Week 1 HW: Principles and Practices version2

Contents

• Week 1 homework
• Week 2 lecture prep

Week1 homework

1. First, describe a biological engineering application or tool you want to develop and why.
   I want to optimize a strain of cyanobacteria for biomanufacturing. Cyanobacteria can be engineered to produce many useful things from atmospheric carbon dioxide, from commodity chemicals to bioactive compounds for pharmaceuticals, but harvesting the products is often energy intensive and expensive, especially at an industrial scale. I am particularly interested in cyanobacterial bioplastics, such as polyhydroxyalkanoates, because this would be a closed-loop carbon cycle for biodegradable plastic.

2. Next, 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: Prevent accidental release that could harm native ecosystems through microbial community shifts or production of commodity chemicals in the natural environment.
  • Subgoal: Include biocontainment systems in all commercially used industrial bioproduction strains.
  • Subgoal: Institute testing standards and protocols to notice any accidental release when it occurs.
• Goal: Increase access to the genetic tools and strains used for cyanobacterial bioproduction to allow more chemicals to be manufactured in this carbon-neutral way.
  • Subgoal: Publish cyanobacterial genetic engineering research (such as new tools, etc.) in open access journals or make PDFs available on personal/lab websites.
  • Subgoal: Enable strain sharing.

3. Next, describe at least three different potential governance “actions” by considering the four aspects below (Purpose, Design, Assumptions, Risks of Failure & “Success”).
   • Policy to require specific risk mitigation and demonstration of effectiveness under realistic application conditions for engineered bacteria approval.
     • Purpose: Currently, engineered bacteria that might affect environment and public health need to be approved by the EPA, FDA, or USDA for commercial use. This new policy would enact specific requirements for approvals for engineered bacteria. Additionally, many publications about genetic biocontainment discuss it as potential risk mitigation, but the effectiveness of the biocontainment is only demonstrated under specific laboratory conditions (i.e. axenic, optimized media, etc.).
     • Design: This would be a change in current federal standards and approval processes. The EPA, FDA, and USDA would need to write and implement new policies, potentially train risk assessors and application managers, and develop testing procedures to ensure compliance. With the overturning of the Chevron doctrine, likely this sort of new policy would require the buy-in of either the companies trying to get their products approved or US Congress to pass new legislation.
     • Assumptions: Companies and reseachers abide by federal regulations regarding testing and approval. Risk assessment is done in good faith, rather than by companies prioritizing profit over safety. Risk assessment is done by trained ecological and biological risk assessors who know what to look for or be aware of.
     • Risks of Failure and Success: This could fail if the requirement is too stringent to allow any new products to be approved. This could also fail if the requirements are too lax, and not all risks are accounted for and mitigated. If experimental conditions do not properly reflect application conditions, what appeared to be effective mitigation in the lab might not be effective mitigation in application.
   • Researchers and inventors could also implement relevant and effective genetic biocontainment in any engineered bacteria used for commercial biomanufacturing.
     • Purpose: For risks around the unintended spread of engineered bacteria or their synthetic genetic constructs, genetic biocontainment can mitigate these risks by preventing proliferation and/or degrading the relevant DNA. By tying the biocontainment system to the intended use of the bacterium, researchers manage risk in a relevant manner, thus ensuring that the bacterium is specific to the intended application and minimizing spread thereby reducing risks.
     • Design: Any developer of an engineered bacteria that could be intentionally or unintentionally released would need to research biocontainment and engineer a system into their bacteria. This would require a change in the current culture of the field, where the risks of engineered bacteria spread and mitigation through biocontainment are sometimes discussed, but mostly considered somewhat niche. If it became common practice to consider application and risks thereof for the products of synthetic biology, I think the design of these sorts of safeguards would be more widespread. Any sort of research requires funding and incentive, so universities, grant funders, and biotech companies would need to start looking for these considerations in proposals to motivate it.
     • Assumptions: Genetic biocontainment is a good strategy to mitigate the potential ecological and public health risks of new synthetic biology products. These risks are limited to ones we think to test (i.e. microbial community shifts, horizontal gene transfer of antiobiotic resistance genes or other functions, proliferation of engineered bacteria in unintended location, local specific bacterial extinction event in the case of a particularly robust engineered bacterium).
     • Risks of Failure and Success: If we rely too heavily on genetic biocontainment, a failure of the genetic system could result in losing that protection against risk. It’s also possible risks would not be seriously considered because we too easily trust biocontainment to minimize the risk.
   • Establish professional society for cyanobacteria-specific or general photosynthetic-organism research to promote resesarch and tool sharing.
     • Purpose: Currently, microalgae research is generally lumped along with all other non-model microbes in synthetic biology. A professional association or conference specific to photobiocatalysis could be a gathering place to collect all relevant tools, protocols, and standards, as well as potentially institute a shared ethics or goal to include improving access to the research and its products.
     • Design: Perhaps a starting point would be to invite cyanobacteria, eukaryotic microalgae, macro-algae, and plant synthetic biologists to a conference on photobiocatalysis, along with industry representatives from companies using or creating engineered phototrophs. This might be best done under the banner of an existing synthetic biology or metabolic engineering professional association (such as the Society for Biological Engineering in the American Institute of Chemical Engineers). If there is enough interest at the conference, attendees could work together to establish a more specific sub-association, or just resolve to discuss access and research sharing at the conference itself.
     • Assumptions: This is a large enough field to host such a specific conference. It might be too niche, but I don’t think so; it might be a conference on the smaller side at first though probably.
     • Risks of Failure and Success: It’s possible industry and start-ups might not want to popularly share their research as there is an economic disincentive.
4. Next, score (from 1-3 with, 1 as the best, or n/a) each of your governance actions against your rubric of policy goals.

