The cultivation of P. tricornutum requires continuous light (8000-11000 lux) at 22° C. Liquid cultures are constantly shaken at 225 rpm in Erlenmeyer flasks containing f/2 medium. Use parafilm for f/2 agar plates to avoid dehydration. For strain maintenance use selective plates and transfer the cells to new plates every four to six weeks.

• Microcarrier preperation

  Transfer 60 mg Tungsten M 10 Microcarrier into a sterile 1.5 Eppendorf cup and add 1 ml sterile EtOH abs. Vortex the suspension 1 min and centrifuge for 1 min with 20000x g at room temperature. Discard the supernant and resuspend the pellet in 1 ml sterile ddH₂O, thereafter centrifuge (1 min/20000 g/RT). Repeat this step and finally resuspend the pellet in 1 ml of sterile ddH₂O. Divide the suspension into 50 µl aliquots. You can store them at -20 °C. ! Vortex the final 1 ml suspension after every aliquot to avoid sedimentation.

• Preperation of P. tricornutum

  You need 100 µl of a 108 cells containing suspension for a single transfection. About one week after inoculation you assess the cell number of your liquid culture by a counting chamber. We used 5 µl of the liquid culture to load a Thoma counting chamber. With the resulting average cell number per small squares, the total cell number were calculated as follows: Cell number= Ø cells/small square * 4 (chamber factor) * 106 * volume of liquid culture (ml) You pelletize the calculated volume by centrifugation (5 min/ 1500 g/RT) and discard the supernant afterwards. Resuspend the pellet in an appropriate volume and transfer 100 µl to the middle of an f/2 agar plate. Use a sterile inoculation loop to spread out the suspension from the center to half the radius of the plate. Incubate the cells for 12-24 hours at conditions given above before you use them for transfection.

• DNA precipitation to the microcarriers

  Transfer 5 µg DNA, 50 µl CaCl₂ and 20 µl Spermidine (0.1 M) into the 50 µl Tungsten-aliquot tube and vortex the mixture 1 min. After 10 min of sedimentation discard the supernant and resuspend the pellet with 250 µl EtOH (HPLC grade). Repeat the vortex-sedimentation-steps. Discard the supernant and resuspent the pellet with 50 µl EtOH (HPLC grade). Now you can use the particles for transfection. The prepareted particles are sufficient for three transfection. ! We recommend to use DNA received by midi-preperation with a minimum concentration of 1000 ng/µl. DNA received by mini-preparation leads to drastical reduction of transfection efficiency.

• Biolistic transfection

  We achieved the transfection by using the Biolistic PDS-1000/He Particle Delivery System from Biorad. Before you can start the transfection procedure, perform a cleaning shot without DNA. Thereafter clean the gun and every single unit you need for transfection with EtOHabs (HPLC grade). After all ethanol evaporated transfer 15 µl of the microcarriers onto the macrocarriers lying on the macrocarrier holders. Assemble the components of the gun by following the producer’s manual. Induce the shot with a vacuum of -25 psi and a pressure of 1350 psi. Release the vacuum immediately after the ripping-event of the rapture-disc. After the bombardment, seal the plates with Parafilm and incubate at 22 °C under continuous light for 24 hours.

• Wash step

  5. Wash step After the incubation use 1 ml f/2-medium to wash the cells from the plates. Spread the cell suspension onto three f/2-zeocin-agar plates equally and thereafter seal the plates with parafilm. The following period of incubation occurs at the conditions given above until the arising colonies are big enough for further analysis (Ø < 1 mm). Then pick the colonies to new selective plates.

For the constructs tagged with GFP we used a Leica TCS SP2 laser scanning microscope (Leica) for the workable confirmation of our biobricks. The protein expression was induced with light or nitrate according to the used promotor.

• Biolistic PDS-1000/He Particle Delivery System (Bio-Rad)
• 1350 psi Rapture Discs (Bio-Rad)
• Macrocarrier (Bio-Rad)
• Macrocarrier Holders (Bio-Rad)
• Stopping Screens (Bio-Rad)
• Tungsten M 10 (Ø 0.7µm) Microcarrier (Bio-Rad)

Solution added to the medium before usage with a dilution of 1:1000.

Solution added to the medium before usage with a dilution of 1:1000.

• For f/2 solid medium: add 1.5 % (w/v) agar
• For selection medium: add 75 µg/ml zeocin