Experiment
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Experiment

We conducted various experiments to test the bio containment system. In particular, we tried and optimized various transformation methods. Below is a list of our experiments. If you want to know the protocol of each experiment, click the button.

  • Making Media

    • YPD agar medium(selective)

    1. Prepare a flask.
    2. Measure and place in a flask.
      YPD agar g or mL
      Dextrose(g) 20
      Peptone(g) 20
      Yeast Extract(g) 10
      Agar(g) 15
      Autoclaved DW(mL) 1000
    3. Autoclave agar-YPD medium.
    4. Let agar-YPD medium cool to ~60°C but avoid further cooling.
    5. If you need G418 plate, add G418 (50 mg/mL stock) and mix it well.
    6. Pour a thin layer (~5 mm) of YPD agar (> 15 mL) into each petri dish.
    7. Let each plate cool until YPD agar get solid.
    8. Store plates at 4°C before use.

    LB agar medium(selective)

    1. Prepare a flask.
    2. Measure and place in a flask.
      LB agar g or mL
      LB Broth(g) 25
      Agar(g) 15
      Autoclaved DW(mL) 1000
    3. Autoclave agar-LB medium.
    4. Let agar-LB medium cool to ~50°C but avoid further cooling.
    5. If you need Kanamycin plate, add Kanamycin (100 mg/mL stock) and mix it well.
    6. Pour a thin layer (~5 mm) of YPD agar (> 15 mL) into each petri dish.
    7. Let each plate cool until LB agar get solid.
    8. Store plates at 4°C before use.
  • Making Electrocompetent Cells (Yeast)

    • Electroporation Buffers

    • Tris-DTT Buffer: Dissolve 0.39 g 1,4-dithiothreitol in a solution of 1 ml 1 M Tris, pH 8.0 (121.1 g Tris base per liter H2O, pH 8.0) and sterilize by filtration. Store at –20 oC.
    • E Buffer: Dissolve 1.2 g Tris base, 92.4 g sucrose and 0.2 g MgCl2 in deionized H2O to a volume of 1 L. Adjust the pH to 7.5 and sterilize by filtration. This solution can be stored at room temperature.

    Electrocompetent cell preparation

    1. Inoculate Yeast colony (freshly streaked on a YPD plate) to 5 mL YPD media with an and grow overnight at 30 degrees Celsius.
    2. Measure absorbance at 600 nm (OD600)
      1. Add 0.4 mL Fresh YPD to a cuvette and zero
      2. Add 0.1 mL Overnight culture to same cuvette and measure OD600
    3. Inoculate a 50-ml culture in YPD media to an absorbance of 0.1 at 600 nm using the overnight culture from Step 1, 2. If OD600 of overnight culture = 2.0, 47.5 mL Fresh YPD + 2.5 mL = 50 mL YPD culture with OD600 = 0.1
    4. Grow cells at 30 degrees Celsius to an absorbance of about for 6 h. Cells must be in early to mid-log growth phase (OD600 = 1.3 ~ 1.5). Using cells in late log or stationary phase substantially decreases transformation efficiency.
    5. Once cells have reached an absorbance of about 1.3 ~ 1.5 at 600 nm, add 500 μL Tris-DTT buffer to the culture. Incubate in a shaking incubator at 30 oC for 15 min. Transformation efficiency is relatively constant for DTT incubation times of 10–20 min, but decreases considerably for incubation over 20 min. (16:48 = )
    6. Pellet cells at 2,500g for 3 min at 4 oC and wash with 25 mL ice-cold E buffer (Wash = Rinse by suspension, Repellet by centrifugation, and Remove supernatant).
      1. Firstly, add 5 mL ice-cold E buffer to break to pellet. Then, add remaining 20 mL and mix well.
    7. Wash cells again with 1 mL ice-cold E buffer. (Wash = Rinse by suspension, Repellet by centrifugation, and Remove supernatant).
    8. Resuspend cells in E buffer to a total volume of 300 μL. Aliquot 20 μL of resuspended cell-DNA mixture per prechilled electroporation cuvette. Keep electroporation cuvettes on ice until pulsed.
    9. Label the prepared Yeast Competent Cells as ‘BY4742 EC’ and store it in deep freezer.
  • Electroporation

    1. DNA to be put into four electroporation cuvettes with 100 ng plasmid
      Cuvette Electroporation Cuvettes 1mm gap
      EP buffer 1M sorbitol
      DNA 100ng/ul
      Cells S. cerevisiae BY4742 EC
      cells and DNA per cuvette Final volume/cuvette: 20ul
      notice on ice
    2. Load cuvette into gene pulser with appropriate setting and electroporate.
    3. Immediately add 1 ml warm (at 30 degrees Celsius) YPD media to the cuvette.
    4. Transfer cells from pulsed cuvettes to a 12 ml Falcon tube. Wash each cuvette with an additional 1 ml of YPD media to recover the remaining cells from the cuvettes.
    5. Shake cells from for 1 h at 30 degrees Celsius.
    6. Pellet cells at 2,500g for 5 min and remove supernatant.
    7. Resuspend in 10 ml YPD media. Plate serial dilutions on YPD-G418 plates to determine transformation efficiency. The backbone-only control should have an efficiency of less than 1% that of the backbone-plus-insert transformations.

