Week 1 HW: Principles and Practices

Project Name: Cellular Fruit Production System

1. Biological Engineering Application & Rationale

Project Overview

I propose the development of a cellular fruit production platform optimized for microgravity environments. Instead of growing whole plants (roots, stems, leaves), this system cultures only the edible cellular tissues (flesh cells from fruits like strawberries or blueberries) within a bioreactor. These cells are then reconstructed into “fruit” with realistic textures using 3D food printing technology.

Technical Approach

This system integrates the following technologies.

1. Plant Cell Culture: Pulp cells harvested from fruits such as strawberries and blueberries are grown as callus (undifferentiated cell clusters) or suspension cultures (cells floating in liquid media). Under appropriate culture conditions (hormones, sugars, vitamins, minerals), these cells proliferate exponentially and produce the same nutrients as the original tissue, including Vitamin C, polyphenols, and flavonoids.
2. Bioreactor Optimization: In microgravity, liquids tend to form spheres due to surface tension, making traditional gravity-dependent stirring difficult. This project utilizes Rotating Wall Vessel (RWV) technology to culture cells with low shear force while maintaining uniform dispersion.
3. 3D Bioprinting: Cultured cells are mixed with a hydrogel (such as algae-derived alginate) and layered to reproduce the shape and texture of the fruit. While this technology is used on Earth for structuring cultivated meat, its adaptation to space environments is currently being researched.

Why is this necessary?

Limitations of Current Space Food Systems

According to NASA research, the primary challenges for long-duration missions (such as a 2–3 year round trip to Mars) include:

• Nutrient Degradation: Micronutrients such as Vitamin C, thiamine, and Vitamin K in retort-packaged or freeze-dried foods can decrease by up to 40–50% after three years of ambient storage (Cooper et al., 2017). This poses risks of scurvy and immune dysfunction.
• Menu Fatigue: Astronauts lose an average of 2–5% of their body weight during long missions, primarily due to anorexia and menu fatigue (Massa et al., 2021). The lack of fresh ingredients significantly impacts psychological well-being.
• Resupply Dependency: The cost of transporting food to the ISS is approximately $20,000 to $40,000 per kg. For Mars exploration, resupply is impossible, making a completely self-contained system essential.

Comparison with Existing Plant Cultivation Systems

While NASA’s Veggie and Advanced Plant Habitat (APH) have successfully grown lettuce, mizuna, and tomatoes, they face the following challenges.

2. Governance / Policy Goals

To ensure this technology contributes to an “ethical” future, the following governance goals are established.

Goal 1: Non-maleficence / Biosafety & Biosecurity

Sub-goal 1a: Planetary Protection

Definition: Preventing Earth-derived biological entities (including GE cells) from escaping into extraterrestrial environments like Mars, which could contaminate local ecosystems or interfere with scientific life-detection surveys.

Context: Per COSPAR’s Planetary Protection Policy (2020), Mars missions are categorized (e.g., Category IVa/IVc). The cells of this system leaking onto the Martian surface could:

• Cause “false positives” in soil sample analysis for life.
• Potentially proliferate in Martian sub-surface ice or liquid water (though unlikely, it cannot be entirely ruled out).

Sub-goal 1b: Crew Safety

Definition: Preventing cultured cells from undergoing unexpected mutations in closed environments that could produce harmful substances, such as new allergens, toxins (alkaloids, lectins), or pathogens (fungal/bacterial contamination).

Context: ISS Veggie experiments confirmed that space-grown crop microbiomes differ from Earth controls (Khodadad et al., 2020). In closed systems, pathogen outbreaks can be fatal.

Goal 2: Equity & Technology Transfer

Sub-goal 2a: Dual-Use Innovation

Definition: Promoting the return of space food technology to solve food security challenges on Earth, such as vertical farming in urban areas, nutrition in extreme environments (deserts, conflict zones), and disaster response.

Sub-goal 2b: Open Science and Global Access

Definition: Ensuring this technology does not become the exclusive property of specific space agencies or wealthy nations, but remains accessible to developing countries and academic institutions.

3. Governance Actions

Option 1: Standardised Biological Containment

Actor: Space agencies (NASA/ESA/JAXA), biosecurity researchers, and the synthetic biology community.

• Purpose: To implement multi-layered biological containment beyond mere physical barriers.
• Design:

1. Auxotrophy: Cells dependent on specific amino acids or rare metals (e.g., Vanadium) not found in the Martian environment.
2. Kill-switch: Genetic circuits where cells undergo apoptosis if a specific signal (light, temperature) is removed.
3. Genomic Barcoding: Inserting synthetic DNA “barcodes” to trace the origin of any leak.

• Assumptions: Kill-switches are redundant enough to resist mutation; Martian conditions are well-understood.
• Risks: Malfunctions could lead to sudden food shortages (failure); over-reliance might lead to neglecting physical containment (“success” byproduct).

