Week 1 HW: Principles and Practices

Week 1: Principles and Practices

Week 1: Save the World or Destroy the World

Part 1. Project Description

Application: Visual-Signaling Engineered Yeast for Real-time Monitoring in Opaque Fermentation Vessels (Onggi, Stainless Steel, etc.)

Why this project? In both traditional brewing (using Onggi) and modern brewing (using stainless steel or plastic tanks), it is nearly impossible to monitor the internal fermentation state—such as abnormal fermentation or contamination—without opening the vessel. Opening the lid risks introducing external contaminants. I want to develop an engineered yeast that converts specific metabolic states into visible signals (color or fluorescence). This “living sensor” will report what is happening inside the “black box” of opaque fermentation tanks in real-time.

One-sentence project goal To build a real-time yeast biosensor that translates internal metabolic data of opaque fermentation vessels into visible biochemical signals.

Part 2. Governance and Policy Goals

• Goal 1. Biosecurity (Non-malfeasance): Prevent environmental persistence of engineered strains by implementing auxotrophy (e.g., Lysine dependency).
• Goal 2. Transparency and Safety: Ensure clear distinction between experimental bio-liquor and consumer products through rigorous labeling and safety protocols.

Part 3 & 4. Governance Actions and Scoring

Scoring Matrix (1 = Best)

Part 5. Priorities and Recommendation

I prioritize a combination of Action 1 (Genetic Kill-Switch) and Action 2 (Mandatory Labeling). Given that brewing environments often lack high-level containment, technical biocontainment (auxotrophy) is the most effective safeguard. This must be reinforced by strict labeling and training to prevent any accidental consumption of the experimental fermentation.

Week 2 Prep: DNA Read, Write, and Edit

Questions from Professor Jacobson

• Polymerase Error Rate: The raw error rate is approx. $10^{-5}$. Compared to the human genome ($3 \times 10^{9}$ bp), this would cause thousands of mutations per division. Biology deals with this via 3’ to 5’ proofreading and post-replication mismatch repair (MMR), bringing the final rate to $10^{-9}$ or $10^{-10}$.
• Coding Diversity: An average 300-aa protein can be encoded by $\approx 3^{300}$ DNA sequences due to codon degeneracy. However, many codes fail in practice due to codon usage bias, mRNA secondary structures hindering translation, and regulatory elements embedded in the sequence.

Questions from Dr. LeProust

• Common Method: Solid-phase phosphoramidite chemistry.
• Length Limit (200nt): Errors (deletions/side reactions) accumulate exponentially with each cycle, making the yield of full-length, error-free oligos negligible beyond this length.
• 2000bp Gene Synthesis: Direct chemical synthesis is impossible at this scale. Instead, shorter overlapping oligos are synthesized and then assembled enzymatically (e.g., Gibson Assembly or PCR assembly).

Questions from George Church

• 10 Essential Amino Acids: Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, Valine, and Arginine.
• The Lysine Contingency: The fact that animals cannot synthesize lysine makes them evolutionarily dependent on external sources. In my project, using Lysine auxotrophy as a biocontainment strategy directly leverages this biological vulnerability to ensure that engineered strains remain tethered to the lab/brewery environment.