Yeast Design

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.

Our yeast cloning was based on pESC vectors. This series of vectors designed for the expression and functional analysis of eukaryotic genes in yeast. These vectors contain a multitude of useful features such as:

E. Coli and Yeast ORIs, allowing to utilize E. Coli for steps such as minipreps, genotyping, and sequencing, which is easier than in yeast.
Antibiotic resistance, for selection in E. coli.
Amino Acid Selection genes- the vectors have 4 complementary varieties, each coding for genes that allow for selection on different drop out media. This was especially useful for creating strains with multiple vectors, as parallel selection of multiple vectors was possible.
GAL1/10 inducible yeast promoter regulating gene transcription opposing orientation. This allowed two genes to be introduced into a yeast host strain in a single expression vector.
Epitope Flags, though we did not utilize this feature in our research, as we did not run western blot or ELISA assays

Before integrating the genes in to yeast expression vectors, we optimized their sequences. As the final goal of our research was to introduce the pathway into plants, we optimized the genes for expression both in S. cerevisiae and Arabidopsis Thaliana.

To construct these vectors, we utilized Gibson Assembly, allowing for the introduction of multiple genes in a single ligation reaction. All of our genes were either synthesized or amplified with 20 bp overlaps, to allow for an efficient ligation reaction. Although the Gal 1/10 promoter was originally part of the vector, we removed during restriction, to allow the introduction of genes on both sides in a single reaction.

We began creating yeast strains, each with a single vector. After that, we had considered utilizing the mat a/a system for creating 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 experiment design. Our final yeast strain was known in the lab as D24 (Dehalogenase, 2,6,6 hydrolase + 2,3,3 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 enzyme from the TCDD degradation pathway.

Team:HebrewU/Design

Yeast Design

Further Reading: