Week 1 HW: Principles and Practices

1. Biological Engineering Application: Phage Therapy for Antibiotic-Resistant Infections

Antibiotic resistance is a growing public health crisis, with an estimated 700,000 deaths annually(O’Neill, 2016). Phage therapy uses bacteriophages (viruses that infect bacteria) to treat these infections. Unlike broad-spectrum antibiotics, phage are precise and can replicate at the site of infection. This new form of therapy can help us combat the increasing pollution of antibiotic resistance bacteria.

The four pillars of engineered phage therapy(Du et al., 2023)

Goals

The primary goal is to create synthetically engineered bacteriophages that can target antibiotic resistant bacteria thus developing an alternative, supplementary therapy to antibiotics, especially in the face of rapidly increasing antimicrobial resistance.

• Sub-goal A: Prevent Genetic Pollution. Ensure that therapeutic phages do not inadvertently spread antibiotic resistance genes (ARG) through horizontal gene transfer.
• Sub-goal B: Ensure Equitable Public Access. Establish infrastructure so that life-saving phage treatments are treated as a public good rather than a niche luxury.

3. Governance Actions

1. Development of a Regulatory and Normative Framework

• Purpose: To ensure that all stakeholders’ concerns are addressed and that this technology is implemented in the safest and most effective manner possible, a multidisciplinary commission comprising molecular biologists, infectious disease specialists, government regulators, and a representative group of citizens should be formed. The commission must develop guidelines to ensure that phages are obligately lytic, free of toxins or resistance genes, and used ethically and equitably in every possible way.
• Design & Assumptions: The commission will establish a regulatory framework to verify that phages are obligately lytic and free of harmful genetic elements. This design assumes that multidisciplinary experts and citizens can reach a consensus on safety and equitable distribution.
• Risks: Failure risks the accidental spread of toxins or resistance genes if screening protocols are bypassed or poorly executed. Success is defined by a transparent, safe, and ethical system that effectively treats drug-resistant infections across all socioeconomic groups.

2. Regulatory Mandate for “Obligate Lytic” Genetic Screening

• Purpose: To prevent the use of lysogenic (temperate) phages, which can integrate into bacterial chromosomes and potentially make infections worse by spreading resistance.
• Design: The FDA would require genomic sequencing and potentially CRISPR-based “tweaks” to ensure every therapeutic phage is an obligate lytic variety.
• Assumptions: Assumes that the distinction between lytic and lysogenic life cycles is technically clear and that synthetic modifications are feasible at scale.
• Risks of Failure & “Success”: Even with lytic phages, horizontal gene transfer can occasionally occur during the lytic cycle, meaning the risk is reduced but not eliminated.

3. “Pull” Incentives (Cash Prizes and Patent Extensions)

• Purpose: To coax pharmaceutical firms into developing phage treatments by offering financial rewards that bypass the difficulty of patenting natural organisms.
• Design: Governments offer cash prizes for successful clinical trials or transferable patent extensions on other profitable drugs in exchange for phage R&D.
• Assumptions: Assumes government agents can accurately assess the “social value” of a new treatment to set appropriate prize amounts.
• Risks of Failure & “Success”: May impose high costs on taxpayers or lead to artificial monopolies on unrelated life-saving medications.

5. Ethical Reflection

A significant ethical concern identified is the complexity of informed consent. Because phage therapy involves infecting a patient with “live” viruses and the science is poorly understood by the public, patients may struggle to provide robust consent compared to simple antibiotic use.

Proposed Governance Action: I propose developing standardized clinical “challenge studies” and physician education frameworks to bridge the information gap. Additionally, we should apply the public-harm principle: since individual antibiotic misuse creates collective harm, governments are ethically justified in enacting coercive policies to regulate phage and antibiotic use to preserve drug efficacy for everyone. # Molecular Biology & Synthetic DNA Homework

References: O’Neill, J. (2016). Tackling Drug-Resistant Infections Globally: Final Report and Recommendations.Review on Antimicrobial Resistance, available from:http://amr-review.org/ Du, J., Meile, S., Baggenstos, J., Jäggi, T., Piffaretti, P., Hunold, L., Matter, C. I., Leitner, L., Kessler, T. M., Loessner, M. J., Kilcher, S., & Dunne, M. (2023). Enhancing bacteriophage therapeutics through in situ production and release of heterologous antimicrobial effectors. Nature Communications, 14(1), 4337. https://doi.org/10.1038/s41467-023-39612-0

Week 2 prep homework questions:

1. Homework Questions from Professor Jacobson

Q1: DNA Polymerase Error Rates and Genome Stability

Nature’s machinery for copying DNA is called polymerase. What is the error rate of polymerase? How does this compare to the length of the human genome? How does biology deal with that discrepancy?

