Engineered Chlamydomonas for Hydrogen Production

Chlamydomonas reinhardtii codon-optimized sequence (GeneArt), coding for the fusion protein Hyd–SOD, was inserted into pChlamy_1 expression vector (GeneArt). The fusion sequence was cloned under the Hsp70A-RbcS2 promoter with an Fd transit peptide sequence for chloroplast delivery [37 (link)]. The plasmid was transformed by electroporation to the nucleus of HydA1–HydA2 double mutant [38 (link)] (DM) according to GeneArt Chlamydomonas Engineering Kit protocol (Life Technologies). For positive clone screening, algae were overlaid with engineered H₂-sensing R. capsulatus and left overnight [39 (link)]. The plates were then scanned using the Fuji FLA-5100 fluorescence imager. A 473-nm laser was used for excitation, whereas 510- and 665-nm filters were used for quantifying GFP luminescence and chlorophyll density, respectively. Genomic DNA isolation was preformed using E.Z.N.A.^® SP Plant DNA Kit (Omega Bio-tek). Immunoblot analysis was used for protein expression verification according to Eilenberg et al. [37 (link)]. Whole cell protein extraction was preformed according to Rühle et al. [40 (link)].