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. Author manuscript; available in PMC: 2021 May 1.
Published in final edited form as: Algal Res. 2021 Mar 5;55:102255. doi: 10.1016/j.algal.2021.102255

Fig. 1. FtOCP expression in Chlamydomonas reinhardtii: transformation, accumulation and localization.

Fig. 1

a) schematic overview of vectors used for transformation of C. reinhardtii with Fischerella thermalis (Ft) OCP (Orange Carotenoid-binding Protein) and C. reinhardtii (Cr) BKT (β-carotene ketolase). Both vectors include Heat shock protein 70/Ribulose bisphosphate carboxylase small chain 2 (Hsp70A/Rbcs2) hybrid promoter. FtOCP vector contains mVenus gene encoding for the YFP (Yellow fluorescence protein) fused to FtOCP, while CrBKT vector contains antibiotic resistance to spectinomycin (AadA, amino-glycoside-3”-adenylyltransferase gene). PsaD (Photosystem I Subunit D) transit peptide (TP) was used to localize both BKT and OCP into the chloroplast. FtOCP and BKT sequences were designed in silico using codon optimization and inserting rbcs2 intron 1. b) YFP fluorescence quantification, expressed as arbitrary unit (a.u.), and western blot analysis showing OCP_YFP accumulation in transformant strains ocp1–4 compared to the background strain UVM4 (UV-mediated mutant 4). c) OCP_YFP cellular localization performed by confocal laser-scanning microscopy. Scale bar (white) represent 5 μm. d) Western blot analysis showing OCP accumulation in transformant ocp1 and ocp2 lines as well as in ocp1–bkt1, ocp1–bkt2, ocp2–bkt1 and ocp2–bkt2 strains.