Option 2: Global Space-Bio Registry

Actor: International regulators (COSPAR, UNOOSA), DNA synthesis companies.

• Purpose: To create a centralised database for tracking all biological entities (DNA sequences, cell lines) sent into space.
• Design: Mandatory registration 180 days before launch, automated risk scoring based on environmental resistance and genetic modification, and expert peer review for high-risk systems.
• Assumptions: International cooperation from all nations and private companies (SpaceX, etc.).
• Risks: Bureaucracy may delay innovation; some actors might bypass the registry (regulatory havens).

Option 3: Open Source Design & Distributed Peer Review

Actor: Academic researchers, open-source communities (iGEM, BioBricks), space engineers.

• Purpose: To promote continuous global improvement and transparency, preventing errors hidden in closed systems.
• Design: Full disclosure of genetic circuits, media recipes, and bioreactor blueprints on platforms like GitHub or Protocols.io. Implementation of “Red Team” exercises and bug bounty programs for security vulnerabilities.
• Assumptions: Linus’s Law; the dominance of “good actors” over malicious ones.
• Risks: Dual-use risk (misuse by malicious actors); potential loss of commercial incentive due to lack of IP.

4. Scoring Matrix

(1 = Best, 3 = Worst)

5. Recommendation

Recommended Strategy: Phased Hybrid Approach

I recommend a phased hybrid strategy to balance innovation with safety:

• Phase 1 (Current - Prototyping): Focus on Option 3 (Open Source) Prioritise open development to gather global expertise and establish a robust, transparent foundation.
• Phase 2 (Ground Testing - ISS Trials): Integrate Option 1 (Containment) Implement biological containment (kill-switches) as the system moves to closed-loop testing in orbit. Perform “Security Sprints.”
• Phase 3 (Mars Missions and Beyond): Full Implementation of Option 2 (Registry) Enforce mandatory international registration and rigorous COSPAR review for planetary-scale missions.

Rationale

• Balance: Autonomy and “repairability” are vital in space. Option 1 alone creates a “lock-in” risk where crews cannot fix their own food systems. Option 2 alone is too slow for early-stage research.
• Risk Management: Governance scales with the level of risk (from BSL-1 labs to the Martian surface).
• Stakeholder Alignment: Academic freedom is protected in Phase 1, while planetary protection is guaranteed in Phase 3.

Homework Questions from 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?

• Error Rate of Polymerase: According to the lecture slides, the error rate for biological synthesis using an error-correcting polymerase is approximately 1 in 10^6.
• Comparison to Human Genome Length: The human genome is approximately 3.2 Gbp ( base pairs) in length. Comparing the two figures reveals a significant discrepancy: if the polymerase error rate is 10^{-6 and the genome is 3.2*10}-9 bp, a single round of replication would theoretically result in approximately 3,200 errors. This number of mutations per cell division would be unsustainable for the organism.
• How Biology Deals with the Discrepancy: Biology employs multiple layers of error correction to bridge this gap:

1. Proofreading: The DNA polymerase itself has an intrinsic proofreading capability (exonuclease activity) that corrects errors during the extension phase.
2. Mismatch Repair (MMR): A separate system, including proteins such as MutS, MutL, and MutH, identifies and repairs mismatches that escape the polymerase’s proofreading. This system recognizes the error (e.g., via MutS), creates a nick (e.g., via MutH), removes the incorrect segment (exonuclease), and re-synthesizes the strand (DNA pol III and ligase).

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?

• Number of Ways to Code: The average human protein coding sequence is approximately 1,036 bp long. This corresponds to roughly 345 amino acids(1036/3). Since the genetic code is degenerate (most amino acids are encoded by multiple synonymous codons, with an average of about 3 options per amino acid), the number of possible DNA sequences for a single protein is astronomical. Approximating the geometric mean of synonymous codons per amino acid as 3, the total number of ways to code for an average protein is roughly 3^345, which is approximately **** distinct sequences.
• Practical Reasons Why Not All Codes Work: While many sequences theoretically encode the same protein, they are not functionally equivalent in practice due to several factors:

1. mRNA Secondary Structure: Different nucleotide sequences can fold into different Minimum Free Energy (MFE) secondary structures. Stable hairpins or other structures (analyzed by tools like NUPACK) can occlude the Ribosome Binding Site (RBS) or interfere with translation initiation and elongation.
2. RNase Cleavage Sites: Certain synonymous sequences may inadvertently create recognition sites for enzymes like RNase III, leading to the cleavage and degradation of the mRNA before it can be translated.
3. Codon Usage Bias: Although not explicitly detailed in the text of the slides, the context of “Recoding” and “tRNA” availability implies that organisms have preferred codons corresponding to abundant tRNAs. Using “rare” codons can slow down translation or cause stalling, affecting protein folding and expression levels.
4. Synthesis Constraints: In the context of “growing” or synthesizing genes, certain sequences (e.g., extreme GC content or repeats) may be chemically difficult to synthesize or assemble error-free.

