Week 1 HW: Principles and Practices

_{Image credit: John Game via Wikimedia Commons}

1. Bio-Engineering Application: Astro-Moss

Project Vision

I propose the development of Astro-Moss, a engineered strain of Physcomitrium patens designed for extreme environmental resilience. By leveraging the high genomic editability of moss, this project aims to create varieties capable of surviving extreme diurnal temperature fluctuations, high ionizing radiation, and desiccation.

Applications

• Extraterrestrial Foundation: Serving as a primary “pioneer” vegetation for Martian habitats to build organic substrate for future crops.
• Functional Bio-textiles: Extracting engineered cells or fibers to create “living” textiles for astronaut apparel that offer self-repairing properties and radiation shielding, contributing to long-term sustainability in space exploration.

2. Governance and Policy Goals

To ensure this application contributes to an ethical future, I have defined the following goals based on the framework of promoting constructive use while minimizing harm:

• Goal 1: Ensure Biosafety and Security. Preventing the unintended release of engineered extremophiles into Earth’s ecosystems or the misuse of resilience genes.
  • Sub-goal 1.1: Biocontainment. Implement failsafes to ensure Astro-Moss cannot survive outside controlled habitats.
  • Sub-goal 1.2: Dual-use Oversight. Screen genetic sequences related to extreme resistance to prevent their use in enhancing harmful pathogens.
• Goal 2: Promote Equity and Global Autonomy. Ensuring that the foundational tools for space colonization are not monopolized by a single entity.
  • Sub-goal 2.1: Open Standards. Establish international protocols for safety data sharing.
  • Sub-goal 2.2: Technology Access. Ensure community biolabs and international researchers can contribute to and benefit from the project.

3. Governance Actions Matrix

4. Detailed Scoring Matrix

Score: 1 (Best/Most effective) to 3 (Least effective/Most problematic)

5. Prioritization, Recommendation, and Ethical Reflections

Target Audience: The United Nations Office for Outer Space Affairs (UNOOSA) and the International Space Exploration Coordination Group (ISECG).

Recommendation Strategy: I prioritize a Hybrid Governance Model that mandates Technical Biocontainment (Action 1) as a prerequisite for mission approval, supported by an International Open-Source Safety Alliance (Action 3) to manage global compliance, safety standards, and equity.

Justification based on Scoring: While DNA Screening (Action 2) provides an immediate defensive layer, our scoring matrix reveals its significant failure in Response and Equity. For a frontier technology like Astro-Moss, we cannot rely on blocking orders alone.

• Action 1 is prioritized because it offers intrinsic safety; a biological kill-switch ensures that even if a containment breach occurs on Earth or a target planet, the organism’s impact is self-limiting.
• Action 3 is essential to prevent the safety requirements of Action 1 from becoming a proprietary wall. By open-sourcing safety modules, we ensure that the Right to Explore remains equitable across all nations.

Ethical Trade-offs Considered:

1. Safety vs. Adaptability: The most robust kill-switch inherently limits the moss’s ability to evolve and survive in truly unknown extraterrestrial variables. I am consciously trading off mission success probability for guaranteed planetary protection.
2. Innovation Speed vs. Regulation: Mandatory international oversight through an Alliance will slow the deployment of Bio-textiles. However, the risk of a Forward Contamination event (accidentally destroying potential Martian microbial life) would set the entire field of synthetic biology back by decades due to the resulting ethical crisis and public outcry.

Assumptions and Uncertainties:

• Assumption of Genetic Stability: This strategy assumes that the complex kill-switch will not be silenced by the high-radiation environments of space. If the mutation rate exceeds our containment design, the technical strategy fails.
• Uncertainty of Extraterrestrial Interaction: We lack data on how engineered genes might interact with potential Martian life. This unknown unknown makes the Biological Footprint Audit (drawing from the DIYBio expert model) a critical necessity to ensure independent, peer-reviewed validation of every mission’s biocontainment integrity.

Week 2 Lecture Prep

Homework Questions from Professor Jacobson

Q1: 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?

Native DNA polymerase has a baseline error rate of approximately 10^{-7 to 10}-8 per base pair. When combined with biological proofreading and mismatch repair mechanisms, the final error rate is reduced to about 10^-10. Given the human genome consists of 3.2 billion base pairs, these high fidelity repair systems ensure that fewer than one error occurs per replication cycle. Biology utilizes these multi layered verification systems to bridge the gap between high data volume and required synthesis accuracy.

Q2: 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?

For a protein of average length, the redundancy of the genetic code allows for over 10^190 different DNA sequence combinations. In practice, the vast majority of these sequences are non functional. Major constraints include codon usage bias leading to translation stalls, the formation of complex mRNA secondary structures that block ribosomal movement, and the accidental creation of regulatory motifs or splice signals that interfere with proper expression.

Homework Questions from Dr. LeProust

Q1: What’s the most commonly used method for oligo synthesis currently?

The most widely utilized method currently is phosphoramidite chemical synthesis.

Q2: Why is it difficult to make oligos longer than 200nt via direct synthesis?

The primary barrier is that the coupling efficiency of each synthesis step never reaches 100 percent. As the nucleotide chain grows, cumulative errors cause the final product yield to drop exponentially. This results in an accumulation of truncated sequences that make the target full length sequence extremely difficult to isolate.

Q3: Why can’t you make a 2000bp gene via direct oligo synthesis?

Direct chemical synthesis of a 2000bp gene results in a yield that is effectively zero. The standard industry approach involves synthesizing multiple shorter oligonucleotide fragments and then using enzymatic assembly techniques to stitch them into a complete gene.

