Search Results for pSRSapI
pSRSapI
$30.00
$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
pRY127d
$30.00
$30.00
From Rosanna Young, Saul Purton lab, University College London, UK, October 2015
CrCD is a conditional negative selectable marker for use in the C. reinhardtii chloroplast that confers sensitivity to 5-fluorocytosine by converting it to toxic 5-fluorouracil (Young and Purton 2014). Plasmid pRY127d contains the crCD gene, with a C-terminal HA tag, inserted into the chloroplast expression vector pSRSapI. CrCD is a modified and codon-optimised version of the E. coli cytosine deaminase (codA) gene. The crCD gene in pRY127d uses the C. reinhardtii psaA exon 1 promoter and 5′ UTR and the C. reinhardtii rbcL 3′ UTR; the flanking regions in the construct target the crCD cassette downstream of psbH by homologous recombination.
This plasmid is called pCD in Young and Purton 2015.
pASapI
$30.00
$30.00
From Saul Purton, University College London, August 2014
Chloroplast DNA sequence containing psbH-trnE2 region with an ‘expression cassette’ engineered into the intergenic MluI site. Cassette comprises of the atpA promoter/5’UTR and rbcL 3’UTR regions separated by a multiple cloning site. Note: the SapI site in the MCS is designed to allow a perfect translational fusion at the ATG of the gene-of-interest.
host strain: DH5α
amp resistant
Economou C, Wannathong T, Szaub J, Purton S (2014). A simple, low cost method for chloroplast transformation of the green alga Chlamydomonas reinhardtii. In: Chloroplast Biotechnology (volume editor: Pal Maliga). Methods in Molecular Biology 1132: 401-411