• See this link if you want examples of what you can include in your answers for each question. Note: the question numbers do not line up, and you can ignore any word requirements in this linked document.
• Please include your answers to the following questions on the “final project” page of your HTG(A)A website.

1. Provide an abstract/narrative/summary for your final project.

This project will engineer Saccharomyces cerevisiae (brewer's yeast) to produce chicken ovalbumin, which represents 54% to 58% of the egg white protein by weight. To achieve this, a codon-optimized chicken ovalbumin gene fused to the α-mating factor (MF) secretion signal will be integrated into a yeast expression vector/system. This will enable the yeast to secrete the ovalbumin directly into the media to simplify downstream processing. In addition to the wet lab experiment, we will also use computational methods to map out the metabolic pathway of this engineered strain and use flux balance analysis to recommend changes to optimize titer.

2. Provide a background and motivation section for your final project.

a. Explain your motivation for pursuing your project

This project is driven by concerns about the impact of large-scale animal agriculture on the environment, food security, and animal welfare.

• Environmental Impact: Precision fermentation can potentially produce ovalbumin with lower land, water, and energy requirements than conventional egg production
• Food Security: This method of egg production would create a stable supply chain for egg proteins that isn't vulnerable to diseases like the avian flu.
• Animal welfare: This eliminates animal welfare concerns around egg production, such as the practice of culling male chicks or the use of battery cage systems.

b. Describing the current state of knowledge related to your project. Try to cite at least 2 peer-reviewed research papers.

There have been multiple papers that have produced ovalbumin with yeast over the last 50 years, with most advancements being in the last few years (ovalbumin-like protein in S.cerevisiae, 1980; bovine beta-lactoglobulin and hen egg ovalbumin by *Trichoderma reesei, 2023;* ovalbumin in engineered S. cerevisiae, 2024). There are two commercial companies (EVERY and Onego bio) which also produce ovalbumin with yeast and have products on the market. Most of the current challenges are around producing animal-free ovalbumin at scale for a low cost (survey), with an expression titer of 2 g/L (paper).

c. Describe how your project is innovative and expand upon the significance of your final project.

This project is innovative because not only does it engineer yeast to produce ovalbumin (which others have already done), but it also explores the metabolic pathway of this method to identify solutions to increasing the expression titer.

3. Aims

For each aim:

1. Create an experimental plan for your final project by including specific methods/tools/technologies/biological concepts for each experiment/analysis step.
2. Present any results/validation of (a part of) your final project. Share what worked, what didn’t work, and why!

4. Describe any unexpected challenge(s) you faced in planning or executing your final project.

• Difficulty in finding an appropriate (and affordable) plasmid backbone
• Low expression made it difficulty to identify if any ovalbumin was produced?

5. Describe the bioethical considerations involved in your project.

6. Describe any future work