Difference between revisions of "Part:BBa K3140003:Design"

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===Design Notes===
 
===Design Notes===
  
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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 the backbone of pUS250, and to the BsaI site in the PsiK gBlock) 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.
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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.
  
 
[[Image:T--Sydney_Australia--PsiM_gblock.png|900px|'''Fig. 1''': The gBlock incorporating part BBa_K3140003 (PsiM), a RBS, and sites for cloning into pET-28c(+) and pUS250.]]
 
[[Image:T--Sydney_Australia--PsiM_gblock.png|900px|'''Fig. 1''': The gBlock incorporating part BBa_K3140003 (PsiM), a RBS, and sites for cloning into pET-28c(+) and pUS250.]]
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===Source===
 
===Source===
  
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Psilocybe cubensis
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The native sequence for the norbaeocystin methyltransferase PsiM (''Psilocybe cubensis'') was obtained from NCBI: GenBank accession [https://www.ncbi.nlm.nih.gov/nuccore/KY984100.1 KY984100.1]
  
 
===References===
 
===References===
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'''1.''' Fricke, J., Blei, F. & Hoffmeister, D. Enzymatic Synthesis of Psilocybin. ''Angew Chem Int Ed Engl'' '''56''', 12352-12355 (2017).

Latest revision as of 06:08, 15 October 2019


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 norbaeocystin methyltransferase PsiM (Psilocybe cubensis) was obtained from NCBI: GenBank accession KY984100.1

References

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