Experiments

  • Material (50μL unit)
    • Marker
    • Pipette
    • Thermal cycler
    • PCR tubes
    • EV71 vp1 mix
      • 25μL Mix (DNA polymerase, dNTP, buffer)
      • 2.5μL EV71vp1 primer (forward)
      • 2.5μL EV71vp1 primer (reverse)
      • 2μL EV71vp1 template
      • 18μL double-distilled water to 50 μL
    • RV vp7 mix
      • 25μL Mix (DNA polymerase, dNTP, buffer)
      • 2.5μL RVvp7 primer (forward)
      • 2.5μL RVvp7 primer (reverse)
      • 2μL RVvp7 template
      • 18μL ddH2O to 50 μL
    • GLBP mix
      • 25μL Mix (DNA polymerase, dNTP, buffer)
      • 2.5μL GLBP primer (forward)
      • 2.5μL GLBP primer (reverse)
      • 2μL GLBP template
      • 18μL double-distilled water to 50 μL
    • GFP mix
      • 25μL Mix (DNA polymerase, dNTP, buffer)
      • 2.5μL GFP primer (forward)
      • 2.5μL GFP primer (reverse)
      • 2μL GFP template
      • 18μL double-distilled water to 50 μL
    • IL12 mix
      • 25μL Mix (DNA polymerase, dNTP, buffer)
      • 2.5μL IL12 primer (forward)
      • 2.5μL IL12 primer (reverse)
      • 2μL IL12 template
      • 18μL double-distilled water to 50 μL
  • Procedure
    • Label PCR tubes: EV71 vp1, RVvp7, GLBP, GFP, and IL12
    • Mix EV71vp1by pipetting all the solutions into the PCR tube labeled “EV71vp1”
    • Repeat step 2 with the other genes mix
    • Quick spin all lipids
    • Put PCR tubes into thermal cycler, set the temperature of each cycle to 95℃ - 56℃ - 72℃, and 4℃ for cooling/preservation
    • Run the thermal cycler
  • Material
    • Electrophoresis gel box
    • Gel casting tray
    • Comb
    • Power supply and electrodes
    • Pipettes
    • Gel
      • Scale
      • 0.5g Agarose
      • 50mL 1xTAE
      • 3μL GelRed
  • Procedure
    • Preparing gel
      • Prepare gel casting tray with comb
      • Weigh agarose, mix with 1XTAE in a flask
      • Heat to dissolve agarose in 1XTAE until the solution became clear
      • Let the solution cool down
      • Add GelRed to stain the gel
      • Pour into gel caster
      • Wait for gel to solidify
    • Electrophoresis
      • Place gel into a gel box, which is filled with 1XTAE
      • Mix loading dye with each DNA sample
      • Load each DNA sample into a well in the gel
      • Load DNA marker into the first well
      • Put the lid onto the gel box, set power supply voltage to 180V, make sure electrodes are placed correctly
      • Run the device for approximately 15 minutes
    • Confirm results
      • Exposed the gel with UV light
      • Take a photo of the result and save it for later use
  • Material
    • Gel with vp1, vp7, GLBP, GFP, and IL12 genes
    • Blade
    • Pipette
    • Centrifuge tubes
    • Purification column
    • 300μL Binding buffer (buffer B2)
    • 500μL Washing buffer
    • 30μL Elution buffer
    • Centrifuge
  • Procedure
    • Using a blade, cut out each part of gel that contains DNA sample
    • Add gel and binding buffer into centrifuge tubes
    • Dissolve the gel at 50℃
    • Centrifuge the mixture at 8000Xg for 30 seconds
    • Transfer mixture into purification column
    • Centrifuge the mixture at 8000Xg for 30 seconds
    • Add washing buffer
    • Centrifuge the mixture at 9000Xg for 30 seconds, suspend liquid in the centrifuge tube
    • Repeat the last two steps
    • Centrifuge at 9000 Xg for 1 minute
    • Transfer purification column to another centrifuge tube
    • Add elution buffer
    • Let stand at room temperature for 1 minute
