Team:WFLA YK PAO/Experiments


Bacteria culture medium preparation
1. LB preparation
1. Add Water 100mL, NaCl 1g, Tryptone 1g, Yeast Extraction 0.5g to a beaker
2. When preparing for solid culture medium, add extra Agar 2.5g
2. Antibiotics solution
1. Add Water 2mL Kanamycin 0.1g to a beaker
2. Add the solution to the culture medium
Plasmid extraction
1. Prepare for buffer SP1, SP2, and SP3, and wash solution
2. Add 500µl butter S to the absorption column, centrifugate for 1 min at 12000g, remove the liquid in the collecting tube, put the absorption column back in the collecting duct
3. Centrifugate 1.5-5ml cultured bacteria solution, centrifugate at 8000g for 2 minutes, remove the supernatant
4. Add 250µl buffer SP1 to the sediment to solve the bacteria
5. Add 250µl buffer SP2 and mix the solution, then keep for 2-4 min at room temperature
6. Add 350µl buffer SP3 and mix the solution
7. Centrifugate at 12000g for 5-10min, pour the supernatant into the absorption column, then centrifugate at 8000g for 30 s, remove the liquid in the collecting tube
8. (Optional) add 500µl Buffer DW1, centrifugate at 9000g for 30 s, remove the liquid in the collecting tube
9. Add 500µl Wash Solution, centrifugate at 9000g for 30 s, remove the liquid in the collecting tube
10. Repeat step 9 once
11. Centrifugate an empty absorption column at 9000g for 1min
12. Put the absorption column in a clean 1.5ml centrifugation tube, add 50-100µl Elution Buffer, put at room temperature for 1 min, centrifugate for 1 min, collect the DNA solution in the collecting tube
Plasmid transfer
1. Add plasmid solution (3 μL) to competent cell
2. Ice-cool for 20min
3. Water bath at 42 centigrade for 1 min
4. Ice-cool for 5 min
5. Shaking incubator for 45? min
6. Centrifugate and keep the pellet (containing bacteria)
IPTG induction
1. Spread plating BL21 with transferred plasmid on the solid culture medium
2. Extracting bacteria from solid culture medium to the liquid LB culture medium.
3. Put it in the shaking incubator
4. Pour the over-night culture solution (seed solution) into the new LB culture medium
5. Test optic density (OD) at 600nm of culture solution
6. Add IPTG (If OD600 ranges from 0.6-0.8)
7. Centrifuge and keep the supernate (containing proteins)
SDS protein separation
1. Aim: prepare the proteins for SDS-PAGE electrophoresis
2. Add SDS solution to lyse cell structure and denature proteins
3. Hot water-bath to accelerate the reaction
4. Centrifugate and extract supernatant to obtain proteins
Nickel column purification
Aim: to obtain pure IsPETase and MHPETase for further decomposition of plastics
Apparatus: Nickel column
a. Wash the nickel column with deionized water
b. Wash the nickel column with Buffer A
c. Add the sample to the nickel column using a syringe and filter and collect the drips with a tube.
d. Pour the liquid collected in the tube back into the nickel column
e. Add buffer A three times
f. Add Buffer B twice and collect the released protein with a tube
Enzyme activity test
Aim: examine the effectiveness of the chosen enzyme on decomposing plastics
Mix 300μL enzyme with one PET granule and 4.7mL glycine-NaOH. The bottle was put into the shakebed for one night. Finally, we used a microscope to observe the degradation effect.