  • LiAc/ssDNA/PEG Transformation
    1. Set the heat bath to 42 degrees and the incubator to 30 degrees.
    2. Prepare the Transformation mix below. Mix as much as possible by vortexing for 10 seconds.
      Reagents volume(μl)
      PEG 3350 (50%(w/v))
      1M Lithium Acetate
      ssDNA (2mg/ml)
      Plasmid
      Autoclaved DW       		240
      36
      50
      0.6
      33.4
      total 360
    3. Thaw the S. cerevisiae competent cells on ice for 10 minutes.
    4. Add the transformation mix to the ep tube containing the competent cells from (1) and vortex for 10 seconds to mix as thoroughly as possible.
    5. Place the EP tube in a heat bath set to 42 degrees Celcius and leave for 40 minutes.
    6. Centrifuge at 13000 rpm, 30s, remove the supernatant (transformation mix) and resuspend the pellet with 1.0 ml YPD liquid medium.
    7. Incubate at 30 degrees Celsius, 250 RPM for more than 2 hours.
    8. Plating on G418 YPD agar plate at 0.1X, 0.01X and incubate in 30 degree incubator.
  • Colony PCR for Yeast Colonies
    1. Sample PCR Template Prep (Yeast Genome Extraction)
      1. Pick different yeast colonies, patch on the plate in order, and release well in a PCR tube with 20 μL UPW (NOT PCR MIX)

        2. *Patch plate = YPD G418

        1. Incubate the patch in a 30 °C incubator for 36 ~ 48h.
        2. Label 'BY4742' and date.
      2. Vortex the inoculated PCR tube for 1 minute.
      3. Spin down by short centrifugation.
      4. Boil the PCR tube in a thermocycler at 98 °C, 10 min => 4 °C.
      5. Use 1μL as the sample PCR Template DNA.
    2. Colony PCR Mix (25 μL mix for 1 sample, 18 colonies)
      PCR Reagents μl
      UPW
      dNTP (2.5mM)
      5X Q5 Buffer
      Q5 DNA Polymerase       		14.25
      2
      5
      0.25       		21.5
      10μM Forward primer
      10μM Reverse primer
      Template DNA       		1.25
      1.25
      1       		3.5
      total volume 25
    3. Thermocycler
      98°C 30s > [98°C 10s => Tm°C 30s => 72°C 50s] x 35 cycle > 72°C 50s > 4°C

    Agarose Gel Electrophoresis

    1. Small tank 1/4 sized gel: Mix 50 ml of 1X TAE buffer and 0.5 g of agarose to prepare a 1% agarose gel. (Prepare appropriate amount for other sizes of gels)
    2. Microwave to completely dissolve the agarose, and cool it in a stirrer.
    3. Before the agarose gel solidifies, add 2.5 µl of 20000X staining star, mix well, insert a small comb into the gel casting tray, and wait until it solidifies.
    4. Put the agarose gel in the tank and pour an appropriate amount of 1X TAE buffer until the gel is submerged.
    5. Mix 5 µL of 100bp plus ladder and 1 µL gel loading dye purple(6X).
    6. Load ladder and PCR products by 5 µl in order (Load ladder in the middle if possible). The remaining 20 µl PCR product will be sent for sequencing.
    7. After 30 minutes of electrophoresis, check the result in a dark room.
  • Golden Gate Assembly
    1. Prepare Golden Gate Master Mix
      SapI(20μL) μl
      Insert + UPW (0.06 pmol each)
      UPW
      Destination Plasmid (0.04 pmol)
      T4 DNA Ligase Buffer(10X)
      T4 DNA Ligase (2000U/μL)
      SapI       		10
      5.5
      0.75
      2
      0.25
      1.5
      total volume 20
      BsaI(15μL) μl
      Insert + UPW (0.06 pmol each)
      UPW
      Destination Plasmid (0.04 pmol)
      T4 DNA Ligase Buffer(10X)
      T4 DNA Ligase (2000U/μL)
      BsaI       		10
      2
      0.3
      1.5
      0.3
      0.9
      total volume 15

      -- 0.1 pmol Gene Fragment (Twist Bio, iDT)

      -- 0.1 pmol Oligo (reverse complements annealed) (Cosmogenetech, iDT)

      - Mass => Mole calculator: https://nebiocalculator.neb.com/#!/dsdnaamt
    2. Mix gently by pipetting up and down 4 times.
    3. Thermocycler
      - [37°C 5 min => 16°C 5 min] x 30 cycles => 60°C 5 min => 4°C
      - If reactions are done overnight, add a 4°C terminal hold to the protocol, but repeat the final 5 min 60°C step the next day before the transformations.
  • Reverse Complementary Oligo Annealing

    • Oligo Annealing Buffers

    1. 1M Potassium Acetate Solution: 1 M Potassium Acetate pH 7.5 (98.15 g Potassium Acetate per liter water, pH 8.0) and sterilize by filtration. Store at room temperature
    2. 2X Oligo Anealing Buffer(2X Duplex Buffer): 200mM Potassium Acetate 10ml, 60mM HEPES 3ml. Adjust UPW to 50ml. Store at room temperature.

    Oligo Doultion & Annealing

    1. Dilute the oligo to 100uM using UPW.
    2. Dispense 25ul each of the forward and reverse primers into an EP tube.
    3. Add 50ul of 2X Duplex Buffer.
    4. Place in a 95-degree Celsius heat block for 5 minutes.
    5. Wait 45 minutes at room temperature before freezing.
  • Heat Shock Transformation
    1. Prepare chemically competent cell (Stable CC) from the deep freezer and label
    2. Leave the chemically competent cell tube in ice for 10 min.
    3. Move to BSC and add Assembly Product 5ul to the tube
      1. If the assembly product was in the refrigerator, heat 60°C, for 5 min in a thermocycler before adding the assembly product to the tube.
    4. Leave the tube for 30 min in ice.
    5. Heat the chemically competent cell. (42°C, 30s)
    6. Leave the tube for 5 min on ice.
    7. Move to BSC and add 900ul LB medium.
    8. Parafilm the EP tube and incubate in a shaking incubator(30°C, 250rpm, 1h).