Team:IngenuityLab Canada/Protocols

Ingenuity Lab - dNANO

 

Project Protocols

Cell Culture and Protein
Purification Protocols
DNA Nano-Wire
Protocols
Ethanol and Isopropanol
Precipitation

Cell Culture and Protein Purification Protocols

HT3 media stock solutions preparation

Stock Solutions:

Stocks for trace mineral solution

  Amount (mg) Volume (mL) Concentration (mg/mL)
ZnSO4 227 10 0.5
Na2MoO42H2O 391 10 >0.5
CuSO45H2O 1975 25 0.05
Co(NO3)26H2O 2490 50 0.05

Trace mineral solution – 50 mL

  Amount (mg) Concentration (mg/ml) Concentration (mg/ml)
Na2EDTA H2O 52 n/a 1.04
H3BO3 143 n/a 2.86
MnCl2 94 n/a 1.80
ZnSO4 n/a 0.5 0.22
Na2MoO4 2H2O n/a 0.5 0.39
CuSO4 5H2O n/a 0.05 0.079
Co(NO3)2 6H2O n/a 0.05 0.049


Prepare fresh iron citrate stock solution – 10 mL

  Amount (mg) Volume (mL) Concentration (mg/ml)
Citric Acid 300 n/a 6
Ferric Ammonium Citrate 300 n/a 6

Stocks for HT3 media:

  Amount (g) Volume (mL) Concentration (mg/ml)
KH2PO4 1.43 50 40
MgSO4 7H2O 3.75 50 75
CaCl2 2H2O 1.8 50 36
NaCO3 1.0 50 20

Fresh kanamycin and glucose – sterile filter.

  Amount (g) Volume (mL) Concentration (g/ml)
Glucose 5 50 0.1
Kanamycin 0.1 10 0.01

HT3 media preparation

  1. HT3 media – 10L
      Amount (g) Volume (ml) Concentration (M)
    NaNO3 15 n/a 1.7e-2
    Iron Citrate Solution n/a 10 3e-5
    KH2PO4 n/a 10 1.75e-4
    MgSO4 7H2O n/a 10 3.04e-4
    CaCl2 2H2O n/a 10 2.45e-4
    NaCO3 n/a 10 1.89e-4
    Trace Mineral n/a 10 n/a
    Glucose n/a 36 2e-3
    ddH2O n/a Bring up to 9990 n/a
  2. pH the media to 6.8 using NaOH/HCl
  3. Sterile filter the media using 0.22-micron sorbitol filter in a 10L transparent carboy.
  4. Add 10ml of stock kanamycin (sterile filtered) into the filtered 9.99 L of media.

Recovery of HT3 cells from glycerol stocks

  1. Obtain HT3 cells stored in HT3 Media + 8% DMSO form nitrogen storage.
  2. Thaw the cells in 42˚C water bath.
  3. After thawing is completed, transfer the culture to Eppendorf Tube.
  4. Centrifuge for 3 min at 7000 rpm using table top centrifuge.
  5. Discard the Supernatant and re-suspend the cells in 1mL of freshly prepared HT3 media.
  6. Inoculate the cells from Step 5 in 49 mL of the HT3 Media in a 250 mL flask using sterile techniques.
  7. Incubate the flask in dark (tin foil wrap) at 30˚C at 100 rpm shaker overnight.
  8. After 24 hours, incubate the 50 mL culture in photosynthetic light 50 μmol/m2s and incubate at 250 rpm.
  9. Measure Absorbance of the culture daily at 730 nm in a 1 ml Cuvette.
  10. After the cells have Optical Density of 0.8, inoculate the 50 mL culture into freshly prepared 450 mL of HT3 Media in a 1 L flask.
  11. Incubate at 30ᵒC at 250 rpm under 50 μmol/m2s photosynthetic light.
  12. Monitor the Optical Density as in Step 9 daily and once reached 0.8 the culture is ready to be inoculated in 10L of media.

Inoculation and Growing of 10 L of HT3 Media

  1. Once the 500 mL cultures in 1 L flask have reached Optical Density of >0.8, inoculate 200 mL of culture into 10L of HT3 Media.
  2. Place the Carboy into the incubator at 30OC with multiple Photosynthetic light surrounding carboy.
  3. Feed filtered (0.2-micron Sorbitol Filter) air into the carboy to induce bubbling and stirring.
  4. Measure the Optical Density daily.
  5. Once the Optical density has reached 1.0, the culture is ready is to be harvest to purify the Photosystem II protein.

Cryopreservation of HT3 Cells

  1. Prepare HT3 Media with 8% DMSO.
  2. Centrifuge 1 mL of Cell culture with Optical Density of 0.8 at 7000 rpm for 3 min.
  3. Re-suspend the cells in 1 mL of HT3 Media containing 8% DMSO.
  4. Place the re-suspended cells in Cyro-freezer compatible tubes.
  5. Store the cells in Cyrofreezer in Liquid Nitrogen for long term storage.