5. Last, drawing upon this scoring, 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 the requirement of risk assessment and mitigation strategies for federal approval of engineered bacteria. I believe this would have the biggest impact in terms of allowing engineered bacteria to be used for public good (such as biomanufacturing) while preventing potential harm (such as ecosystem destabilization by permanently altering native microbiome in instances of escape). The development of genetic biocontainment tools and implementation thereof becoming regular practice in the field of engineered microbes would be awesome, but I think would be harder to bring about and would take longer - although it might actually have more impact. The establishment of a professional society could help institute such norms. Starting a new conference would probably be easiest in terms of discovering feasibility - proposing it to a handful of host organizations would rapidly identify whether this is currently worth pursuing or if it would need to be worked on for a while first.

References:

1. Chemla, Y; Sweeney, CJ; Wozniak, CA; et al. Engineering Bacteria for Environmental Release: Regulatory Challenges and Design Strategies. Authorea. July 05, 2024. DOI: 10.22541/au.171933709.97462270/v2
2. George, DR; Danciu, M; Davenport, PW; et al. A bumpy road ahead for genetic biocontainment. Nature Communications, 15(650). January 20, 2024. DOI: 10.1038/s41467-023-44531-1
3. Schmelling, NM; Bross, M. What is holding back cyanobacterial research and applications? A survey of the cyanobacterial research community. Nat Commun 15, 6758. August, 8, 2024. DOI: 10.1038/s41467-024-50828-6

Week2 Lecture Prep

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?
   Polymerase error rate: $1 : 10^{6}$. The human genome is around 3.2 Gb, or $3.2 * 10^{9}$ basepairs. Biological polymerases are error-correcting; they have have proofreading mechanisms. There are also mutation repair mechanisms.
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?
   The average human protein is encoded within 1036bp. This might be answerable based on the last slide titled “Fabricational Complexity”, but I couldn’t quite figure out what these formulas are supposed to be calculating without explanation. So instead, we can do some back-of-the-napkin math together. 1036bp is $1036/3 \approx 345$ codons, or 344 amino acids (because of the stop codon at the end), assuming that the 1036bp figure doesn’t include introns. Most amino acids have either 4 or 2 codons that can encode for it, although a couple have more or less. We’ll average it out to approximately 3 codons per amino acid. I imagine that not all amino acids are used at the same frequency in human proteins, but I don’t actually know what it is off the top of my head, so we’re just going to go with what we have. Each possible DNA sequence for an amino acid sequence includes every combination with all possible codons for each amino acid. So assuming an average human protein has 344 amino acids, and the average number of codons per amino acid is 3, then there are $3^{344} = 1.3 E164$ different ways to code for an average human protein. In practice, not all tRNAs are synthesized at the same frequency, so it might take unreasonably long for certain codons to be recognized during chain extension; and during DNA replication, errors can be made and some errors will be more tolerable than others due to codon wobble.

