From Rosanna Young and Saul Purton, University College London, UK, July 2016

pSRSapI is a vector for expressing transgenes in the C. reinhardtiii chloroplast (Wannathong et al., 2016). The transgene should be inserted into the expression site using SapI and SphI restriction enzymes (or their isoschizomers LguI and PaeI, respectively). It will then be flanked by the C. reinhardtii psaA exon 1 promoter and 5′ UTR (perfect scarless fusion at the ATG start codon) and the C. reinhardtii rbcL 3′ UTR. The external flanking regions in the construct include an intact psbH gene and target the expression cassette downstream of psbH by homologous recombination.

Ampicillin selection in E. coli. Minimal medium for restoration of psbH gene in C. reinhardtii, selecting for phototrophic growth. The recipient cell line must therefore be a psbH mutant such as CC-5168 cw15 ∆psbH [strain TN72].