FIGURE 1.

Generation of Chlamydomonas lines overexpressing SBP1. (A) SBP1 construct used for transformation. The 2172 bp SBP1 ORF (exons shown as black boxes), interrupted by all native SBP1 introns (thin lines), was domesticated to generate a level 0 module for the MoClo strategy. Using MoClo, the SBP1 ORF was equipped with the HSP70A-RBCS2 promotor and the RPL23 terminator (pAR and tRPL23, respectively, white boxes) and with a 3xHA-tag or without a tag (mStop) (gray box), giving rise to two level 1 constructs. These were combined with another level 1 construct containing the aadA gene conferring resistance to spectinomycin (light gray box) to yield the final level 2 constructs for transformation. (B) Screening of transformants overexpressing SBP1. The UVM4 strain was transformed with the level 2 constructs shown in (A). Total cell proteins from 12 spectinomycin resistant transformants recovered with each construct were extracted and proteins corresponding to 1.5 μg chlorophyll were analyzed by immunoblotting using anti-HA or anti-SBPase antibodies. The transformant number is given on top of the lanes. Transformants exhibiting highest expression levels for SBP1-3HA or SBP1-mStop (red) were used for further analysis.