Photosystem II Purification

    Culture Harvest

  1. Grow HT3 cells in BG-11 media supplemented with glucose and Kanamycin in 10L carboy under 50 uE m-2s- 1 at 30ᵒC with air bubbling for 3 days.
  2. Check the Optical Density (OD) at 730nm and harvest only when the OD is >0.8
  3. Harvest culture using the Floor-model centrifuge; cell pellets by re-suspending them with Buffer A (50mM MES-NaOH, pH 6.0, 10mM MgCl2, 5 mM CaCl2, 25% glycerol)
  4. Combine washed cell pellets and centrifuge at 12,000gs for 10 minutes.
  5. Resuspend cells in Buffer A; Determine chlorophyll A (chlA) concentration with 80% (v/v) acetone and check the Oxygen Evolving Activity.
  6. Oxygen Evolution

    Refer to oxygen evolution protocol


    7. Cell Lysis and Clarification

  7. Cells were lysed with inhibitors added and through 2 passes at 35 psi.
  8. Centrifuge the sample from the cell disrupter rotor and resuspend the pelleted Thylakoid membranes
  9. Bring the resuspended Thylakoid membranes to to 0.8% (w/v) dodecyl maltoside (DDM)


    10. Thylakoid Membrane Isolated and Solubilization

  10. Incubate sample for 20 minutes in the cold room with gentle stirring.
  11. Removed unsolubilized material by centrifugation at 12,000g for 20 min. Decant the solubilized Thylakoid membrane extract

    12. Affinity Chromatography Purification

  12. Load the extract on pre-equilibrated GE Ni2+ Sepharose Binding Buffer (Buffer A + 0.04% (w/v) DDM), Elution buffer (Buffer A + 0.04% (w/v) DDM + 200mM L-Histidine).
  13. Elution PSII fractions with 35% Elution Buffer.
  14. Pool fractions in the center 90% of the target peak at 673nm wavelength
  15. Concentrate sample in 15mL Amicron 100kd MWCO centrifuge concentrator by centrifuging and remove L-histidine by diluting sample and concentrating twice.
  16. Collect the PSII sample and aliquot in PCR tubes to be stored in -80ᵒC Cyrofreezer

    17. Proteoliposomes Preparation

      Making Liposomes:

    1. In a glass vial measure out Escherichia coli Total Lipid Extract and dissolve it in chloroform to a concentration of 40mg lipid /ml solvent stock
    2. From the stock, transfer a volume of the solution mix to another glass vial and mix with chloroform to a concentration of 20mg/ml.
    3. For a small volume of 2ml or less of the 20mg/ml, evaporate the chloroform using dry nitrogen gas in a gas fume hood until there a thin lipid film.
      • for larger volumes, use round bottom flask in rotatory evaporator to prepare a thin lipid film.
    4. Rehydrate the lipid film from step 3 by suspending the lipid film in buffer (5mM MES, 50mM KCl, 200µM Pyranine, 2mM MgCl2 pH 6.5) to a final concentration of 15.34mg/ml.
    5. Vortex until the film emulsifies into the buffer
    6. Sonicate the emulsified solution for 5-10 minutes.
    7. Freeze the emulsified solution for 5 minutes at -80ᵒC.
    8. Transfer the solution to a 42ᵒC water bath for 5 minutes; Repeat (Step 7-8), the freeze-thaw cycle twice
    9. Using Avanti Polar Lipid Extruder, extrude through a 0.4µm nucleopore polycarbonate membrane 21 times to form liposomes.
    10. The liposome can be stored in -80ᵒC until needed for use.

      11. Preparing the Proteoliposome:

    11. Mix buffer (5mM MES, 50mM KCl, 2mM MgCl2, 2mM CaCl2 pH 6.52) and liposome solution to a concentration of 4mg/ml,
    12. Add detergent at saturated (Rsat) and solubilized (Rsol) concentration.
      • For CHAPS, Rsat is 3.0mM and Rsol at 7.0mM
      • Photosystem II protein concentration to 2.3mg/ml,
    13. Incubate the detergent and liposome mix overnight (~10 hours)
    14. Add photosystem II protein to the lipid-detergent solution and incubate for 30 minutes at 4ᵒC mixing gently on a rotating mixer.
      • Keep all the samples in dark from this point on.

      15. Detergent Removal Step:

    15. After protein-lipid-detergent incubation, add 80mg of BiobeadsTM suspeneded in buffer and incubate for 1 hour at 4ᵒC on the rotator).
      • 20grams of BiobeadsTM SM-2 Resin dissolved in buffer (5mM MES, 50mM KCl, 2mM MgCl2, 2mM CaCl2 pH 6.52), degassed and stored in 4ᵒC for use.
    16. Repeat (Step 14) for 3 detergent additions; after the third addition, add 240mg of BiobeadsTM.
    17. Remove the BiobeadsTM from the proteoliposomes by SPN Column Protein Assay and centrifuge in bench-top micro-centrifuge at 1000g for 1 minutes to separate BiobeadsTM from solution.

