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.
Multi Cultivator MC1000
Cultivation experiments conducted with the Multicultivator MC1000 from PSI were set up as followed; All cultivation tubes, hoses, glassware and media was autoclaved at 121°C for 40 min at 4 bar.OD measurements were made at 680 nm and 720 nm. To blank, the media was filled, under a biosafty cabinet, into the cultivation tubes and measurments were made. Cultures were measured in a Photometer and normalized to OD 1 [R.U.] and were then given to the blanked media to dilute them to OD ~0,1 [R.U.].After filling in the cultures, all hoses wer conected as discribed in the MC 1000 manual and the experiments were started.
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 |
Colony Screening PCR
To check if a construct was successfully transformed into C. reinhardtii, a colony PCR can be used as a screening method. The goal of the colony PCR is to amplify the inserted DNA fragment from the transformed C. reinhardtii genome through a PCR. This allows us to assess if the transformation was successful. If a DNA band is visible with the expected length on the electrophoresis gel, the transformation was probably successful. Sequencing of the amplified DNA fragment is necessary to assure the success of the transformation.
Preparation of DNA:
- For each Plate with Chlamy that you are going to screen, take one V-Bottom 96-well-plate
- Put 40 µl of resuspended cell solution in each well of the V-Bottom plate
- Centrifuge at 2500 g for 10 min and discard supernatant immediately
- Add 20 µl dilution buffer to each well and resuspend the pellet. Incubate 5 min at room temperature.
- Centrifuge at 4000 g for 10 min
Preparation of DNA:
It is important to calculate the mastermix total volume and the volume of the components according to the amount of wells with DNA you want to screen. A volume 18 µl mastermix and 2 µl DNA per well is recommended.| Total volume | 100 µl |
|---|---|
| Phire Plant MM (2x) | 50 µl |
| screen FW primer (100 µM) | 0.5 µl |
| screen RV primer (100 µM) | 0.5 µl |
| Betaine (5M) | 20 µl |
| H2O | 30 µl |
PCR:
| PCR cycling | |||
|---|---|---|---|
| initial denaturation | 98 °C | 5 min | |
| [denaturation | 98 °C | 10 s< | |
| annealing | 65 °C | 10 s | |
| elongation | 72 °C | 100 s] | x40 |
| final extension | 72 °C | 2 min | |
| storage | 8 °C | ∞ |
Fluoreszenz Screening
After having obtained successful results of the integration of YFP into a C. reinhardtii clone from the colony PCR, it can be screened for fluorescence. If the YFP protein was expressed correctly, the clone should display a signal.
- For each clone to be screened, place 160 µl of resuspended cell culture in a 96-well plate
- Measure using a fluorescence plate reader. We used the TECAN Plate Reader Infinite 200 Pro, a monochromator
- For YFP, measure using an excitation wavelength of 514 nm and read the emission at 527 nm
E. coli Protocols
Cultivation
Media & Plates
To be able to work with E. coli., you will need medium. This medium is essential for any experiment regarding E coli. The medium we used for E. coli is LB medium.
LB Medium
For 1 L LB medium: 20 g LB Powder in 1 L dest. water, autoclaveLB Plates
For 1 L LB medium with Agar: 25 g LB Powder with 13 g agar-agar in 1 L dest. water, autoclave.| Antibiotics | Working Concentration |
|---|---|
| Spectinomycin | 50 µg/ml |
| Ampicillin | 100 µg/ml |
| Carbenicillin | 100 µg/ml |
Overnight Cultures
Cultivation of single colonies is done overnight for 8-16 hours, depending on desired culture density. Use a snap-cap culture tube or 25 ml-flasks with cotton tops.
- Pipet 5 ml of LB-Medium in the culture tube. If required, add the working concentration of antibiotics.
- Working under sterile conditions, pick single colonies from a growth LB-plate with a sterile toothpick or pipet tip. Drop the toothpick or tip into the culture tube.
- Place the tube in an incubator at 37°C while shaking at 220 rpm.