pSRSapI
$30.00
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].
Wannathong T, Waterhouse J, Young R, Economou C, Purton S (2016) New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii. Applied Microbiology & Biotechnology 100 (12): 5467-77
Young R, Purton S (2016) Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnology Journal 14 (5): 1251-60
Young R, Purton S (2014) Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression. The Plant Journal 80 (5): 915-25