Team:York/Protocols

YORK
YORK

On this page, the QWACC team would like to share with you the protocols that we have been using over the summer. This library of protocols has been developed alongside our supervisors for the purpose of this study. Here you can find the exact methods we use to generate our data and results. We share them in the interest of reproducibility.

Chlamydomonas transformation

  1. Preparing Chlamydomonas batch culture for transformation

Inoculation to produce a 4L culture:

        1. Inoculate 50 mL of TAP with a single, fresh colony from a TAP streak plate.

Use a 250 mL conical flask for efficient aeration and mixing while shaking.

        1. Grow, while continuously shaking, for 5 days at 50 – 100 µE until culture density reaches ~ 1 x 10^7 cells/mL.
        2. Transfer 50 mL into 400 mL of fresh TAP.
        3. Grow while shaking for another 2 days at 50 – 100 µE until culture density reaches ~ 8-9 X 10^6 cells/mL.
        4. Inoculate 4L of fresh TAP with all 450 mL Chlamydomonas, grow while bubble air and continuously stirring for ~30 hrs at 100 µE until culture density reaches 2-4 X 10^6 cells/mL.

Ensure the final inoculation uses an exponential-phase culture.

For smaller volumes (up to 1L), this step can be done in a conical flask with continuous shaking.

The high volume is in case the culture does not reach the desired concentration, a larger volume of cells can be spun down for transformation.

 

  1. Transformation

Work with open cultures should be performed in an appropriate flow hood to decrease contamination risk.

Use 50 mL Chlamydomonas, at 2-4 x 10^6 cells/mL, per transformation.

Each transformation requires 5 x 10^7 cells.

Materials to prepare

        1. TAP Sucrose, 40mM sucrose -- 15mL per transformation
        2. Selection plates

Plate preferences depend on type of screening that will be performed

We use rectangular plates (250 mm x 250 mm)

1.5% TAP agar with appropriate antibiotic

Label on the side to avoid interference with the colony picker camera during selection

        1. DNA insert cassettes digested with appropriate enzyme
        2. Electroporation cuvettes
        3. Centrifuge tubes
        4. Glass L-Shaped spreaders
        5. Pipette tips and pipettes

 

Harvesting

      1. Aliquot Chlamydomonas culture into centrifuge tubes
      2. Pellet Chlamydomonas culture using centrifuge (4 min at 1000x g)
      3. Discard supernatant and avoid disturbing cell pellet

Electroporation

  1. Use TAP 40 mM sucrose to dilute cell pellet to 2x10^8 cells/mL.
  2. Add 250µL of Chlamydomonas culture per electroporation cuvette.
      1. Incubate at 16oC for 5 mins.
      2. Prepare recovery tubes (15 mL falcon tubes) with 8mL TAP sucrose.

Steps 6-8 should be performed as quickly as is possible, especially step 8.

Before electroporation, dry the cuvette.

      1. Add transformation cassette DNA (14.5ng/kb cassette per 250µL) and mix briefly.

Do not mix by repeatedly pipetting, use pipette tip to mix by stirring.

      1. Electroporator should be set to 800V, 25µF.
      2. Administer pulse and immediately add the mixture from the cuvette to the recovery tube.

Plating and growth

  1. After all electroporations are complete, transfer recovery tubes to a shaker, and grow for 6 hours in the dark.
  2. Centrifuge the recovery tubes for 4 min at 1000x g.
  3. Discard most of the supernatant, leaving about 500µL.
  4. Resuspend the pellet in the residual supernatant and  spread on a transformation plate using a glass spreader.  
  5. After all plates are adequately dry, place them upside-down in stacks in plastic bags, and return to growth room.
  6. Incubate transformation plates at 2-10 µE for 1-2 days.
      1. Increase the light intensity to 25-50 µE for 8-12 days

This should yield >2mm colonies that can be used for selection

  • ABOUT US
  • We are an overgraduate team representing the University of York.
  • Our Quicker Way to Analyse Co-Cultures (QWACC) is our entry into the Hardware track for iGEM 2017.
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