Homework Questions from George Church

Q1: What are the 10 essential amino acids in all animals and how does this affect your view of the “Lysine Contingency”?

The ten essential amino acids for animals are phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, histidine, arginine, leucine, and lysine.

Lysine contingency as a biological safety measure is fundamentally fragile. Since lysine is already a naturally essential amino acid for animals, engineering an organism to be unable to synthesize it merely replicates a natural state rather than adding a secure safeguard. In real world environments, engineered organisms can easily bypass this restraint by consuming lysine from common plants or animal tissues. Effective biocontainment should instead rely on synthetic dependencies not found in nature. An example is semantic containment where organisms require non canonical amino acids provided only in a controlled lab setting to ensure they cannot survive in the wild.

Week 2: DNA Read, Write, & Edit

DNA Design Challenge

3.1 Protein Choice

I chose pHluorin (superecliptic pHluorin, SEP) because it makes pH changes visible as a clear signal. pH is a broadly meaningful indicator across scales: it can reflect cellular and bodily conditions (e.g., local acidity in compartments or stress-related microenvironments) and also environmental conditions (e.g., water/soil toxicity or chemical shifts). This makes SEP useful not only as a biosensing concept, but also as an interaction metaphor. Even small changes in conditions can switch what becomes visible.

>AAS66682.1 superecliptic pHluorin [synthetic construct]
MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQC
FSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHK
LEYNYNDHQVYIMADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKD
PNEKRDHMVLLEFVTAAGITHGMDELYK

3.2 Reverse Translate: Protein (amino acid) sequence to DNA (nucleotide) sequence.

To obtain the precise nucleotide sequence for superecliptic pHluorin (SEP), I performed a targeted search and retrieval process using the NCBI:

1. Protein Identification: I accessed the official NCBI Protein record for superecliptic pHluorin (SEP) under accession number AAS66682.1.
2. Nucleotide Record Location: On the protein record page, I utilized the “Nucleotide sequence from coding region” link found under the Related information sidebar. This link redirected me to the NCBI Nucleotide (nuccore) entry AY533296.1, titled “Synthetic construct superecliptic pHluorin mRNA, complete cds”.
3. CDS Feature Selection: Within the nuccore page, I specifically identified the CDS (coding region) segment to isolate the exact sequence responsible for protein translation.
4. FASTA Retrieval: I displayed the sequence in FASTA format (ensuring the header line starts with >) to maintain compatibility with downstream design tools.
5. Sequence Capture: I copied the nucleotide CDS sequence (bases 1..717) as the foundational DNA template for the SEP protein.

>lcl|AY533296.1_cds_AAS66682.1_1 [protein=superecliptic pHluorin] [protein_id=AAS66682.1] [location=1..717] [gbkey=CDS]
ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATG
GGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTAT
TTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTAACTTATGGTGTTCAATGC
TTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTAC
AGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGA
TACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAA
TTGGAATACAACTATAACGATCACCAGGTGTACATCATGGCAGACAAACAAAAGAATGGAATCAAAGCTA
ACTTCAAAATTAGACACAACATTGAAGATGGAGGCGTTCAACTAGCAGACCATTATCAACAAAATACTCC
AATTGGCGATGGGCCCGTCCTTTTACCAGACAACCATTACCTGTTTACAACTTCTACTCTTTCGAAAGAT
CCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGG
ATGAACTATACAAATAA

• 3.2 & 3.3 Reverse Translation & Optimization: The sequence was reverse-translated and codon-optimized for E. coli expression to ensure high yield while avoiding Type IIs restriction sites (BsaI, BsmBI, BbsI).
• DNA Sequence:

Prepare a Twist DNA Synthesis Order

I designed an expression cassette to be synthesized as a clonal gene.

• Design Components: J23106 Promoter, B0034 RBS, Optimized sfGFP, 7xHis Tag, and B0015 Terminator.
• Vector Selection: pTwist Amp High Copy. This circular backbone allows for direct transformation into E. coli, speeding up the experimental cycle by 1-2 weeks.

Figure 1: Annotated sfGFP expression cassette designed in Benchling.

Part 5: DNA Read/Write/Edit

Inspired by Marina Otero Verzier’s theory, this documentation explores a move away from the Cartesian enclosure of traditional data centers. I propose using Engineered Moss as a living fabric and information archive for extraterrestrial exploration.

5.1 DNA Read: Material Entanglement

• Technology: Nanopore Sequencing.
• Vision: Reading the Moss. This transcends binary retrieval; it is an act of “touching a leaf” to listen to the archive. Nanopore allows us to decode how cosmic radiation and human interaction have “co-written” the DNA, creating a material entanglement where the building, the document, and the environment are one.

5.2 DNA Write: The Breathing Library

• Technology: Enzymatic DNA Synthesis.
• Vision: Encoding human collective memory into moss spores.
• Theory: This breaks the “myth of endless growth.” Moss archives are non-hierarchical; as they flourish on alien regolith, they capture CO2 and produce oxygen. It is an architecture that “makes breath possible”—an archive that is both alive and gives life.

5.3 DNA Edit: Ontological Freedom

• Technology: CRISPR/Cas9.
• Vision: Editing moss for extreme desiccation and radiation tolerance.
• Ethics: This edit is not an extractive tool for profit. We grant the moss ontological freedom to express its agency in a new world. It is an act of solidarity and cohabitation between species, allowing the “Data Forest” to germinate in the inconceivable futures of space.