LeProust:

1. What’s the most commonly used method for oligo synthesis currently?
   Phosphoramidite synthesis.
2. Why is it difficult to make oligos longer than 200nt via direct synthesis?
   There are side reactions that occur, causing the accumulation of errors (incorrect bases).
3. Why can’t you make a 2000bp gene via direct oligo synthesis?
   I think this is because of the side reactions in Q2, right? Like, the accumulation of errors limits oligo synthesis to around 200 bases in practice. Also, oligos are single-stranded DNA; a 2000bp gene is double-stranded, and therefore you’d either need to synthesize both strands and ligate them together, or synthesize one strand and use it as a template for PCR or something.

Church:

3. Given the one paragraph abstracts for these real 2026 grant programs sketch a response to one of them or devise one of your own: BioStabilization Systems - ARPA-H \

Biologic therapeutics are critically important for a number of diseases, but require careful and specific conditions at all points on the supply chain to maintain efficacy. Specifically, cell therapies and biologics require extreme cold to prevent degradation, thus making biologics inaccessible to people who don’t live near a specialized medical center. To solve this problem, we propose to express biologic therapeutics in extremophiles from abyssal marine sediment, which demonstrated little cell proliferation in low-oxygen environments but regained metabolic activity when incubated with oxygen. We predict that the faster cell turnover period at warmer temperature, oxygen-rich, and high-nutrient conditions will allow us to engineer these bacteria to produce the biologic therapeutic molecules. Once production is achieved, we will seal the cells into low-oxygen capsules for transport, which we predict will slow their metabolic rate enough to preserve the goal product until oxygen is provided again. If successful, this research could expand access to biologic therapeutics to anywhere that can aseptically incubate microbes at room temperature and purify the molecules therein.

References:

1. Morono, Y; Ito, M; Hoshino, T; et al. Aerobic microbial life persists in oxic marine sediment as old as 101.5 million years. Nat Commun 11, 3626. 2020. DOI: 10.1038/s41467-020-17330-1
2. Suzuki, Y; Webb, SJ; Kouduka, M; et al. Subsurface Microbial Colonization at Mineral-Filled Veins in 2-Billion-Year-Old Mafic Rock from the Bushveld Igneous Complex, South Africa. Microb Ecol 87, 116. 2024. DOI: 10.1007/s00248-024-02434-8

Personal notes/drafting

abstract formula:

• 1 sentence on the broad problem: Biologic therapeutics are critically important for a number of diseases, but require careful and specific conditions at all points on the supply chain to maintain efficacy.
• 1-2 sentences on the specific problem: How to transport cell therapies and biologics at room temperature, _{decentralizing medicine}
• 1 sentence on the broad goal: We aim to express biologic compounds in extremophiles from the deep subsurface where energy and nutrients are limited.
• 2-3 sentences on methods: aerobic microbes from oxic abyssal marine sediment that proliferated at 10C with provision of nutrients and higher conc O2; might need to consider eukaryotic protein folding in prokaryotes; low O2 environment - maybe sealing the cells (post-therapeutic production, pre-shipping) into an airtight capsule would prevent metabolic activity including the breakdown of said therapeutics?
• 1 sentence on future work: maybe also try extremophiles found within old rock samples
• 1 sentence on conclusion/impact: expands access to biologics, especially to under-resourced communities