Week 1 HW: Principles and Practices

1. Describe a biological engineering application or tool you want to develop and why.

Administration of broad-spectrum antibiotics, while clinically necessary to solve acute infections, leads to the gut microbiome imbalance, a state known as ‘dysbiosis’. This phenomenon is characterized by a decline in microbial alpha diversity and the depletion of obligate anaerobes, which are critical for the production of short-chain fatty acids (SCFAs) and for maintaining gut homeostasis. This ecological instability also facilitates the expansion of opportunistic microbes, notably Clostridioides difficile, by eliminating the protective mechanism of colonization resistance. Furthermore, the persistent presence of residual antibiotic concentrations in the distal gastrointestinal tract exerts a potent selective pressure for horizontal gene transfer of resistance determinants, thereby enriching the intestinal resistome and contributing to the global crisis of multidrug resistance.

To mitigate these risks, I want to develop a living drug designed to prevent dysbiosis caused by diverse antibiotic classes. My goal on this project is to engineer a single strain or a defined consortium capable of degrading at least three distinct classes of antibiotics simultaneously. This living drug implements a kill switch, ensuring non-survival outside the human gut or without a specific synthetic inducer. While it secretes neutralizing enzymes, the genes for them are not transferable to other bacteria.

By protecting the gut microbiota, this tool can achieve three primary clinical endpoints:

• Maintenance of the epithelial interface to prevent the translocation of inflammatory markers
  
• Reduction of secondary infections due to antibiotics
  
• Mitigate antimicrobial resistance gene transfer
  

2. Describe one or more governance/policy goals related to ensuring that this application or tool contributes to an “ethical” future, like ensuring non-malfeasance (preventing harm). Break big goals down into two or more specific sub-goals.

GOAL 1: GENETIC CONTAINMENT AND ENVIRONMENTAL MONITORING

• Government and policies must ensure the containment of the synthetic genes to prevent their intentional or unintentional release to the environment.
• Biocontainment mechanisms in the design of the synthetic circuit should be compulsory. These mechanisms should ensure that the living drug will not survive without the absence of a synthetic inducer or will not transfer its synthetic gene to other bacteria via horizontal transfer.
• Policies should establish a protocol that monitors the environment (i.e., household microbial ecology) of the person receiving the living drug

GOAL 2: PREVENTION OF MISUSE AND BIOSECURITY

• Policies should prevent the use of the drug to protect a pathogen from treatment. The genetic sequences of high-efficiency neutralizing enzymes should be safeguarded from bioterrorists and actors who aim to create antibiotic-proof pathogens.

GOAL 3: INFORMED CONSENT

• Policy must require a fully informed consent form for patients, detailing the ecological nature of LBPs.
• Policy should require manufacturers to have a rescue drug so that physicians can immediately stop the adverse reactions of the living drug.

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

ACTION 1: MONITORING OF DNA SYNTHESIS COMPANIES

• Purpose: The law or policy ensures the proprietary rights of the genetic sequences used in the synthetic circuit.
  
• Design: When the genetic sequences become accessible in a directory, any order for these sequences from DNA synthesis providers by an unverified lab or person would trigger a comprehensive investigation.
• Assumptions: The bad actors will avail the services of legitimate synthesis companies, rather than utilizing the black market.
• Risks of Failure: Flagging the synthetic sequences might actually highlight their utility to bioterrorists who want to create antibiotic-resistant pathogens.
• Risks of Successes: Ordering the necessary genetic parts from the DNA synthesis providers would make things difficult for legitimate academic research and companies involved in curing antibiotic resistance and antibiotic-induced dysbiosis.

ACTION 2: MANDATORY RESCUE DRUGS

• Purpose: Rescue drugs should be able to neutralize the engineered strain when patients experienced adversed effects.
  
• Design: The drugs should not be a target of the engineered therapeutic, and it can eliminate them 100%.
• Assumptions: The engineered strain will not develop immediate resistance to the rescue drugs during the treatment window.
• Risks of Failure: The chassis will acquire resistance to this rescue drug, which can cause unstoppable infection.
• Risks of Successes: It limits the use of the living drug to only patients who are not already resistant to the rescue drugs, narrowing the patient pool.

ACTION 3: MICROBIAL IMPACT STATEMENT

• Purpose: The microbial impact statement aims to inform patients about the biological properties of the living drug and to ask for consent
  
• Design: It is a document that explains the biological nature of the living drug, including its residence time, mechanism of action, and potential side effects.
• Assumptions: Transparency increases trust rather than fear of GMOs.
• Risks of Failure: Patients might misunderstand GMOs and refuse treatment due to stigma.
• Risks of Successes: It could lead to “secondary discrimination,” where employers or insurance companies demand to see a patient’s Microbial Impact Statement to ensure they are not carrying engineered microbes.

4. score (from 1-3 with, 1 as the best, or n/a) each of your governance actions against your rubric of policy goals.

5. Describe which governance option, or combination of options, you would prioritize, and why. Outline any trade-offs you considered as well as assumptions and uncertainties.

I WOULD PRIORITIZE ACTION 1 AND ACTION 2.

Action 1 prevents the synthetic gene circuit templates from reaching unverified or bad actors who plan to intentionally release these genes to the environment or used it to develop multi-drug resistant strains. While this action prevents the template from spreading, Action 2 is a backup plan to ensure that any unintentional strains derived from the synthetic circuits can be easily killed.