    • Centrifuge at 9000 Xg for 1 minute
    • Collect the liquid in the centrifuge tube
  • Material
    • Marker
    • Pipette
    • Thermal cycler
    • PCR tubes
    • vp7-vp1 overlap extension PCR mix (50μL)
      • 43μL Mix
      • 2μL vp1 template
      • 2μL vp7 template
      • 1μL RV vp7 forward primer
      • 1μL EV71 vp1 reverse primer
      • 1μL ddH2O
    • IL12-GFP overlap extension PCR mix (50μL)
      • 43μL Mix
      • 2μL IL12 template
      • 2μL GFP template
      • 1μL IL12 forward primer
      • 1μL GFP reverse primer
      • 1μL ddH2O
    • GLBP-vp7-vp1 overlap extension PCR mix (50μL)
      • 43μL Mix
      • 2μL GLBP template
      • 2μL vp7-vp1 template
      • 1μL GLBP forward primer
      • 1μL vp1 reverse primer
      • 1μL ddH2O
    • GLBP-vp7-vp1-GFP overlap extension PCR mix (50μL)
      • 43μL Mix
      • 2μL GLBP-vp7-vp1 template
      • 2μL GFP template
      • 1μL GLBP forward primer
      • 1μL GFP reverse primer
      • 1μL ddH2O
    • GLBP-vp7-vp1-IL12-GFP overlap extension PCR mix (50μL)
      • 43μL Mix
      • 2μL GLBP-vp7-vp1 template
      • 2μL IL12-GFP template
      • 1μL GLBP forward primer
      • 1μL GFP reverse primer
      • 1μL ddH2O
  • Procedure
    • Label PCR tube
    • Prepare mix by pipetting all the solutions and water in to the labeled PCR tube
    • Spin all prepared solutions
    • Put PCR tubes into thermal cycler, set the temperature of each cycle
    • Run the thermal cycler
  • Refer to 1.2
  • Refer to 1.3
  • 2.1.1 Preparing the LB culture medium
    • Material:
      • Liquid medium:
        • 10g Tryptone
        • 5g Sodium Chloride
        • 2.5g Yeast extract
        • 500mL ddH2O
      • Solid medium:
        • 10g Tryptone
        • 7.5g Agar
        • 5g Sodium Chloride
        • 2.5g Yeast extract
        • 500mL ddH2O
    • Procedure:
      • Weigh the above material into 500mL conical flask.
      • The prepared solution needs to be sterilized in an autoclave for later use.
      • Pour the solid medium into petri dishes and let it for solidify
  • 2.1.2 E.coli (DH5α) Cultivation
    • Material:
      • DH5α culture
        • LB culture medium
        • Petri dish
        • Kana antibiotic
    • Procedure:
      *High sanitary standards must be followed, sterilize hands and apparatus frequently and perform inside ultra-clean table near alcohol burner.*
      • For liquid culture:
        • In 20mL test tube, add 5mL of liquid LB culture medium, 50μL of DH5𝛼 culture, and 5 μL of Kana antibiotic.
        • Place into shaking incubator under 37℃ for bacteria to grow overnight
      • For solid culture:
        • In 20ml test tube, add 50mL of LB solid culture medium and 50μL of Kana antibiotic
        • Pour into petri dish
        • Let them cool down
  • Material:
    • Plasmid Mini-PREPS Kit from Sangon Biotech (Shanghai) Cooperation (containing sp1, sp2, sp3 solution)
  • Procedure:
    • Centrifuge the DH5𝛼 solution at 12,000 Xg for 1 minute to separate and collect the bacteria
    • Remove supernatant
    • In order to lysis the bacteria, carry out alkaline lysis:
      • Resuspend: Add 250 μL of the sp1 solution from the kit into the centrifuge tube, gently tilt the tube for 5-10 times to mix the solution
      • Lyse: Add 250 μL of the sp2 solution, gently tilt the tube for 5-10 times to mix the solution. Set still at room temperature for 2 minutes.
      • Neutralize: Add 350 μL of the sp3 solution, mix gently. Set still at room temperature for 2 minutes.
