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.
Benchling Design Benchling Design

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.