Team:Sydney Australia/Part Collection
Part Collection
The parts we’ve made this year form two broad collections. The Psilocybe cubensis inspired collection, and the fluorescence collection. For each collection we will describe how we recommend expressing the parts and replicating or improving upon our results.
The Psilocybe cubensis Collection
All these parts are inspired by the genes that Psilocybe cubensis uses to generate psilocybin from tryptophan. We wish to preface the following by stating that we strongly encourage that you check your country or state’s regulations before attempting to order any of our constructs. Furthermore, the use of our constructs may require further licensing and approval. These parts have been generated in the interest of medical research.
Parts in this collection:
- PsiD - Tryptophan decarboxylase from Psilocybe cubensis - BBa_K3140000
- PsiK - 4-hydroxytryptamine kinase from Psilocybe cubensis - BBa_K3140002
- PsiM - Norbaeocystin methyltransferase from Psilocybe cubensis - BBa_K3140003
- PsiH - Cytochrome P450 monooxygenase from Psilocybe cubensis - BBa_K3140001
- PsiH (codon optimised) - Cytochrome P450 monooxygenase from Psilocybe cubensis - BBa_K3140004
For individual expression:
- PsiD, PsiK, PsiM:
- We recommend the use of the pET-28c(+) plasmid. We used TOP10 cells which are a K12 derivative for DNA purification and BL21 cells with a supplementary pGro7 plasmid for protein purification. We recommend shaking after induction at 15C for increased soluble protein expression.
- For PsiK, please remember that PsiK phosphorylates 4-hydroxytryptophan so will require supplementary phosphate in its media to most efficiently convert its substrate into its product.
- For PsiH and PsiH (codon optimised)
- We recommend the use of the pCW-CYP26A plasmid, but first you will need to remove the human cytochrome P450 2A6 as we describe in our ‘Experiments’ section. Alternatively you could order a P450 reductase sequence (human, fungal, or other) and try co-ligating that into a plasmid of your choice.
- We recommend the use of DH5-alpha cells with a supplementary pGro7 plasmid.
- P450s are membrane bound and thus insoluble. Bear that in mind when doing protein work. Many P450s are also very tricky with their media requirements so we recommend following the initial steps of the P450 indole assay protocol as supplied by Dr Elizabeth Gillam when trying to induce this enzyme.
For co-expression:
- We recommend using Golden Gate cloning to transform PsiD, K, and M into an inducible plasmid such as pUS250.
- Your chosen PsiH can remain in a separate plasmid or you can try to co-transform PsiH and your chosen P450 reductase into your golden gate plasmid also.
- We recommend the use of DH5-alpha cells with a supplementary pGro7 plasmid.
- We recommend merging the methods of PsiH and PsiD/K/M together, following the indole assay steps up until the induction step, then incubating post-induction at 15C.
The Fluoroprotein Collection
All these parts are fluoroproteins, and whilst some of them might be brighter than others (like a lot seriously) we have added all of them to the registry in case one of them suits your specific purpose.
Parts in this collection:
- VVD36-C73A - Truncated VIVID fluoroprotein derived from Neurospora crassa - BBa_K314000
- VVD36-C73A-CH1 (rank order harmonised) - BBa_K3140006
- VVD36-C73A-CH2 (nearest frequency harmonised) - BBa_K3140007
- VVD36-C73A-CH3 (relative proportion harmonisation) - BBa_K3140008
- VVD36-C73A-CH4 (codon optimised) - BBa_K3140009
- VVD36-C73A-CH4-M130I - BBa_K3140010
Expression suggestions:
- We recommend expression in the pK18 plasmid in TOP10 (K12 derivative) cells.
- Please bear in mind, however, that the blue/white selection of pK18 will not work with the VIVID protein. VIVID is small enough that the LacZ activity of the plasmid is not sufficiently reduced with insertion so some positive colonies may be blue or turn blue over time.