      18. Removing Pyranine from Assay:

    18. Desalt the proteoliposomes on the FPLC using a 5ml Hi-trap column in buffer (5mM MES, 50mM KCl, 2mM MgCl2, 2mM CaCl2 pH 6.52)
    19. Pool fractions in the center 90% of the target peak. (1mL per well)

      20. Oxygen Activity:

    20. Run Oxygen Evolution on the liposome (refer to the oxygen evolution protocol. Use the buffer liposome was prepared in)

      21. Protein Activity:

    21. Prepare each 5ml of stock solution so that each of come down to the following final concentrations when aliquoted to 200µl assay buffer (5mM MES, 50mM KCl, 2mM MgCl2, 2mM CaCl2 pH 6.52)
      • 300µM DCBQ, 0.2µM Valinomycin, 2mM Ferricyanide, 20mM DPX
      • 300µM DCBQ, 0.2µM Valinomycin, 2mM FeCY, 20mM DPX, 1.5µM CCCP
      • 50 µM DQ, 0.2µM Valinomycin, 2mM FeCY, 20mM DPX
      • 50µM DQ, 0.2µM Valinomycin, 2mM FeCY, 20mM DPX, 1.5µM CCCP
    22. Add 175µl of assay buffer and 25µl of proteoliposomes to a 200µl total volume in each well of 96 well plate.
    23. Use the plate reader to check florescence at 460nm and 404nm.
    24. Take an initial reading at time 0
    25. Expose the plate under UV light for set period of time and take reading after each time point.
    26. Add CCCP at the 18 minutes' time mark and take a reading.
    27. To convert the fluoresce value to pH, use the ratio of 460nm/404nm and put it in a Pyranine standard curve.
      • Pyranine Standard Curve can be created using 1µM pyranine in buffer at a range of pH between 3-9 and using the ratio of fluoresce value at 460nm/404nm

    CCCP: Carbonyl cyanide m-chlorophenyl hydrazine
    DPX: p-xylene-bis-pyridinium bromide
    FeCY: Ferricyanide
    DQ: decylubiquinone
    DCBQ: 2,5-Dichloro-1,4-benzoquinone

    </div>

    DNA Nano-Wire Protocols

    DNA origami folding buffer preparation

    1. Stock - 1M MgCl2 – 100 mL
       Amount (g)Volume (mL)Concentration (M)
      MgCl24.761501
    2. 20x folding buffer - 100mL
       Amount (g)Volume (mL)Concentration(mM)
      Tris-HCl1.576 100
      EDTA0.5845 20
      MgCl2 – from 1M stock-0.022220
    3. pH the 20x folding buffer at 8.0 using NaOH/HCl
    4. Sterile filter the solution
    5. Store at room temperature

    Folding reaction protocol

    1. Re-suspend the staple oligo in nuclease free water with final concentration of 100 mM of each staples. Store in -20˚C after use
    2. Prepare a concentrated cocktail mix with all the staples with final concentration 460 nM
    3. Total folding reaction is 50 μL
    4. Mix DNA scaffold (single stranded), staple strands and capture strand (Poly A) with concentrations listed below:
      1. Scaffold: 10 nM
      2. Staple strand (concentration of each strand): 100 nM
      3. Capture strand: 10 nM

      5. Note: The ratio of scaffold DNA, staple DNA (individual) and capture strand should always be 10:100:10, respectively

    5. Add 2.5 uL of the 20 x folding buffer to the reaction mixture created in step 5
    6. Bring the volume up to 50 μL
    7. Thermo-cycler setup
      1. Heat at 80˚C for 5 min
      2. 80OC to 60˚C temperature decrease linearly over the course of 80 minute. (4 min/OC)
      3. 60OC to 24˚C over the course of 14 hours and 52.2 minute with decrease rate of 24.7 min/OC
      4. Hold at 24˚C for infinity
    8. Confirm by modified agarose gel analysis

      9. Note: Continue further if positive result obtained from agarose gel analysis

    9. Purify the DNA origami sample using the 100 kDa MWCO Amicon Ultra 0.5 mL filters
      1. Load sample on the column and fill 450 μL of 1x folding buffer to get total volume of 500 μL
      2. Centrifuge at 14000g for 5 min using table top centrifuge
      3. Discard the filtrate and add folding 450 μL of 1x folding buffer and centrifuge at 1400g for 5 min
      4. Carefully place the column inverted in a fresh Eppendorf tube
      5. Centrifuge at 1000g for 3 min to get purified DNA orgami solution
      6. Determine concentration and image it with TEM