SDS-page gel electrophoresis
1. Make SDS-page gel
1. Add 2.7ml lower layer gel solution and lower layer gel buffer
2. Add 60µl coagulant
3. Pour into the mold and wait until it solidifies
4. After 5-10 min after the gel solidifies, remove the liquid above it
5. Add 0.75 ml upper layer gel solution and colored upper layer gel butter
6. Add 15µl coagulant
7. Pour into the mold and wait until it solidifies
2. Insert proteins into the gel
1. Mix 5µL of loading buffer with 10µl sample
2. Insert 10µL of sample in each hole and 5µl of the marker in the remaining two holes
Overlapping PCR
1. Use PCR to amplify copies of IsPETase.
1. Template: pET28a-IsPETase
2. Primers: IsPETase-F & Overlap-R
2. Use PCR to amplify copies of MHETase.
1. Template: pET28a-MHETase
2. Primers: Overlap-F & MHETase-R
3. Agarose gel electrophoresis + Gel DNA recovery
4. Use the overlap part to link the gene for IsPETase and MHETase & amplify copies of linked Is-MH
1. Template: pET28a-IsPETase & pET28a-MHETase
2. Primers: IsPETase-F & MHETase-R
Agarose gel electrophoresis
1. Make Agarose gel
1. 0.5g Agarose
2. 50ml TAE Buffer
3. Put in microwave for 1 minute
4. Add nucleic dye for 1 µL
5. pour into the mold and wait until it solidifies
2. Insert genes into the gel
1. Mix 10µL of loading buffer and the product of PCR
2. Insert 30µL of sample in each hole and 2µL of the marker in the remaining two holes.
Enzyme cut and plasmid reconstruction
1. Add EcoR1 and Not1 into the sample to cut the Is/MH sequence
2. Insert the Is/MH gene into the plasmid and connect the inserted gene with the vector gene using T4 ligase
3. Transfer the recombinant sequence to DH5α
4. Spread plating of bacteria on solid LB culture medium
Test for enzyme activity
1. Add 5 PET microparticles into the 50ml culture solution containing genetically modified bacteria
2. Put in the shaking incubator overnight
3. Observe the PET microparticles under 400x optical microscope
Xuhui Lab
Plasmid extraction
Aim: to obtain the pET28a plasmid with ISPETase and/or MHETase from the originally cultured strain, for transfer into BL21(DE3) E.coli strain
Apparatus: Diamond plasmid kit
Bacteria strain: DH5α (TOP10) - a designed strain for amplifying a plasmid
1. Balance pH using buffer S, centrifugate to immerse
2. Add buffer SP1 (containing RNase) to already centrifugated cultured bacteria; mix thoroughly
3. Add an alkaline buffer SP2 to obtain DNA;; invert the centrifugation tube several times and wait for 2 minutes to complete reaction (destroy nucleoids); add buffer SP3 to balance pH for selection of plasmid
4. Centrifugate to eliminate cell debris; add wash solution to purify by centrifugation (and let the sample get rid of genome-related DNA (this step repeats for one time).
5. Add elution buffer, centrifugate to collect plasmids, refrigerate at -20 degrees celsius for storage
Gel electrophoresis
Aim: to identify whether the plasmid contains the wanted gene
Preparations: 1% agarose solution as gel immersed in TAE buffer (EB added to dye); keep pre-cut and cut samples to identify the presence of plasmids and the wanted gene segment; EcoR I and Xho I enzymes are selected to cut off the gene segment
Principles: DNA is negatively charged in dissolved form; they move across the same medium at different rates given the same voltage, according to their relative molecular weights; linearized (enzyme-cut) samples move faster than cyclic samples of the same length.
Plasmid transfer
Aim: to induce the E. coli BL21 to absorb in the engineered plasmid
Principles: transfer the plasmid into the bacteria and then prevent it from receiving other plasmids
1. Competence E. coli stored in -80 centigrade placed on ice
2. Add the plasmids and wait for 30 minutes for them to be absorbed
3. Hot activation at 42 centigrade for 90 seconds and immediately put on ice for 3 minutes to remove competence
4. Culture the bacteria
Ni column protein purification
The aim of this procedure is to purify pet28a-IsPETase and pet28a-MHETase. The basic principle behind this is that the Imidazole component in the buffers would competitively replace His-tag on pET28a, causing the target proteins to be washed down for collection. The following steps make up the whole procedure: First, Rinse the column with 10mL H2O. Second, equilibrate the column with a 10ml Solution buffer. Then proceed to load the sample and Re-equilibrate the column. After, load the 10ml wash buffer, collect the run-through. Finally, load 10ml Elution Buffer and collect run-through. Rinse the column and keep the system with 6mL 20% ethanol under room temperature.
Enzyme activity testing
The aim of this step is to test the effect of IS-PETase&MH-PETase on microPET degradation. First, we mixed 10µg/mL enzyme with 5mg PET granule under 30 ℃. Then we proceeded to add 50mM Glycine buffer (pH 9.0) and centrifuged the system under 1000 rpm. The system was then heated to 85℃ and kept for 10min. Finally, we used a microscope to observe the degradation effect.