TRADE-OFFS

• Implementing Action 1 will hinder the progress of legitimate researchers in synthetic biology
• There will be challenges in public acceptance if people feel the security is handled behind the scenes without their involvement.

ASSUMPTIONS AND UNCERTAINTIES

• We assume that bad actors are using legitimate synthesis companies, and not the black market.
• We assume that the rescue drug will always be 100% effective. There is an uncertainty in whether the living drug could evolve resistance to the rescue drug.

AUDIENCE FOR RECOMMENDATION

This governance strategy is best targeted at:

• public and private health agencies
• public and private regulatory bodies
• manufacturers of the living drug
• consumers of the living drug

REFLECTION ON THE THINGS I LEARNED IN CLASS

Ethical concerns:

1. Dual-Use Risk - the same genetic sequences you use for other’s benefit could be repurposed to promote bioterrorism and cause harm.
2. Coercion risk - monitoring vs surveillance
3. Synthetic biology products might be too novel for existing regulations
4. Equity - commercialization of synthetic biology that could address advanced issues will be prominent in wealthy nations, while the risks may be outsourced to developing regions

Governance actions:

1. Implement a mandatory international protocol where all commercial DNA synthesis providers must screen orders against a secure database
2. Establish a legal framework that treats biological monitoring data as temporary. It should be illegal to use safety-related biological data for non-medical purposes.
3. Regulatory bodies should move toward a product-based rather than a process-based evaluation. This allows for a more flexible process that updates as the technology evolves.
4. Strengthen and enforce the Nagoya protocol

WEEK 2 LECTURE PREPARATIONS

Homework Questions by Professor Jacobson

1. 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?

• The estimated spontaneous mutation rates of DNA polymerases are in the range of 10^-6 to 6^-7 errors per base pair(Roberts et al., 1988). These numbers are negligible compared to the entire length of the human genome of 3.055 billion base pairs (Nurk et al., 2022). It means that you have at most 300 mutations in replicating the whole genome.

• Biology deals with the errors by the following mechanisms (Kunkel & Bebenek, 2000):

  1. Exonuclease proofreading - DNA polymerases have a 3’ to 5’ exonuclease activity to correct errors made by them during replication
  2. Post-replication repair - replication errors can be modulated by various forms of repair operating after replication, including mismatch repair and several types of excision repair

2. 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?

There are 61 codons that code for 20 amino acids. Most amino acids are degenerate, which means that multiple codons can code for one amino acid. Let’s say that the average human protein is around 450 amino acids, the number of possible DNA sequences is about 10¹⁵⁰.

However, some of the codes do not work to code for the protein. Depending on the organism, there is codon usage bias, wherein different organisms have different tRNA abundances. Hence, a certain amino acid will preferentially be coded by a codon with the most abundant number of tRNA. Another reason is that the mRNA, which is the direct template for protein synthesis, can form secondary structure that prevents it from being translated.

REFERENCES

Kunkel, T. A., & Bebenek, K. (2000). DNA replication fidelity. Annual review of biochemistry, 69(1), 497-529.

Nurk, S., Koren, S., Rhie, A., Rautiainen, M., Bzikadze, A. V., Mikheenko, A., … & Phillippy, A. M. (2022). The complete sequence of a human genome. Science, 376(6588), 44-53.

Sun, M. (2022). Investigation of DNA Polymerase Epsilon and Apobec Mediated Mutagenesis Using In Vivo and In Vitro Models (Doctoral dissertation, Tulane University).

Homework Questions by Dr. LeProust

1. What’s the most commonly used method for oligo synthesis currently?

Solid-state phosphoramidite synthesis is currently most commonly used method for oligonucleotide synthesis. The process involves coupling the 3’-terminal nucleoside residue to a universal solid support as a phosphoramidite building block in the first cycle of oligonucleotide synthesis, after which the chain assembly continues (Guzaev, 2013).

2. Why is it difficult to make oligos longer than 200 nt via direct synthesis?

The coupling efficiency of the synthesis decreases as the oligonucleotide grows longer. When the oligo reaches a length of 200 nucleotides, it already consists of truncated sequences rather than the full-length product.

3. Why can’t you make a 2000 bp gene via direct oligo synthesis?

Because of the increasing chemical damage and physical tangling, in addition to the decreasing coupling efficiency, as the length of the synthesized oligo increases.

REFERENCES

Guzaev, A. P. (2013). Solid‐phase supports for oligonucleotide synthesis. Current protocols in nucleic acid chemistry, 53(1), 3-1.

Homework Questions from George Church

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

The 10 essential amino acids, according to Lopez et al. (2024):

1. Histidine (His)
2. Isoleucine (Ile)
3. Leucine (Leu)
4. Lysine (Lys)
5. Methionine (Met)
6. Phenylalanine (Phe)
7. Threonine (Thr)
8. Tryptophan (Trp)
9. Valine (Val)
10. Arginine (Arg)

The “Lysine Contingency” is a concept popularize by Jurassic Park, where scientist engineered the dinosaurs to unable to produce lysine. Without a manual supplement provided, the dinasaurs would theoretically die, preventing them from surviving in the wild.

As lysine is one of the essential amino acids of animals required to survive, scientists do not need to put an extra effort to engineer them in that way because it is a natural state of almost all complex animals, including dinosaurs. Besides, lysine is one of the most abundant amino acids in nature which can be taken by these dinosaurs as part of there diet.

REFERENCES

Lopez, M. J., & Mohiuddin, S. S. (2024). Biochemistry, essential amino acids. In StatPearls [Internet]. StatPearls Publishing.