Homework Questions from Dr. LeProust 1. What’s the most commonly used method for oligo synthesis currently?

The most commonly used method for oligonucleotide synthesis is the Phosphoramidite method.

• This chemistry was developed by Marvin Caruthers in 1981 and remains the gold standard for DNA synthesis today.
• The synthesis cycle consists of four main steps: De-blocking (Detritylation), Coupling, Capping, and Oxidation.
• This method is the foundation for modern high-throughput synthesis platforms, including the silicon-based platform used by Twist Bioscience, which miniaturizes the reaction to increase throughput and reduce costs.

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

Synthesizing oligos longer than 200 nucleotides (nt) directly is difficult primarily due to coupling efficiency limits and the accumulation of errors, which lead to exponentially lower yields.

• Yield Decay: DNA synthesis is a cyclical process. The final yield (Y) is determined by the coupling efficiency (E) raised to the power of the sequence length (n): Y=E^n. Even with a very high industrial efficiency of 99.8%, the yield for a 200nt oligo drops significantly, and for longer sequences, it approaches zero rapidly.
• Side Reactions: As the chain grows, side reactions such as depurination (loss of purine bases due to acid exposure) accumulate, damaging the DNA.
• Purification Challenges: Separating the full-length product (n) from failure sequences (like n-1 or n-2) becomes increasingly difficult as the length increases because their physical properties become too similar.

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

It is practically impossible to create a 2000bp gene via direct oligo synthesis because the theoretical yield would be effectively zero.

• Mathematical Limitation: Applying the yield formula Y=E^{n, attempting to synthesize a 2000bp sequence directly—even with 99.5% efficiency—would result in a yield of $0.995}{2000} \approx 0.004%$. It is statistically unlikely to recover any full-length, error-free molecules.
• Assembly Requirement: Instead of direct synthesis, long genes are constructed using Gene Assembly. This involves synthesizing shorter oligonucleotides (e.g., oligos of ~50–100 bases) and stitching them together using methods like PCR assembly or enzymatic ligation (e.g., Gibson Assembly) to form the full 2000bp sequence.

Homework Question from George Church Topic: Essential Amino Acids and the Scientific Validity of the “Lysine Contingency”

1. The Ten Essential Amino Acids in Animals For many animals—particularly mammals, including humans and rats—the following ten amino acids are classified as nutritionally essential. These compounds cannot be synthesized de novo by the organism, or cannot be synthesized at a rate sufficient to meet metabolic demands; therefore, they must be obtained through dietary intake.

1. Arginine
2. Histidine
3. Isoleucine
4. Leucine
5. Lysine
6. Methionine
7. Phenylalanine
8. Threonine
9. Tryptophan
10. Valine

2. Scientific Perspective on the “Lysine Contingency” The “Lysine Contingency,” as presented in the film Jurassic Park, is described as a fail-safe mechanism intended to prevent genetically resurrected dinosaurs from escaping and surviving off the island. The narrative explains that “through genetic engineering, the dinosaurs were altered to be unable to synthesize the amino acid lysine; consequently, unless administered lysine by the park staff, the animals would slip into a coma and die” [3].

However, based on the fact that Lysine is included in the aforementioned list of essential amino acids, it is evident that this premise contains significant scientific contradictions:

• Natural Absence of Biosynthetic Capability: All animals, including vertebrates, inherently lack the metabolic pathway required to synthesize lysine. Therefore, even without any genetic intervention, these animals are physiologically incapable of synthesizing lysine and rely entirely on exogenous (dietary) sources for survival [3][4].
• Ineffectiveness as a “Safety Measure”: While the assertion that the animals would die without lysine is accurate, this vulnerability is not the result of genetic engineering but rather a fundamental biological constraint. In the natural world, animals maintain survival by consuming lysine-rich foods (e.g., meat, soy, plants). Consequently, even if the dinosaurs were to escape the island, they could readily sustain themselves by preying on flora and fauna containing lysine, rendering this “safety measure” functionally ineffective [4].

Conclusion: From an academic standpoint, it can be concluded that the “Lysine Contingency” misrepresents a naturally occurring physiological trait—specifically, the dependence on essential amino acids—portraying it either erroneously as the product of advanced genetic control or deliberately embellishing it for dramatic narrative effect.

References:

[1] Study.com. “Essential & Nonessential Amino Acids | Roles & Differences.” (Accessed 2026-02-11).

[2] Nutrena Feeds. “What are Essential Amino Acids in Protein?” (Accessed 2026-02-11).

[3] Jurassic Park Wiki. “Lysine contingency.” (Accessed 2026-02-11).

[4] Roanoke Gaming. “METABOLIC CATASTROPHE Behind Jurassic Parks Lysine Contingency Explained.” YouTube. (Accessed 2026-02-11).