Coding

Part:BBa_K3140003:Design

Designed by: Fahad Ali   Group: iGEM19_Sydney_Australia   (2019-10-12)
Revision as of 06:05, 15 October 2019 by Fahad3li (Talk | contribs)


PsiM - Norbaeocystin methyltransferase from Psilocybe cubensis


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part was intended for use in pET-28c(+) for initial analysis of protein expression, and in pUS250 for final expression of a polycistronic construct incorporating PsiD, PsiK, and PsiM. To achieve this, the part was ordered in a gBlock containing BsaI sites (complementary to a BsaI site in the PsiK gBlock and to a BsaI site in the backbone of pUS250) for Golden Gate cloning, and with EcoRI and HindIII sites to enable traditional restriction cloning into pUS250. As there is a ribosomal binding site in the pET-28c(+) backbone, but not in pUS250, a RBS sequence was added to the gBlock upstream of the EcoRI site, so that it would not incorporate into pET-28c(+). The RBS sequence used was the consensus Shine-Dalgarno sequence.

Fig. 1: The gBlock incorporating part BBa_K3140003 (PsiM), a RBS, and sites for cloning into pET-28c(+) and pUS250.

Source

The native sequence for the 4-hydroxytryptamine kinase PsiK (Psilocybe cubensis) was obtained from NCBI: GenBank accession KY984099.1

References

1. Fricke, J., Blei, F. & Hoffmeister, D. Enzymatic Synthesis of Psilocybin. Angew Chem Int Ed Engl 56, 12352-12355 (2017).