Transformation of C. reinhardtii
Plasmids were delivered to the C. reinhardtii chloroplast using
microprojectile bombardment [35] with a Biolistic
PDS-1000/He Particle Delivery System (Bio-Rad, Hercules, USA). Cells
were grown to a cell density of 2 x 106 cells/mL
(early mid-log phase), harvested by centrifugation, and plated on 1.5%
TAP agar plates at concentration of 1 x 108cells/plate (bombardment plates hereafter). Gold DNAdel carrier
particles (Seashell Technology) were coated with the appropriate plasmid
DNA prior to bombardment of the plates. Plates were incubation in low
light (~10 μE) overnight and then the lawn of cells
harvested in 1 mL of TAP medium and plated on 1.5% TAP+Spc plates at a
concentration of 5 x 107 cells/plate. Plates were
incubated in ~50 μE light at 25°C until colonies
appeared and were large enough to re-streak (~ 2 weeks).
Several colonies for each strain were re-streaked to single colonies on
TAP+Spc plates several times to obtain homo-transplastomic lines. To
induce loop out of the selection cassette, strains were re-streaked to
single colonies twice on TAP+5-FC plates. Integration of foreign DNA and
homoplasmy of the plastome was checked by PCR analysis of genomic DNA
extracted from single colonies using the Chelex
method.[36] Primers used in the analysis are
detailed in Supplementary Table 1 .