Team:Sydney Australia/Demonstrate

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Demonstrate

What We Achieved

We have demonstrated that two out of the four components of our system work together as expected under realistic conditions. When we cloned PsiD, PsiK, and PsiM into an inducible plasmid, pUS250 in our case, and ran a resting cell assay (see our Experiments page for more details on that protocol) in a phosphate-containing buffer, PsiD and PsiK can catalyse the conversion of their respective substrates (Figure 1). This data showed that two of our parts functioned together in vivo. Additionally, despite an inability to characterise PsiM confidently, our system demonstrated an ability to create baeocystin, the psychoactive precursor to psilocybin. Together these results suggest that our system can catalyse the conversion of 3 of the 5 intermediates in the biosynthetic pathway

Figure 1: LCMS data from pUS387 phosphate buffer assay showing the production of tryptamine from endogenous tryptophan, catalysed by PsiD, and norbaeocystin from 4-hydroxytryptamine, catalysed by PsiK.

PsiD, and to a lesser extent PsiK, have also been shown to work in a cell-free enzyme-only system (Figure 2).

Figure 2: LCMS data from PsiD (left) and PsiK (right) single cell assays.

Thus we can confidently say that two major components of our project have been demonstrated to work under real world conditions and that our system produces psychoactive compounds.