Composite

Part:BBa_K2423008:Design

Designed by: Oscar Broström   Group: iGEM17_Uppsala   (2017-10-01)
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UGTCs2 with BBa_J04500


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 758
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 931
    Illegal AgeI site found at 1286
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The design process started with taking the sequence found on (2). Then we located the ORF and codon optimized it using IDT's codon optimizing tool. We generated ORFs until they did not contain EcoRI, XBaI, SpeI and PstI restriction sites. After that we wanted to add a his-tag, but we did not know whether we should put it on the N- or C-terminus and since there is no structure for the protein we performed homology modeling to get a structure. With our homology model we were able to identify which terminus that would yield the most optimal place to put the his-tag, which was the N-terminus. A lac-inducible promoter was chosen so that protein would be easy to overexpress. BBa_J045000 was used as could be found in the iGEM standard kit.


Source

The gene of interest had previously been worked on by Moraga et al. (2004) (1), where they were able to clone the cDNA of UGTCs2. They published the sequence that they used on GenBank with AY262037 as the accession number (2).

References

1. Moraga AR, Nohales PF, PĂ©rez JAF, GĂłmez-GĂłmez L. Glucosylation of the saffron apocarotenoid crocetin by a glucosyltransferase isolated from Crocus sativus stigmas. Planta. 2004 Oct 1;219(6):955–66.

2. Crocus sativus glucosyltransferase 2 (GLT2) mRNA, complete cds. 2003 Nov 1; Available from: http://www.ncbi.nlm.nih.gov/nuccore/AY262037.1