Figure 1.

Strategy to clone genes into a chloroplast expression vector whilst preventing their expression in Escherichia coli. The gene of interest ( GOI ) is redesigned such that one or more tryptophan (TGG) codons are altered to TGA, indicated by asterisks. These changes can be integrated into the codon‐optimized gene design prior to ordering the synthetic gene and integrating it into the Chlamydomonas reinhardtii chloroplast genome. A tRNA gene based on the C. reinhardtii plastidial trnW, but with the anticodon sequence altered to recognize UGA, is also introduced (trn W _UCA ). This enables readthrough of the GOI in C. reinhardtii. Flanking regions amplified from chloroplast DNA allow targeted integration of the constructs into the chloroplast genome by homologous recombination; the target site is a neutral region downstream of either psbH or psaA exon 3, depending on the construct. The psbH gene can be used for selection in a psbH mutant recipient strain.