      • Centrifuge at 12000 Xg for 8 minutes, and transfer the supernatant in to a purification column
    • Add 500μL of wash buffer from the kit, centrifuge at 9,000 Xg for 30 seconds, Remove the solution at the collection column. Repeat this step for 2 times.
    • Transfer the purification column into a new centrifuge tube. Add 50 μL of elution buffer (from the kit) into the column. Set still at room temperature for 1 minute, and then centrifuge at 9,000 Xg for 1 minute. Collect the DNA solution in the centrifuge tube.
    • Test for concentration of the pET28a plasmid using the Nanodrop.
  • Material:
    For PCR tube #1:
    • 20 μL pET28a plasmid
    • 5 μL Cutsmart buffer
    • 1 μL EcoR1
    • 1μL XhoI
    • 23 μL ddH2O
    For PCR tube # 2:
    • 20 μL vp7-vp1 DNA fragment
    • 5 μL Cutsmart buffer
    • 1 μL EcoR1
    • 1μL XhoI
    • 23 μL ddH2O
    For PCR tube # 3:
    • 20 μL GLBP-vp7-vp1-GFP DNA fragment
    • 5 μL Cutsmart buffer
    • 1 μL EcoR1
    • 1μL XhoI
    • 23 μL ddH2O
    For PCR tube # 4:
    • 20 μL GLBP-vp7-vp1-IL-12-GFP DNA fragment
    • 5 μL Cutsmart buffer
    • 1 μL EcoR1
    • 1μL XhoI
    • 23 μL ddH2O
  • Procedure:
    • Measure and add materials into 4 PCR tubes separately
    • Insert DNA fragment into thermal cycler at 37℃for 30 minutes
    • Insert pET28a into thermal cycler at 37℃for 2h
  • 2.3.1 Gel electrophoresis to assess the digested pET28a
    • Refer to 1.2
  • 2.3.2 DNA fragment extraction
    • Refer to 1.3 except melting the gel
  • 2.4.1 DNA ligation
    • Material:
      • 15 μL digested plasmid from previous step
      • 2 μL of digested DNA segment from previous step
      • 2μL T 4 ligase buffer
      • 1 μL T4 ligase
    • Procedure:
      • Measure concentration and add all solution into a PCR tube
      • Place into thermal cycler at 25℃for 30 minutes
  • 2.4.2 Transformation using heat shock method
    • Material:
      • 10 μL of product from DNA ligation
      • 50 μL of E.Coli DH5𝛼
    • Procedure:
      *High sanitary standards must be followed, sterilize hands and apparatus frequently and perform inside ultra-clean table near alcohol burner.*
      • Keep competent cells DH5𝛼 on ice for 5 minutes
      • 10 μL of product from DNA ligation, and 50μL of E.Coli DH5𝛼 into a sterilized 2.0 mL centrifuge tube
      • Bath on ice for 30 minutes
      • Heat shock for 90 seconds in water bath at 42 ℃
      • Bath on ice for 3 minutes
      • Add 940 μL of LB culture medium, place into shaking incubator to let grow for 30 minutes
      • Centrifuge at 9000 Xg for 1 minute, remove the supernatant
      • Spread the remaining precipitate onto solid LB culture medium with ampicillin, incubate the plate at 37℃ overnight
  • Material:
    • Colony PCR (prepare amount for 10 copies)
      • 10 μL Mix (DNA polymerase, dNTP, buffer)
      • 1.0 μL EV71vp1 primer (forward) or GLBP primer (forward)
      • 1.0 μL EV71vp1 primer (reverse) or GFP primer (reverse)
      • 1 colony on plate as template
      • 8 μL ddH2O
    • Gel:
      • 50mL 1xTAE
      • 0.5g Agar
      • 5μL GelRed
  • Procedure:
    • PCR:
      • Prepare the solution, distributing into 8 1.5mL PCR tubes
      • Select 8 colonies using 2.5μL pipette inside ultra-clean table
      • Pipette each colony into individual tubes. Mix well and centrifuge at 8000 Xg for 30 seconds
      • Place into thermal cycler to undergo PCR cycles for 95℃ - 56℃ - 72℃, and 4℃ for cooling/preservation
    • Gel electrophoresis:
      • Place prepared gel into gel box, which is filled with 1XTAE
      • Mix loading dye with each DNA sample
      • Load each DNA sample into a well in the gel
      • Load DNA marker into the first well
      • Put the lid onto the gel box, set power supply voltage to 180V, make sure electrodes are placed correctly
      • Run the device for approximately 15 minutes
      • After 15 minutes, expose the gel with UV light
      • Take a photo of the result and save it for later use
  • Material:
    • 10 μL of product from DNA ligation
    • 50 μL of E.coli BL21 (DE3)
  • Procedure: Refer to 2.34
    Results of concentration of OD600 tested with the Nanodrop after 3.5 hours of bacterial growth:
    0.277, 0.249, 0.226, 0.254, 0.235, 0.326
    Results of concentration of OD600 tested with the Nanodrop after 4.5 hours of bacterial growth:
    0.426, 0.412, 0.430, 0.432, 0.465, 0.512
    OD600 value between 0.4-0.6 is optimal for IPTG-induced protein expression.
