Team:HebrewU/Design

HebrewU HujiGEM 2018





Based on the model our team created, we identified 8 enzymes we believed would play a key role in catalyzing the degradation of TCDD. Though our plan is to host this pathway in plants, for a number of reasons we decided to first examine our pathways effectiveness in Saccharomyces cerevisiae (bakers' yeast). First and foremost, they are easier to work with than plants. They have shorter generation times, and they require cheaper and less sensitive growth provisions. Additionally, due to their ability to utilize plasmids, creating expression vectors for yeast was more straight-forward; it did not require genome editing, as is the case with plants. Though all of the aforementioned qualities apply to bacteria as well, it was important that we worked explicitly with eukaryotic organisms. Yeast, unlike bacteria, contain central molecular systems such as membrane-anchored organelles and post-transcriptional editing. As such, testing our pathway in yeast gave us an accurate understanding of how our pathway would behave in plants.






We began creating yeast strains, each with a single vector. After that, we had considered utilizing the mat alpha/a system for creating a strain with more than one vector, but as we found our transformation method extremely efficient, we chose to execute secondary transformations on yeast cells already contain a separate vector.
Parallel to these transformations, we engineered control strains that contained the empty vector, with no extra enzymatic activity, but allowed for these stains to be grown on the same selective Drop Out media. These strains were critical for creating useful control groups for our experimental design.

Our final yeast strain was known in the lab as D24 (Dehalogenase, 2,6,6 hydrolase + 2,2,3 dioxygenase , and 44a dioxygenase) and has a corresponding control strain with 3 vectors allowing for growth on -Leu, Trp, His Dropout media, but containing none of the enzymes from the TCDD degradation pathway.



Further Reading: