Chlamydomonas reinhardtii Transformation and Growth

Chlamydomonas reinhardtii UVM4 cells (Neupert et al., 2009 (link)) were grown in Tris-Acetate-Phosphate (TAP) medium (Kropat et al., 2011 (link)) on a rotatory shaker. For transformation, cells were grown at a light intensity of 100 μmol photons m^–2 s^–1 to a density of 5 × 10⁶ cells/ml and collected by centrifugation at 4000 × g for 2 min. 5 × 10⁷ cells were mixed with 1 μg DNA linearized with NotI and transformed by vortexing with glass beads (Kindle, 1990 (link)). Vortexed cells were diluted twofold with TAP and 2.5 × 10⁷ cells were spread onto TAP agar plates containing 100 μg ml^–1 spectinomycin. Plates were incubated over-night in the dark and then incubated at 30 μmol photons m^–2 s^–1 for about 10 days. For growth curves, cells were inoculated in 100 ml TAP medium and grown at 150 μmol photons m^–2 s^–1 to a density of about 8 × 10⁶ cells/ml. 100 ml TAP or Hepes-Minimal-Phosphate (HMP) medium (5 mM Hepes-KOH instead of 20 mM Tris, no acetate) were then inoculated with 3 × 10⁵ cells/ml in triplicates for each strain and growth was monitored by cell counting using the Z2 Coulter Particle Count and Size Analyzer (Beckmann). The culture volume is the summed cell volume of all cells in 1 ml medium. For mass spectrometry analyses, samples were harvested 22 h after inoculation (early log phase).