  • Material:
    • 505.5 μL of 1M IPTG solution
    • Pipette
    • 6 tubes of pET28a vp7-vp1 BL21 (DE3)
  • Procedure
    • Add 3 μL of IPTG to tube 1
    • Add 7.5 μL of IPTG to tube 2
    • Add 15 μL of IPTG to tube 3
    • Add 30 μL of IPTG to tube 4
    • Add 150 μL of IPTG to tube 5
    • Add 300 μL of IPTG to tube 6
    • Incubate all pET28a vp7-vp1 BL21 (DE3) at 18℃ overnight
  • Material:
    • Containers
    • HisA
      • 1.41g Disodium phosphate
      • 14.61g Sodium chloride
      • 0.68g Imidazole
      • 500mL ddH2O
    • HisB
      • 0.705g Disodium phosphate
      • 7.305g Sodium chloride
      • 8.51 g Imidazole
      • 250 mL ddH2O
    • Tris-HCl
      • 6.06 g Tris
      • 500mL ddH2O
    • IPTG
      • 0.477g IPTG
      • 2 mL ddH2O
    • Coomassie brilliant blue staining and destaining solution
      • 400mL Ethanol
      • 100mL Acetic acid
      • 0.2 g Coomassie blue G-250 (staining)
      • 500mL ddH2O
    • 6M HCl
  • Procedure
    • Prepare each mixture in a separate container
    • Adjust the pH of HisA, HisB, and Tris-HCl by adding HCl to them gradually
    • When adding HCl, make sure to constantly confirm current pH value
    • HisA and HisB need to be adjusted to 7.4, and Tris-HCl to 8.0
  • Material:
    • 6 tubes IPTG induced pET28a vp7-vp1 BL21 (DE3)
    • 25mL Tris-HCl
    • Centrifuge
  • Procedure
    • Centrifuge IPTG induced pET28a vp7-vp1 BL21 (DE3) at 6000 rpm, 10 minutes
    • Add 15 mL Tris-HCl to each tube (Perform this step at the ultra-clean table)
    • Centrifuge IPTG induced pET28a vp7-vp1 BL21 (DE3) at 6000 rpm, 10 minutes
    • Add 10mL Tris-HCl to each tube (Perform this step at the ultra-clean table)
    • Centrifuge IPTG induced pET28a vp7-vp1 BL21 (DE3) at 6000 rpm, 10 minutes
  • Material:
    • Ice
    • 6 tubes of centrifuged IPTG induced pET28a-vp7-vp1 BL21 (DE3) bacteria
    • 10 mL Tris-HCl
    • Ultrasonic cell crusher
  • Procedure
    • Keep all IPTG induced pET28a vp7-vp1 on ice throughout whole experiment
    • Add 10 mL Tris-HCl to each tube
    • Clean the probe of ultrasonic cell crusher
    • Transfer 1 tube to ice box in the cell crusher
    • Adjust height of platform until a desirable height
    • Ultrasonic crush for 15 minutes
    • Repeat steps above for all 6 tubes
  • Material:
    • Pipette
    • Ni column
    • 50 mL centrifuge tube
    • ddH2O
    • 10 mL HisA
    • 1mL HisB
    • Buffer A
    • Ni-NTA resin
    • Crushed IPTG induced pET28a vp7-vp1 BL21 (DE3) bacteria
  • Procedure
    • Prepare filtration instrument by inserting Ni column half into centrifuge tube
    • Use Ni-NTA resin to wash beads in the Ni column
    • Use ddH2O to wash column
    • Add buffer A to beads
    • Purify supernatant of IPTG induced pET28a vp7-vp1 BL21 (DE3) bacteria by pipetting it into Ni column
    • Repeat the previous step 10 times
    • Add 3 mL HisA to column
    • Wait until all HisA has dripped into centrifuge tube, add another 3mL of HisA
    • Wait until all HisA has dripped into centrifuge tube, add another 4mL of HisA
