Team:British Columbia/Accomplishments

Contents 1 Wet Laboratory Achievements 2 In vitro assay production of monoterpene in bacteria 3 Geranyl Pyrophosphate (GPP) Assay 4 GC-MS Chromatogram 5 Diterpene production in yeast 6 Mountain Pine Beetle & Yeast Co-culture 7 Blue Stain Fungi & Yeast Co-culture Co-culture 8 Modeling Achievements 9 Monoterpene Production Model 10 Beetle Epidemic Model

Wet Laboratory Achievements

---

In vitro assay production of monoterpene in bacteria

Geranyl Pyrophosphate (GPP) Assay

Alpha-pinene and Beta-pinene synthases were purified using His SpinTrap Ni-affinity columns and were assayed in vitro with GPP. Using gas chromatography-mass spectrometry (GCMS), we confirmed the synthesis of alpha and beta pinene monoterpenes from our enzyme assays.

GC-MS Chromatogram

Diterpene production in yeast

Mountain Pine Beetle & Yeast Co-culture

To prove the effectiveness of our theoretical model of release of Saccharomyces cerevisiae into the wild, we investigated whether wild-type yeast will survive during the transporation process (via the mountain pine beetles) from plate of origin (plate with yeast products; simulating our trapbox) to the next media plate. Beetles were incubated with yeast and were challenged with diferent amount of times away from the next media plates (in empty plates for 0, 10, 24, and 36 hours of time; simulating the transfer period when beetles leave the trapbox and fly off to the next tree). Results were analyzed qualitatively for the presence of GFP after growth on selective media plates from each time challenge. Based on these results, we can conclude that pine beetles do can carry wild-type yeast and transfer them onto the next media plate. It appears that our yeast product can survive on the beetles for 36 hours away from media plate. However, it cannot be determined whether the amount of yeast products on the beetles declines with time.

Blue Stain Fungi & Yeast Co-culture Co-culture

To determine whether terpene producing Saccharomyces cerevisiae can grow with and/or inhibit the growth of , we co-culture blue stain fungi with diterpene producing yeast and wild-type yeast in the same media. However, results or inclusive Say we're optomizing protocol for co-culturing

Modeling Achievements

---

Monoterpene Production Model

We modeled a simplified and modified version of the mevalonate pathway that describes our engineered yeast cells. We created a series of differential equations to model each chemical reaction that were based on first-order Michaelis-Menten kinetics. Enzyme constants were estimated/found using literature values and the simulations were conducted using MATLAB.

Our simulation showed that 17.10% more beta-pinene is produced and 17.00 % more alpha-pinene is produced when K6R-hmg2 and erg20-2 are used instead of HMG2 and ERG20 alone in a yeast cell. For manufacturing purposes, sensitivity analysis was performed and it was determined that the pathway could be improved to increase the production of monoterpenes by increasing the concentration of the enzymes K6R-HMG2 and IDI1 for DMA-PP. In particular, the tripling the [K6R-HMG2] increases the [GPP] by 8.7000 times. The tripling the [K6R-HMG2] and [IDI1] for the production of DMA-PP increases the [B-pinene] by 7.3166 times and 1.3052 times respectively.

Beetle Epidemic Model

We employed a cluster-based modeling to predict the spread of mountain pine beetle infestation from year 2011 up to 2020 based on cluster analysis. In addition, we identified strategic subpopulation centres for deployment of synthetic products.

Synthase Model

We constructed 3D structure models of alpha-pinene, beta-pinene, and limonene synthase to identify amino-acid mutations near the substrate-binding site that would improve monoterpene production.

Human Practices Achievements

---

High School Mentorship

Synthetic Biology Gone Wild

Public Perceptions

Special Considerations

---