

Mix the sample homogenously, filter through a 0.45 μm membrane filter, and then analyze by HPLC.

Goal: Determain the rate to degrading PET by using PETase and MHETase

Materials:

Process

1. Cut 5 plastic film, the size is 1cm time 1cm.
2. Put them on cultivating base, make sure to make it flat.
3. Add 100ml of MHETase and 100ml of Ser/isPETase/dsPETase/Ser on the plastic, make sure not to make the plastic film curve and drip these enzyme equally.