Part:BBa_K2423005:Design
CaCCD2 with BBa_J04500
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1939
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 837
Source
Originally, the group enzymes (CCD2) was discovered by Frusciante et al. (2014) (1) through deep transcriptome analysis. More specific information about CaCCD2 came approximately one year later by Ahrazem et al. (2015) (2). The sequence (mRNA reverse transcribed to cDNA) they found there has the GenBank accession number KP792756.
Design Notes
When designing this part we initially took the coding sequence from KP792756 (3) and codon optimized it for Escherichia Coli using IDT's tool. We repeatedly used it until there were no restriction sites for EcoRI, XbaI, SpeI and PstI. The choice of BBa_J04500 came from that we wanted to be able to induce the production of our protein using IPTG and that it was in the iGEM starter kit making it readily available to us. A his-tag was added to be able to purify the protein using immobilized affinity chromatography (IMAC). The placement of the his-tag (N-terminal) was based on the homology model we created (4).
References
1. Frusciante S, Diretto G, Bruno M, Ferrante P, Pietrella M, Prado-Cabrero A, et al. Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis. Proc Natl Acad Sci USA. 2014 Aug 19;111(33):12246–51.
2. Ahrazem O, Rubio-Moraga A, Berman J, Capell T, Christou P, Zhu C, et al. The carotenoid cleavage dioxygenase CCD2 catalysing the synthesis of crocetin in spring crocuses and saffron is a plastidial enzyme. New Phytol. 2016 Jan 1;209(2):650–63.
3. Crocus ancyrensis carotenoid cleavage dioxygenase (CCD2) mRNA, complete cds. 2015 Nov 4; Available from: http://www.ncbi.nlm.nih.gov/nuccore/KP792756.1
4. Modeling iGEM Uppsala 2017; Available from: http://2017.igem.org/Team:Uppsala/Model