Strains and culture conditions
C. reinhardtii strain CC-1690 was acquired from The Chlamydomonas
Resource Center (University of Minnesota) and used as the parental cell
line for all transformants with the exception of the CrCD transformant
which was described previously.[32] Strains were
maintained on 1.5% agar plates containing TAP
medium[33] at 25°C and a light intensity of
~50 μE. Where appropriate, spectinomycin (Spc:
Sigma-Aldrich; S4014) and 5‑fluorocytosine (5-FC: Sigma-Aldrich; F7129)
were added to agar plates at a concentration of 300 μg
mL-1 and 5 mg mL-1, respectively. 20
mL liquid cultures were prepared from freshly grown agar plates (2-3
days) in 50 mL Erlenmeyer flasks and incubated at 25°C,
~50 μE with shaking at 120 rpm.
For growth tests on plates (‘spot tests’), liquid cultures were grown to
mid-log phase (~ 3 x 106) before being
normalised by optical density at 750 nm to the lowest measured sample.
Dilutions were then prepared at 1:10 and 1:100 in TAP medium and 5 μL of
each dilution spotted onto TAP agar plates supplemented with either Spc
or 5-FC. Plates were incubated for 1–2 weeks to allow spots to develop.