Team:Sydney Australia/Design
Project Design
The Brief
The design goal for Usyd's iGEM team this year was simple. Design a one cell system capable of producing psilocybin using genes found in the biosynthetic pathway of psilocybe cubensis - psiD, psiH, psiK and psiM.
This firstly involved the integration of the decarboxylase (psiD), kinase (psiK) and methyltransferase (psiM) genes from the pathway into one plasmid backbone, each with their own ribosomal binding site but all under the control of one strong promoter. The monooxygenase (psiH) we hoped to integrate in another plasmid to co-express alongside the other genes. More on that below.
Once we obtained a functional Magi.coli system we could work on improving its efficiency for scaled production of psilocybin.
Leveraging Codon Harmonization
We also aimed to leverage an investigation into codon harmonization to further increase our chances of producing functional Psi proteins in E. coli , particularly PsiH. Codon harmonization revolves around conserving codon rarity between your donor and host organism during heterologous gene expression. The idea being that rare codons may serve a particular function in translation possibly contributing to the proper folding of protein. We decided to model how several harmonization methods might improve expression of our psi genes. We applied our harmonization methods to the fluorescent protein VVD gene derived from another fungus, Neurospora crassa, and used fluorescence to monitor protein expression in systems containing different codon harmonized VVD genes
Building Accessibility to Codon Harmonization
Finally one of the key things we wanted to accomplish was improving the accessibility of codon harmonization for future teams. We decided it would be best to build software that could easily apply our harmonization methods to other genes. This software would allow teams to generate codon harmonized gene sequences to suit their chassis organisms.
Design Summary
Designing our parts
Psi Parts
WT sequences for all psi genes were downloaded and scanned for unwanted restriction sites. These sites were removed along with any cryptic promoters and strong RBS binding sites within the gene.
VVD Parts
All VVD parts were based on the native VVD protein from Neurospora crassa. The VVD36 coding sequence was generated by removing the first 36 amino acid residues and appending a start codon to the start of this truncated sequence. The cystine residue at position 73 (TGC) was then changed to alanine by changing the codon to GCC to yield VVD36-C73A.
Choosing our Plasmids
PsiH expression vector
The PsiH protein we wish to express from Psilocybe cubensis is a cytochome P450 monooxygenase. These enzymes are known for being notoriously tricky to express functionally in E. coli and often require specific expression conditions or molecular chaperones. The also need a partner enzyme, a P450 reductase. It would be possible to clone in a P450 reductase from Psilocybe cubensis alongside PsiH into any given vector, but the P. cubensis P450 reductase is not well characterised and also has never been expressed in E. coli - a bit of a gamble! pCW is an already well-characterised and frequently used plasmid which can co-exist with numerous other plasmids. The plasmid contains a human P450 gene and a cytochrome p450 reductase gene already! We hoped to co-opt this system by excising the existing p450 and replacing it with our own. This would allow us to express PsiH protein alongside the existing P450 reductase gene.
Pet28 for individual PsiD/K/M Expression
pET-28c(+) is a tightly regulated plasmid, perfect for strong and controllable expression of our Psi proteins. Our genes can be inserted in frame with an N-terminal 6xHIS tag which will prime our proteins for Nickel affinity column purification and enable us to characterize our proteins in vitro.
pUS250 for golden gate cloning of Psi D/K/M
pUS250 has a tightly regulated cumate promoter system upstream of its first multiple cloning site. This plasmid is exceptional when screening for positive recombinant colonies during Golden Gate cloning, as it contains a blue chromoprotein between its golden gate-suitable cloning sites (BsaI). pUS250 is also an in-house plasmid which we like to use every chance we get!