    • Wait until all HisA has dripped into centrifuge tube, add add 1mL HisB
    • Collect the elution buffer for estimate the protein purity and size
  • 4.1.1 Preparation of the SDS-PAGE gel
    • Material:
      • 2.7 mL separating gel solution
      • 2.7 mL separating gel buffer
      • 0.75 mL stacking gel solution
      • 0.75 mL stacking gel buffer
      • 75 μL accelerated coagulant
      • SDS-PAGE gel mold
    • Procedure:
      • Set up the apparatus inject water and wait for 5 minutes to ensure no leaking
      • Mix 2.7 mL of separating gel solution and 2.7 mL of separating gel buffer together, mix well
      • Add 60 μL of accelerated coagulant to previous solution, mix well
      • Inject the solution for separating gel into glass mold, sealing with water, wait to solidify for 10 minutes
      • Pour out water, dry remaining water with filter paper
      • Combine 0.75 mL of stacking gel solution and 0.75 mL of stacking gel buffer, mix well. Add 15 μL of accelerated coagulant.
      • Inject the solution for stacking gel into glass mold, insert comb
      • Wait for 15 minutes to solidify
  • 4.1.2 Performing SDS-PAGE
    • Material:
      • SDS-PAGE gel
      • Electrophoresis gel box
      • Gel caster
      • Power supply and electrodes
      • Protein samples
      • 10~180 kDa TureColor Prestained Protein Marker
      • SDS-PAGE loading buffer
    • Procedure:
      • Place gel into gel box, which is filled with SDS-PAGE buffer solution
      • Prepare each protein sample into proportion of 50 μL protein with 10 μL loading buffer
      • Load each protein sample into separate wells in the gel
      • Load protein marker into the first well
      • Put the lid onto the gel box, set power supply voltage to 180V, make sure electrodes are placed correctly
      • Run the device for approximately 60 minutes
  • 4.1.3 Coomassie brilliant blue staining and destaining
    • Material:
      • Coomassie brilliant blue staining solution
      • Coomassie brilliant blue destaining solution
    • Procedure:
      • Soak SDS-PAGE gel from previous step into Coomassie blue staining dye, stain gel using shaking incubator for 30 minutes
      • Destain with Coomassie blue staining destaining solution, soak and shake overnight
      • Confirm result by exposed with light
      • Take a photo of the result and save it for later use
  • Material:
    • ELISA
    • 6 tubes IPTG induced pET28a-GLBP-vp1-vp7-GFP in BL21 (DE3)
  • Procedure:
    • Inoculate the single colonies of the engineered strains to 10mL of fresh LB medium (containing antibiotics), and incubated at 37°C, 220 rpm for 12h
    • Inoculate 1 mL of bacterial cultured medium to 100mL of fresh LB medium (100mg/L Ampicillin) and incubated at 37°C, 220 rpm until the 0D600 was around 0.6
    • Different IPTG concentration including 0.1 mmol/L, 0.25 mmol/L, 0.5 mmol/L, 1mmol/L, 5mmol/L, and 10 mmol/L were added to the medium
    • Detect GFP intensity in cultivation time from 0h to 8h
    • Save data and analyze
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