Team:Bilkent UNAM Turkey/Protocols

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Growth Conditions

Tris Acetate Phosphate (TAP) medium

from Gorman, D.S., and R.P. Levine (1965) Proc. Natl. Acad. Sci. USA 54, 1665-1669.

This is probably the most widely-used medium at present for experimental work.

Following solutions are prepared. 

   1. TAP salts
    
   NH4Cl	 	15.0 g
   MgSO4 . 7H2O	 	4.0 g
   CaCl2 . 2H2O	 	2.0 g
   water to 1 liter	 

   2. phosphate solution
    
   K2HPO4	 	28.8 g
   KH2PO4	 	14.4 g
   water to 100 ml	 

   3. Hutner's trace elements


To make the final medium, mix the following: 

   2.42 g Tris
   25 ml solution #1 (salts)
   0.375 ml solution #2 (phosphate)
   1.0 ml solution #3 (trace elements)
   1.0 ml glacial acetic acid
   water to 1 liter

   For solid medium, add 15 g agar per liter

Autoclave.

For Tris-minimal medium omit the acetic acid and titrate the final solution to pH 7.0 with HCl

Reference: http://www.chlamy.org/TAP.html

== Hutner's trace elements ==


Hutner et al. (1950) Proc. Am. Philos. Soc. 94, 152-170

This mixture is used both in TAP and in the Sueoka high salt medium.

For a detailed analysis of how well this trace elements solution meets the nutritional requirements of C. reinhardtii, see Merchant et al. (2006) Biochim. Biophys. Acta 1763, 578-594.


For 1 liter final mix, dissolve each compound in the volume of water indicated.

The EDTA should be dissolved in boiling water, and the FeSO4 should be prepared last to avoid oxidation. compound amount water EDTA disodium salt 50 g 250 ml ZnSO4 . 7 H2O 22 g 100 ml H3BO3 11.4 g 200 ml MnCl2 . 4 H2O 5.06 g 50 ml CoCl2. 6 H2O 1.61 g 50 ml CuSO4 . 5 H2O 1.57 g 50 ml (NH4)6Mo7O24. 4 H2O 1.10 g 50 ml FeSO4. 7 H2O 4.99 g 50 ml

Mix all solutions except EDTA. Bring to boil, then add EDTA solution. The mixture should turn green. When everything is dissolved, cool to 70 degrees C. Keeping temperature at 70, add 85 ml hot 20% KOH solution (20 grams / 100 ml final volume). Do NOT use NaOH to adjust the pH.

Bring the final solution to 1 liter total volume. It should be clear green initially. Stopper the flask with a cotton plug and let it stand for 1-2 weeks, shaking it once a day. The solution should eventually turn purple and leave a rust-brown precipitate, which can be removed by filtering through two layers of Whatman#1 filter paper, repeating the filtration if necessary until the solution is clear. Store refrigerated or frozen convenient aliquots. Some people shorten the time for formation of the precipiate by bubbling the solution with filtered air.

If no precipitate forms, the solution is still usable. However, you might want to check the pH in this case and adjust it to around 7.0 using either KOH or HCl as needed.

To prepare sulfur-free trace elements for hydrogen generation, the sulfate salts can be replaced with equimolar chloride salts (ZnCl2 10.0 g; CuCl2 . 2 H2O 1.00 g; FeCl2 . 4 H2O, 3.60 g). . Reference: http://www.chlamy.org/trace.html

Transformation of Chlamydomonas with Glass Beads

contributed by Karen Kindle

  • 1. Grow cells in SGII+NH4NO3 until they reach a density of 1-2 x 10^6/ml.
  • 2. Spin down cells at moderate speed (5000 rpm for 5 min in a GSA type rotor).
  • 3. Resuspend in 1/100 volume SGII+KNO3 and allow to shake at room temperature for 2-4 hours.
  • 4. If desired, treat cells with gamete autolysin (prepared as described in The Chlamydomonas Sourcebook*) for approximately 45 minutes. I usually resuspend the cells in about 1/25 the original volume in autolysin. Spin and wash once with SGII+KNO3 before transformation. Optional: Test effectiveness of lysis by sensitivity to 0.05% NP-40. (Count duplicate samples +/- detergent in hemacytometer.)

Note: Several recent experiments suggest that autolysin treatment increases the transformation rate of cw-15 mutants.

  • 5. Work in lots of a few tubes at a time. Add 300 microliters cells, 100 microliters 20% polyethylene glycol, 1-2 micrograms DNA (linearized DNA generally transforms somewhat more efficiently than supercoiled), and 300 mg sterile glass*** beads. Vortex for 15-30 seconds at top speed on a Fisher Vortex Genie 2 mixer; plate cells immediately on an SGII+KNO3 selective agar**. Depending on age and moisture of plates, parafilm immediately or after 24 hours; and put in the light.

Colonies should be visible in about 6 days.

* The protocol is followed except that the culture supernatant of the mated gametes is filtered through a 0.22-0.45 micrometer nitrocellulose filter. I don't know how much of the activity (if any) is lost in this protocol, but the requirement for sterility to my mind outweighs any loss in this step. After the filtration, the supernatant is frozen in 50 ml lots at -20 deg C.

** Use washed agar to remove traces of ammonia and other impurities.

***Glass beads are approximately 0.5 mm in diameter and are available from Thomas. We wash them with acid, rinse them with water until the pH is neutral, dry them, and then weigh 300 mg into glass culture tubes and bake for approximately 2 hours at 400 deg F.

 

Different Protocol to Transform Chlamy with Glass Beads.

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.

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