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.

1. 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
2. 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.
3. 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.
4. Prepare a 24-well plate with 600 µL TAP medium in each well.



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%	+/-



5. Transformtation
1. Choose electroporation conditions for the desired strain according to Table 1
2. 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).

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

3. 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.
4. 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.
5. Seal recovery plate with parafilm and incubate overnight with gentle shaking (110 rpm) under continuous light (50 µmol m^-2 s^-1).
6. 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.

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:

1. For each Plate with Chlamy that you are going to screen, take one V-Bottom 96-well-plate
2. Put 40 µl of resuspended cell solution in each well of the V-Bottom plate
3. Centrifuge at 2500 g for 10 min and discard supernatant immediately
4. Add 20 µl dilution buffer to each well and resuspend the pellet. Incubate 5 min at room temperature.
5. Centrifuge at 4000 g for 10 min

Preparation of DNA:

PCR:

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.

1. For each clone to be screened, place 160 µl of resuspended cell culture in a 96-well plate
2. Measure using a fluorescence plate reader. We used the TECAN Plate Reader Infinite 200 Pro, a monochromator
3. For YFP, measure using an excitation wavelength of 514 nm and read the emission at 527 nm

1. Thaw 50 µl competent E. coli cells on ice and add 1-5 µl plasmid. Add the same amount of water to ONE tube, as a control with no expected growth
2. Mix carefully, do not vortex/resuspend
3. Incubate on ice (20-30 min)
4. Heat shock: 30-60 sec, at 42 °C
5. Incubate on ice (2 min)
6. Suspend the cells in 1 ml LB Medium and incubate for 45 min at 37 °C
7. Centrifuge cells at 100 g for 4 min at 4 °C
8. Dispose most of the supernatant
9. Put 50-100 µl of cell suspension onto a petri dish with LB medium with antibiotics
10. Grow overnight at 37 °C