Week 1 HW: Principles and Practices

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Process of Researching the project:

I looked at some iGem projects to gain inspiration and a sense of the types of topics that people have addressed. https://video.igem.org/c/2025_presentations/videos

First, describe a biological engineering application or tool you want to develop and why.

Vitiligo is an autoimmune condition characterized by the selective loss of melanocytes, leading to depigmented skin lesions. A growing body of evidence suggests that elevated oxidative stress in melanocytes precedes and exacerbates immune-mediated destruction by increasing cellular damage and inflammatory signaling.

My project proposes an oxidative-stress–responsive genetic circuit in melanocytes that activates cytoprotective pathways only under pathological redox conditions and automatically deactivates once oxidative stress resolves. By enhancing stress resilience rather than inducing pigmentation, this approach aims to reduce melanocyte vulnerability while minimizing unintended cosmetic or immune effects.

Goals:

  1. Prevent Harm: Prevent the technology from causing biological, social, or psychological harm. • A1. Prevent biological harm from uncontrolled gene expression, persistence, or immune disruption. • A2 Prevent social harm, including stigmatization, cosmetic misuse, or reinforcement of colorism. • A3 Prevent misuse or repurposing beyond therapeutic, supportive contexts.

  2. Promote Safe and Constructive Innovation: Enable beneficial research and translation without unnecessary restriction.

    • B1. Encourage disease-aligned, upstream interventions (stress resilience vs cosmetic alteration). • B2. Avoid chilling effects on legitimate academic research. • B3. Support iterative learning and transparency around failures.

  3. Respect Autonomy, Equity, and Trust • C1. Incorporate patient perspectives into design and deployment decisions. • C2. Avoid one-size-fits-all assumptions about desirability of treatment. • C3. Ensure accessibility and avoid disproportionate burdens on marginalized groups.

Next, describe at least three different potential governance “actions” by considering the four aspects below (Purpose, Design, Assumptions, Risks of Failure & “Success”).

Governance Action 1: Mandatory Design-Layer Safeguards for Stress-Responsive Therapeutics

  • Purpose: Require that stress-responsive therapeutic circuits incorporate reversibility, thresholding, and containment as baseline design criteria.
  • Design: Circuits must be inducible and auto-deactivating.
  • Assumptions: design constraints meaningfully reduce harm, researchers can implement safeguards without prohibitive cost, reviewers can competently assess circuit-level safety claims.
  • Failure risks: Smaller labs may be excluded due to resource burden.

    Governance Action 2: Patient-Guided Scope Limitation via Advisory Committees

  • Purpose: Require early-stage patient advisory input for disease-facing synthetic biology projects, particularly for visible or identity-linked conditions.
  • Design: Structured advisory committees (e.g., Vitiligo Patient Advisory Committee).
  • Assumptions: Diversity of patient views can be captured.
  • Risks of Failure & “Success”: Overrepresentation of certain demographics, Projects may narrow scope so much that some beneficial uses are delayed.

    Governance Action 3: Transparency & Post-Research Monitoring Norms

  • Purpose:Establish norms for transparent disclosure of assumptions, limitations, and misuse risks, even for preclinical tools
  • Design: Public documentation of design assumptions and failure modes, Scenario analysis of misuse or unintended deployment.
  • Assumptions:Transparency deters misuse.
  • Risks of Failure & “Success”: Sensitive details could be misused.

    3. Scoring Governance Actions Against Policy Goals

    (1 = best, 3 = weakest, n/a = not applicable)

    Governance Action / Policy GoalOption 1
    Design Safeguards    		Option 2
    Patient Governance    		Option 3
    Transparency
    A. Prevent Biological Harm122
    • A1 Biological safety132
    • A2 Social harm212
    • A3 Misuse prevention221
    B. Promote Constructive Use211
    • B1 Disease alignment112
    • B2 Not impede research211
    • B3 Learning from failure221
    C. Autonomy & Trust312
    • C1 Patient voicen/a12
    • C2 Respect diversity212
    • C3 Equity221–2

    Prioritize a combined strategy centered on Option 1 (Design Safeguards) and Option 2 (Patient Governance), with Option 3 (Transparency) as a supporting layer. Option 1 scored strongest on preventing biological harm (A1) and performed well on misuse prevention. Because the MelanoGuard system directly manipulates cellular stress-response pathways, technical harm prevention must occur at the level of the biological system itself, not solely through external oversight. Option 2 scored best on social harm prevention (A2), autonomy and trust (C), and constructive use (B). Vitiligo is a visible, identity-linked condition, so ethical failure is more likely to occur at the social level than the molecular level. Option 3 scored highest for misuse detection (A3) and learning from failure (B3), but weaker on direct harm prevention and trust building.

