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Team:Bilkent UNAM Turkey/Protocols
From 2011.igem.org
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.
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.