• Error Rate: The error rate of a standard DNA polymerase (without proofreading) is approximately $1$ in $10^{4$ to $1$ in $10}5$. However, with intrinsic proofreading mechanisms ($3’$-$5’$ exonuclease activity), the error rate is improved to approximately $1$ in $10^{6$ to $1$ in $10}7$.
• Comparison to Human Genome: The human genome is approximately 3.2 billion base pairs ($3.2 \times 10^{9$ bp) in length. This creates a massive discrepancy; an error rate of $10}{-6}$ would result in thousands of errors ($\approx 3200$) every time the genome is replicated.
• Dealing with the Discrepancy: Biology resolves this through post-replication error correction mechanisms, such as Mismatch Repair (MutS, MutL, MutH), which identify and fix errors missed by the polymerase. This reduces the overall error rate significantly (typically to around $10^{-9}$ or lower), ensuring genomic stability over generations.

Q2: Protein Coding Complexity

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?

• Ways to Code: An average human protein corresponds to a gene sequence of approximately $1036$ base pairs, translating to roughly $345$ amino acids. Because the genetic code is degenerate (64 codons for 20 amino acids), there are multiple ways to encode most amino acids. Assuming an average degeneracy of 3 options per position, the number of ways to code for a single protein is astronomical, roughly $3^{345}$.
• Practical Limitations: Many of these sequences fail to produce the desired protein efficiently due to “fabricational complexity”:
  • Secondary Structure: DNA or mRNA strands may form stable secondary structures (like hairpins) that interfere with transcription or translation.
  • GC Content: Sequences with extreme GC content (very high or very low) are difficult to synthesize and replicate.
  • Repeats and Homopolymers: Long runs of identical bases (e.g., $>30$ bp) can cause slippage errors.
  • Codon Usage Bias: Organisms prefer specific codons for efficient translation; using rare codons can stall ribosomes.

Refrences: O’Neill, J. (2016). Tackling Drug-Resistant Infections Globally: Final Report and Recommendations. Review on Antimicrobial Resistance, available from: http://amr-review.org/

2. Homework Questions from Dr. LeProust

Q1: Modern Oligo Synthesis

The most commonly used method currently is solid-phase phosphoramidite chemistry. This involves a cycle of deprotection, coupling, capping, and oxidation to build the oligonucleotide chain nucleotide by nucleotide on a solid support.

Q2: Limits of Direct Synthesis (>200nt)

The difficulty arises from the coupling efficiency of the chemical reaction. The yield of the full-length product decays exponentially with length according to the formula: $$\text{Yield} \approx (1 - 1/N)^N \approx 37%$$ (where efficiency is less than 100%). As length increases, cumulative yield drops drastically, and side reactions (like depurination) or truncated failure sequences accumulate, making purification difficult.

Q3: Challenges with 2000bp Gene Synthesis

Due to the exponential decay of yield, the probability of synthesizing a correct, continuous 2000bp strand purely by sequential base addition is effectively zero. Instead, longer genes are constructed by synthesizing shorter oligonucleotides and “stitching” them together using methods like PCR assembly or enzymatic assembly (e.g., Gibson Assembly).

3. Homework Question from George Church

What are the 10 essential amino acids in all animals and how does this affect your view of the “Lysine Contingency”?

• 10 Essential Amino Acids: The amino acids that animals cannot synthesize de novo are: Arginine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, and Valine.
• View on the Contingency: In Jurassic Park, the “Lysine Contingency” was a safeguard where dinosaurs were engineered to be unable to produce Lysine. However, since Lysine is already an essential amino acid for all animals, the “engineering” was scientifically redundant. Animals naturally lack the enzymes to make Lysine from Aspartate. The “contingency” was actually just a strict dietary control of a nutrient that is naturally essential, rather than a novel genetic modification.