Phaeodactylum tricornutum expressing GFP under the light inducible promoter was grown for one week in a climate chamber. After that we put it into complete darkness for four days, for degradation of GFP and minimize the amount left in the cells. Thereafter we grew it for 24 hours under different light conditions to test the light inducible promoter.

Five conditions were tested:

		Quantum fluence rate [µmol/ m2s]		wavelength [nm]		intensity [µW/cm2]
Blue light		50		471		1275
Green light		19		571		380
Red light		50		673		1050
White light		-		standard neon tube		1580
darkness		-		-		-

After exposure the cells were harvested in darkness in 1 ml aliquots at 13,000 rpm and shock frozen in liquid nitrogen. Proteins were extracted as described above. With the extracted proteins we performed a Western blot (see below) to investigate if the promoter works and to quantify the expression of GFP under the different conditions.

To identify our proteins we performed Western blots. In a Western blot proteins can be detected by antibodies. Therefore a first antibody binds specifically to the protein of interest and a second antibody that is specific to the first antibody is added for detection. The second antibody is fused with a reporter enzyme (e.g. horseradish peroxidase) that allows detection of chemiluminescence after treatment with a chemiluminescence agent.

In order to blot proteins a SDS gel electrophoresis was performed and afterwards blotted onto a nitro cellulose membrane. To accomplish this four Whatman filter papers were soaked into membrane buffer and a nitro cellulose membrane, also soaked in membrane buffer, were stacked. On this the SDS gel was placed and covered with with four Whatman filter papers soaked with gel buffer. Then the proteins were transferred with 0.8 mA/cm² for two hours.

To prevent unspecific binding of the first antibody, the membrane was rotated for at least one hour in 5 % milk powder in TBST at 4 °C. The primary antibody was dissolved in 2 % milk powder in TBST (dillution: 1:500 anti GFP, anti IgG 1:10,000, all antibodies obtained by Santa Cruz biotechnology) and added to the membrane. Afterwards it was washed two times in TBST for five minutes. The secondary antibody was dissolved in 2 % milk powder in TBST (anti anti GFP 1:10,000, anti anti IgG 1:10,000), added to the membrane and incubated for one hour. Afterwards it was washed with TBST for three times for five minutes each. After incubation with a detection agent, containing Luminol, p-Coumaric acid and H2O2, the detection was performed with a Chemocam Professional from INTAS science imaging.

Sample preparation:
• Resuspend pellet in 1 ml water
• Measure OD (600 nm)
• Take the volume corresponding to a certain OD
• Protein preparation of the taken sample with the predefined OD
  • Cell culture is centrifuged for 5 minutes (full speed)
  • Discard supernatant
  • Add 150 µl sodium hydroxide
  • Incubate 10 minutes on ice
  • Add 850 µl bidest. water
  • Add 200 µl 70 % TCA and incubate for 20 min on ice
  • Centrifuge for 15 minutes at 4 °C (20000xg)
  • Wash pellet with aceton and centrifuge for 15 minutes at 4 °C
  • Repeat the latter step until the chlorophyll is washed out
  • Dry pellet at room temperature
  • Resuspend in 8 M urea-buffer
  • Shake the samples for 15 min at 60 °C
  • Load samples on a 12 % SDS gel
• Load samples on the gel
• Run gel for at least 1 h (120V)
  • Mini-Protean Tetra Cell (Biorad)
• Blotting on a nitrocellulose membrane over night (20 V)
• Submerge blot in 4 % milk powder in TBST
• Incubate for 2 h at 4 °C
• Incubate membrane for 1-2 h with the milk and the primary antibody against GFP and tubulin (dilution: 1:1000 GFP, 1:2000 tubulin)
• Wash membrane in TBST for 10 min (3 times)
• Incubate for 1 h at 4 °C with the secondary antibody in milk-TBST (goat anti mouse HRP)
• Wash for 10 min at 4 °C in TBST (3 times)
• Detection in western imager station (chemocam)
  • ECL-solution:incubate membrane for 1 min in ECL solution