Team:Bilkent UNAM Turkey/Protocols

Protocol.

1. Grow cells in appropriate medium (permissive) to a density of ~2 x 10⁶/ mL.

2. Collect cells by centrifugation in sterile centrifugation bottles at room temperature (3500 g x 10 min). Discard supernatant.

3. Resuspend cells in 1/25 – 1/50 initial volume in selective medium with a cotton-plugged pipet. Transfer to a sterile centrifugation tube.

4. Collect cells by centrifugation at room temperature (3500 g x 10 min). Discard supernatant

5. Resuspend cells at approximately in 1/70 initial volume in selective medium (approximately 3.0 x 10⁸ cells / mL.

Count a 1/100 dilution with the hemacytometer under the microscope. Adjust the volume to obtain a concentration of 2. x 10⁸ cells / mL.

6. To a tube containing 0.3g glass beads (sterilized by baking) add:

- 0.3 mL cell suspension.

- ~ 0.5 – 1.0 ug DNA.

7. Vortex at full speed for 15 seconds.

 

8. Pour the contents of the tube on a selective plate, gently tilt and rotate the plate to spread the medium evenly.

9. Allow the liquid to dry (protect from light to avoid phototactic movements of the cells). Seal the plates with Parafilm (Micropore tape for minimal medium), and incubate under appropriate conditions for selection. (If cells were grown under auxotrophic conditions (acetate, dark) and will be selected for photoautotrophic growth (minimal, light), put the plates in dim light for 16 – 24 hours before transferring to light. Colonies will appear within 1-3 weeks depending on the selection applied.)

References:

Kindle, K. (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc. Natl. Acad. USA 87: 1228-1232.

B. Electroporation method for nuclear transformation of Chlamydomonas reinhardtii

Materials

- Cell-wall deficient host cell strain

- Sterile centrifugation bottles and tubes

- Electroporation cuvettes

- Plates with appropriate solid medium for selection of the transformants

- DNA with selection marker

- TAP, 40mM sucrose

- TAP, 40mM sucrose, 0.4% PEG 8 000 - Starch 20%

Starch 20% preparation

20 g starch in a centrifuge tube

Wash with ethanol 100%

Wash with water

Repeat 2 times

Resuspend in 100 ml Ethanol 70%

Aliquots of 20 ml and keep at room temperature

The day of transformation, centrifuge an aliquot 1 minute at 1 000 rpm

Wash 4 times with TAP + sucrose 40 mM

Resuspend in 20 ml of TAP + sucrose 40 mM + PEG 8 000 0.4%

Protocol

1. Grow 250 ml of cells to a density of 2 x 10⁶ cells/ml

2. Collect cells by centrifugation at room temperature at 3 500 rpm for 5 minutes in sterile centrifugation bottles. Discard supernatant

 

3. Resuspend in 1.25 ml of TAP, 40mM sucrose

4. Incubate on ice 10 minutes

5. Transfer 250 μl of cells in a cuvette containing 1 μg of DNA

6. Incubate at room temperature 5 minutes

7. Electroporate 0.75 kV, 25 μF, no R, 6 msec

8. Incubate at room temperature 10 minutes

9. Add 1 ml of starch 20% and pour the contents of the cuvette on a selective plate, gently tilt and rotate the plate to spread the medium

10. Allow the liquid to dry (protect from light), seal the plates with parafilm and incubate under appropriate conditions for selection of transformants.

C. Chloroplast transformation of Chlamydomonas reinhardtii

Materials.

- Host cell strain.

- Sterile liquid growth medium (permissive for the host cell line). (Approximately 10 mL of culture / transformation plate.)

- Sterile liquid growth medium (corresponding to selective conditions). (This will be used to wash the cells by centrifugation before transformation. Use appropriate medium(eg minimal) depending on the selection for transformants that will be applied.)

- Sterile centrifugation bottles and tubes.

- Sterile, cotton-plugged 5 mL pipets.

- Plates with appropriate solid medium for selection of the transformants.

- DNA with selection marker. (1ug / uL, 1.0 ug per sample, sufficient for up to 7 plates).

- 100 mg/mL tungsten powder in sterile 50 % glycerol. (25 uL per sample)

- 2 M CaCl₂, sterile (25 uL per sample)

- 100mM spermidine (base), filter sterilized. (10 uL per sample)

- Filter holders for Helium gun.(Sterilize by washing with Ethanol, air dry in sterile hood)

- Sterile microfuge tubes and tips.

 

Protocol.

1. Grow cells in appropriate medium (permissive) to a density of ~2 x 10⁶/ mL.

2. Collect cells by centrifugation in sterile centrifugation bottles at room temperature (3500 g x 10 min). Discard supernatant.

12

3. Resuspend cells in 1/30 initial volume in selective medium with a cotton-plugged pipet. Transfer to a sterile centrifugation tube. (Steps 3 and 4 can be omitted if the media for the culture and for selection on the plates are compatible).

4. Collect cells by centrifugation at room temperature (3500 g x 10 min). Discard supernatant

5. Resuspend cells in 1/30 initial volume in selective medium (approximately 6 x 10⁷ cells / mL).

6. Plate 0.3 mL of cell suspension evenly on selective plate.

7. Allow the liquid to dry (protect from light to avoid phototactic movements of the cells).

8. Sonicate the tungsten suspension briefly (the tube is attached with a stand and clamp so as to touch the tip of the sonication probe immersed in a beaker of water).

9) In a sterile microfuge tube placed on ice, add in order:

- 25 uL 100 mg/mL tungsten (in 50 % glycerol)

- 2 uL DNA (0.5 mg / mL)

- 25 uL CaCl₂, 2 M.

- 10 uL Spermidine base, 0.1 M.

10. Incubate on ice for 10 min.

11. Spin 1-2 min in microfuge.

12. Remove 25 uL of the supernatant. Resuspend the rest by vortexing and a brief sonication (2-3 sec) as above.

13. Apply 8 uL to a filter holder, attach to Helium outlet. Place a plate in the apparatus and proceed with bombardment. (Parameters that can be optimized include: Helium pressure, opening time of the valve, pressure in the chamber, distance from the sample holder to the plate).

14. Seal the plates with Parafilm (Micropore tape for minimal medium), and incubate under appropriate conditions for selection. (If cells were grown under heterotrophic conditions (acetate, dark) and will be selected for photoautotrophic growth (minimal, light), put the plates in dim light for 16 – 24 hours before transferring to light. A ring of colonies will appear within 1-3 weeks depending on the selection applied.)

References.

Boynton et al (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240: 1534-1538.

Finer et al. (1992) Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Reports 11: 323-328.