    Lecture 2 Slides 1 Q/A

    Nature’s machinery for copying DNA is called polymerase.

  • What is the error rate of polymerase?
    1:10⁶ (error:base-pairs)
  • How does this compare to the length of the human genome. How does biology deal with that discrepancy?
    Human genomee is ~3.2 X 10⁹ base pairs. That would be 3200 errors. Biology has error correction and has a proof reading step and a post-replication mismatch repair.
  • How many different ways are there to code (DNA nucleotide code) for an average human protein? In practice what are some of the reasons that all of these different codes don’t work to code for the protein of interest? Average human protein is ~1036 BP. Each codon is 3 BP, so 1036/3 = 345 amino acids. On an average there are 3 synonymous codons per amino acid. so ~3^345 different ways. There are physical and structural consntraints.

    Lecture 2 Slides 2 Q/A

    1. What is the most commonly used method for oligo synthesis today?
      all commercial oligos today are made using phosphoramidite chemistry.
    2. Why is it difficult to make oligos longer than ~200 nt by direct synthesis?
      Error accumulation per synthesis cycle and errors compound exponentially.
    3. Why can’t you make a 2000 bp gene via direct oligo synthesis?
      Direct synthesis scales exponentially poorly, not linearly and errors would be everywhere

    Lecture 2 Slides 3 Q/A

    What are the 10 essential amino acids in all animals and how does this affect your view of the “Lysine Contingency”? 1. Histidine 2. Isoleucine 3. Leucine 4. Lysine 5. Methionine 6. Phenylalanine 7. Threonine 8. Tryptophan 9. Valine 10. Arginine

    So, #4 shows that all animals have in Lysine. Microbes and plants can synthesize all 20 canonical amino acids. This is why lysine biosynthesis pathways exist in bacteria but not animals. In animals (including humans) Lysine is always externally supplied, Cells already operate under lysine dependency. Therefore, Lysine contingency does NOT inherently restrict survival and is not a meaningful containment mechanism in animal hosts. Lysine contingency can function as containment in microbes, but only if: The environment lacks lysine, No cross-feeding occurs, No bypass pathways evolve. Lysine contingency should never be used alone and should be combined with the following so that animals can’t supply it, Microbes can’t evolve it easily and Environment doesn’t contain it:

    •	Genetic code recoding
•	Non-standard amino acid dependence
•	Codon reassignment
•	Virus resistance
•	Metabolic isolation

    Knowing that lysine is one of the 10 essential amino acids in all animals makes it clear that lysine contingency alone is not a robust biocontainment strategy; it only becomes meaningful when combined with genetic code engineering or dependence on non-standard amino acids. Because lysine is essential to all animals and ubiquitously available through diet and environment, “lysine contingency” by itself is biologically weak and only functions as a safety mechanism when embedded in a larger framework of genetic code recoding and metabolic isolation.

    [Given slides #2 & 4 (AA:NA and NA:NA codes)] What code would you suggest for AA:AA interactions?

    Collapse AA pairs into a small set of symbols:

    •	HΦ: hydrophobic packing (Leu/Ile/Val/Phe…)
•	HB: hydrogen-bond pairing (polar donors/acceptors)
•	SB: salt bridge (Asp/Glu ↔ Lys/Arg/His; pH-dependent)
•	π: aromatic stacking / cation–π (Phe/Tyr/Trp with Lys/Arg)
•	SS: disulfide bond (Cys–Cys)
•	M: metal coordination (His/Cys/Asp/Glu and specific metals)
•	G/P: geometry breakers (Gly flexibility, Pro rigidity)
•	X: steric/charge clash (strongly unfavorable)

    Now an AA:AA “codeword” is something like:

    •	(Asp, Lys) → SB
•	(Cys, Cys) → SS
•	(Phe, Tyr) → π
•	(Leu, Ile) → HΦ

    ChatGPT prompts:

    1. Tell me about NRF2–KEAP1 pathway and the role it plays in Vitiligo
    2. Are there any scientific issues with my explanation: “Vitiligo is is an autoimmune disease where oxidative stress in Melanocytes triggers immune system to attack them causing pigmentation. The idea I’m considering is developing a oxidative stress sensing mechanism that regulates a genetic circuit which turns on the protective mechanism in melanocytes until oxidative stress is active, and turns off the the protection when no oxidative stress is detected.”
    3. Create a narrative based on the slides (for all the slides from lecture 2)
    4. What’s Lysine Contingency
    5. If Lysine is already produced endogenously, why is used as a biocontainment strategy?