Protocols
Collapse all
Expand all
Chlamydomonas Protocol
Cultivation
Media
To be able to work with C. reinhardtii, you will need medium. This medium can be in either liquid or solid form and is essential for any experiment regarding C. reinhardtii. The media we used for C. reinhardtii is TAP medium.
| Media | 0,5 L | 1,8L |
|---|---|---|
| 40 x TAP/TA/TAPi | 12,5 ml | 20 ml |
| 40 x Beijerink Salts | 12,5 ml | 20 ml |
| Trace Elements (revised) | 3,5 ml | 5,6 ml |
| H2O | 471,5 ml | 754,4 ml |
| For Plates: + Agar (1,8 %) | 7,2 g | 14,4 g |
Tap Medium should be adjusted to 7,0 - 7,2 pH and autoclaved.
Stocks
| 40 x TAP 1L | autoclave, store at 4°C |
|---|---|
| Tris (121,14 g/mol) | 96,8 g (0,8 M) |
| 1 M (K) PO4 | 40 ml (40 mM) |
adjust pH 7.0-7.2 with conc. AcOH (~44ml)
| 40 x TA 1L | autoclave, store at 4°C |
|---|---|
| Tris (121,14 g/mol) | 96,8 g (0,8 M) |
adjust pH 7.0-7.2 with conc. AcOH (~44ml)
| 40 x TAPi 1L | autoclave, store at 4°C |
|---|---|
| Tris (121.14 g/mol) | 96,8 g (0,8 M) |
| 1 M K2HPO3 120.08 g/mol | 40ml (40mM) |
adjust pH 7.0-7.2 with conc. AcOH (~44ml)
| 1 M (K)PO4 | MW(g/mol) | conc. | 420 ml |
|---|---|---|---|
| K2HPO4 ∙3 H2O | 228,22 | 1 M; 228 g/l | ≈0,6 M; 136 g/l |
| K2HPO4 | 136,09 | 1 M; 136 g/l | ≈ 0,4 M; 55,08 g/l |
250 ml 1 M K2HPO4 + ca. 170 ml KH2PO4 , adjust to pH 7, autoclave, store at 4°C
| 1 M (K)PO3 | MW(g/mol) | conc. | 1 l |
|---|---|---|---|
| K2HPO3 | 120.086 | 1 M; 120.086 g/l | ≈1 M; 120.086 g/l |
K2HPO3 was obtained by Haihang co. ltd. (13977-65-6) Autoclave, store at 4°C
| 40 x Beijerinck salts 1 L | autoclave, store at 4°C |
|---|---|
| NH 4Cl | 16 g |
| CaCl2 ∙2H 2O | 2 g |
| MgSO 4 ∙7H 2O | 4 g |
Trace Elements (revised)
To make the trace elements following publication was used: Kropat, J., Hong-Hermesdorf, A., Casero, D., Ent, P., Castruita, M., Pellegrini, M., … Malasarn, D. (2011). A revised mineral nutrient supplement increases biomass and growth rate in Chlamydomonas reinhardtii. The Plant Journal, 66(5), 770–780. http://doi.org/10.1111/j.1365-313X.2011.04537.x
Additives
| Additive (optional) | Working Concentration |
|---|---|
| Arginin | 100 µg/ml |
| Paromomycin | 10-12 µg/ml |
| Hygromycin | 10 µg/ml |
| Ampicillin | 100-500 µg/ml |
Cultures
Generally, all C. reinhardtii strains we used are stored as stocks in agar-tubes at room temperature of and a light intensity of 150µE . To start a liquid culture, these were picked and resuspended in TAP medium, placed in a shaking incubator at 110 rpm and 150 µE. Cultures were subsequently split to remain at a cell density of 10^7 . Successfully transformed clones were plated on TAP-agar plates (with 10 µg/ml antibiotics) and incubated in a light incubator under continuous light conditions at room temperature.
Transformation (Electroporation)
For the nuclear transformation of our C. reinhardtii strains, we chose the electroporation method. Transforming of L1-constructs was performed as a co-transformation with a plasmid conferring antibiotic resistance to select for cells that were transformed successfully.
- Grow a C. reinhardtii culture in TAP medium, shaking at 110 rpm unter alternating temperature and light cycles (14 h 40-60 µmol photons m-2 s-1 at 25 °C and 10 h darkness at 18 °C). Grow cells for 10 days, diluting the culture every 48-72 h to keep them in the logarithmic growth phase. The cell density for transformation should be around 1-2 x 10^6 cells/ml
- Harvest cells and start transformation 1-2 h before switching from light to dark conditions. Centrifuge cells (2500 g for 10 min at RT) and resuspend pellet in ME-Suc buffer to a cell density of 1 x 10^8 cells/ml. Aliquot 500 µL of the cell suspension in 2 ml Eppendorf tubes.
- Heat shock cells, then incubate for 30 min at 40ºC in a thermomixer, while gently mixing at 350 rpm. Let cells recover for 30 min at RT.
- Prepare a 24-well plate with 600 µL TAP medium in each well.
- Transformtation
- Choose electroporation conditions for the desired strain according to Table 1
- One electroporation requires: 38 µL of heat-shocked cells, 0.5 g of antibiotic-resistance marker plasmid and 0.5 µg of the plasmid to be transformed. Combine cells with plasmids for 5 electroporations (see Table 2 below).
- Fill a 2 mm electroporation cuvette with 40 µL of cell-mixture. Carefully monitor the resistance of the solution and keep it around 400-500 Ω.
- To collect the transformed cells after electroporation, transfer 600 µL fresh TAP medium from the recovery plate into the cuvette, then place mix back in the well. Repeat this process with the remaining electroporation mix. Use the same cuvette for all 5 electroporations of the mixture.
- Seal recovery plate with parafilm and incubate overnight with gentle shaking (110 rpm) under continuous light (50 µmol m-2 s-1).
- The following day, place cells from one well (600 µL) gently onto one TAP-Agar plate with 10 µg/ml antibiotics. Incubate the sealed plates at room temperature under continuous light for 7-14 days until single colonies are visible to the naked eye.
| Strain | Cell wall | Voltage | pulse length | interval | No. of pulses | decay rate | polarity |
|---|---|---|---|---|---|---|---|
| Strain | |||||||
| CC-3403 | no | 200 V | 8 ms | 50 ms | 2 | 40% | + |
| UVM-4 | no | 200 V | 8 ms | 50 ms | 2 | 40% | + |
| SAG32-11b | yes | 300 V | 12 ms | 50 ms | 1 | 40% | + |
| 2. Transfer pulse (same for all strains) | |||||||
| all strains | 20 V | 50 ms | 50 ms | 5 | 40% | +/- |
| resistance marker plasmid (1 µg/µl) | 5x 0.5 µg | 2.5 µ |
| L1-construct plasmid (1 µg/µl) | 5x 0.5 µg | 2.5 µ |
| cells (1 x 10 <8 cells/ml) | 5x 38 µl | 190 µl |