    DNA origami agarose gel analysis

    1. Measure out 0.3 grams of agarose
    2. Dissolve in 30 mL of 0.5x TBE buffer
    3. Microwave for 30 seconds with swirling at 15 second mark
    4. Once cooled to lukewarm temperature add 330 μL of MgCl2 (11mM final) to the mixture
    5. Add EtBr to final concentration of 0.5 μg/mL and swirl until complete homogenous mixture is present
    6. Insert the comb into apparatus and pour the gel. Let it solidify over 5 min
    7. Fill apparatus with 0.5x TBE buffer containing 11 mM MgCl2
    8. Mix the sample with DNA loading dye load them on the gel

      9. Note: Do not let the gel outside for extended period of times because it can dry out. Always have it submerged in TBE buffer after solidification

    9. Load the samples and run at 60 V for about 2-3 hours

    PCR reaction set up

    1. PCR reaction mix (50 μL) – protocol obtained from NEB
      1. 10 ng template DNA
      2. 0.5 μM forward primer (IDT technologies)
      3. 0.5 μM reverse primer (IDT technologies)
      4. Phusion polymerase HF buffer (NEB)
      5. 0.5 μL of phusion polymerase (NEB)
      6. Fill up to 50 μL with nuclease free H2O
    2. Thermo-cycler set up
      1. Initial denaturation: 98˚C – 45 seconds, 25-35 cycles
      2. Denaturation: 98˚C – 30 seconds
      3. Annealing: 63˚C – 30 seconds
      4. Extension: 72˚C - 1 minutes and 45 seconds*
      5. Final extension: 10 minutes
      6. Hold: 4˚C

      * Steps b-d: 25-35 cycles

    Agarose gel for DNA origami analysis

    1. TBE buffer (5X) 1L
       Amount (g)Volume (mL)Concentration
      Tris base54- 
      Boric acid27.5- 
      0.5 M EDTA-20 mL1mM
    2. Agarose gel composition – 1% agarose (w/v)
      1. Agarose – 0.30 grams
      2. Mix agarose with 30 mL 0.5x TBE buffer and add MgCl2 to 11 mM final concentration in 125 mL flask
      3. Heat in microwave for 30 seconds. If agarose isn’t dissolving repeat process
      4. Setup gel apparatus
      5. Gently swirl the mixture of agarose mixture until lukewarm
      6. Add EtBr (10 μL) to the mixture and pour the mixture
      7. Let it solidify for 5 minutes
      8. Load DNA origami sample
      9. Apply 120 volts and run for approximately 45 minutes
      10. View the gel under UV light

    Agarose Gel Composition – 1%

    1. Agarose – 0.30 grams
    2. Mix agarose with 30 mL 1x TAE buffer
    3. Heat in microwave for 30 seconds. If agarose isn’t dissolving repeat process
    4. Setup gel apparatus
    5. Gently swirl the mixture of agarose mixture until lukewarm
    6. Add EtBr (10 μL) to the mixture and pour the mixture
    7. Let it solidify for 5 minutes
    8. Load DNA samples
    9. Apply 120 volts and run for approximately 45 minutes
    10. View the gel under UV light

    Ethanol and Isopropanol Precipitation

    Ethanol precipitation

    1. Adjust the salt concentration using sodium acetate (NaOAc, 0.3M, pH 5.2, final concentration)
    2. Add 2 - 3 volumes of ice cold 100% ethanol. Mix by pipetting
    3. Stored in -80˚C for 1 hour. To allow for DNA precipitation
    4. Centrifuge at 15,000 x g for 30 minutes at 4˚C. To pellet the precipitated DNA
    5. Decant the supernatant, carefully, using a pipette tip. Do not disturb the DNA pellet
    6. Wash the DNA pellet twice, using 70% ethanol. Be careful not to disturb the pellet
    7. Air dry at room temperature to evaporate residual 70% ethanol
    8. Re-dissolve your DNA pellet in suitable buffer solution or neutral pH deionized water that is nuclease-free

    Isopropanol precipitation

    1. Adjust the salt concentration using sodium acetate (NaOAc, 0.3M, pH 5.2, final concentration)
    2. Add 1 volumes of 100% isopropanol. Mix by pipetting. Room temperature
    3. Centrifuge at 15,000 x g for 30 minutes at 4˚C. To pellet the precipitated DNA
    4. Decant the supernatant, carefully, using a pipette tip. Do not disturb the DNA pellet
    5. Wash the DNA pellet twice, using 1 mL of 70% ethanol. Be careful not to disturb the pellet
    6. Air dry at room temperature to evaporate residual 70% ethanol
    7. Re-dissolve your DNA pellet in suitable buffer solution or neutral pH deionized water that is nuclease-free