This procedure is to amplify the IsPETase and MHETase DNA. The reaction system consists of 50µL Primestar, 1µL cDNA, 1µL forward primer, 1µL reverse primer, 19µL ddH2O, and 3µL DMSO. The whole PCR process is made up of three stages: DNA denaturation, primer annealing, and DNA synthesis. During the first stage, the system was heated to 96℃ for 6min plus 10s to break the double-strand structure of the target DNA strands. Then cooled down to 66℃ for 15s, the system entered the second stage during which primers annealed to the single-stranded templates. Finally, the system was heated to 72℃ and kept for 90s, during which DNA polymerase bind to the primers and started DNA synthesis. This whole process was repeated for 35 cycles and finally extended for 90s under 72℃.
T4 Ligase- enzyme ligation
In the experiment of our group, the function of T4 is slightly different from the other group’s. They used the T4 ligase enzyme to connect the target gene and the plasmid vector to obtain the recombinant vector. However, we utilize the T4 ligase both to connect the gene of PETase and MHETase together and to link this recombinant gene onto the plasmid vector. The material required for this step is:
-T4 DNA ligase 2µL
-DNA 0.03pmol:IsPETase: 0.02µg; MHETase: 0.04µg
-Vector 0.3pmol (0.106µg)
-ddH20 16µL (as the whole system is 20µL)
The solution with ligase should be put under 16 ℃ for 2 hours.
1. Remove BL21(DE3) competent cell from -80oC and thaw on ice.
2. Add 100 ng plasmid DNA into BL21(DE3) strain and mix gently.
3. Incubate tube on ice for 30 minutes.
4. Heat shock tube at 42 oC for ~ 90 seconds without shaking.
5. Replace tube on ice for 3 minutes.
6. Add 100 μl room temperature LB medium.
7. Shake and incubate tube at ~ 200 rpm for 60 minutes at 37 oC.
8. Spread on LB agar plate containing 50 μg/ml kanamycin.
9. Incubate the plate upside down at 37 oC overnight.
Pilot expression
1. Pick three single, well-isolated colonies and inoculate it into 4 ml LB medium containing 50 μg/ml kanamycin, respectively.
2. Incubate the cells in shaker at 37oC with shaking at 200 rpm.
3. When the OD600 value reaches 0.6 ~ 0.8, two of the three tubes are added IPTG with the final concentration of 0.5 mM IPTG for induction at 15 oC for 16 h and at 37 oC for 4 h, respectively, and the last one tube as the negative control.
4. Detect the protein expression and solubility using both SDS-PAGE and western blot.
Sample preparation
1. Harvest cell pellet from 450 μl culture, and lyse with 300 μl lysis Buffer (50 mM Tris, 150 mM NaCl, 5% glycerol pH 8.0) using sonicator for 1 minutes.
2. Whole cell lysate: Mix 50 μl 5 x loading buffer to 100 μl cell lysate as the sample of whole cell lysate. Heat the sample at 100 oC for 10 minutes, and centrifuge at 15,000 rpm for 5 minutes.
3. Supernatant and pellet of cell lysate: Centrifuge the remained 200 μl cell lysate at 15,000 rpm for 10 minutes, and collect the supernatant and pellet of cell lysate, respectively
4. Mix 90 μl 5 x loading buffer to 180 μl supernatant as the sample of supernatant of cell lysate. Resuspend all of the precipitation with 150 μl 5 x loading buffer as the sample of pellet of cell lysate. Heat the samples at 100 oC for 10 minutes, and centrifuge at 15,000 rpm for 5 minutes before loading into the gel.
MHETase Enzyme Activity Test
MHETase Reactions were performed in triplicate over a 1 d time course at 30°C. Reaction mixtures contain 50 nM recombinant GST-MHETase, 5 mM MHET in 90 mM NaCl, 10% (v/v) DMSO, 45 mM sodium phosphate, pH 7.5. Reactions were terminated by adding an equal volume of methanol. The reactions using inactive GST-MHETase (obtained by boiling for 10 min) were used as blank controls.
The activity of MHETase was indicated by the decline absorbances at a wavelength of 240 nm.