DNA read/write/edit

1. DNA Read

I want to sequence the DNA of the firefly luciferase because I want to create a bioart installation that displays bioluminescent menstrual blood.

For sequencing a single known gene like firefly luciferase, Sanger sequencing is recommended. Sanger is first-generation, adapted for small, defined targets. It is fast and cost effective.

The input would be the firefly luciferase gene from a plasmid containing it or amplified directly from a DNA library.

Essential preparation steps: PCR amplification, purification, quantification, and primer selection.

Sanger sequencing steps: (1) Sequencing PCR reaction (2) Capillary electrophoresis (3) Base calling.

The output is a chromatogram file containing: the raw fluorescence trace (coloured peaks, one per base) a called nucleotide sequence (~600–900 usable bases per read) a quality score for each base position

2. DNA Write

I would like to synthesize a genetic construct based on the firefly luciferase gene from Photinus pyralis.

Rationale

Luciferase is one of the most widely used reporter genes in molecular biology because it catalyzes a light-producing reaction. I am interested in using this gene because bioluminescence creates a striking visual effect while also symbolically transforming menstrual blood, something culturally stigmatized, into a glowing, living artwork.

The project sits at the intersection of synthetic biology, feminist bioart, and molecular sensing. Rather than treating menstrual blood as medical waste, the installation would reframe it as biologically active and aesthetically meaningful material.

DNA Construct Design

I would synthesize a plasmid containing:

  • Firefly luciferase gene (luc2)
  • Promoter
  • Biosensor regulatory elements responsive to hormones

Firefly luciferase sequence:

1 ctgcagaaat aactaggtac taagcccgtt tgtgaaaagt ggccaaaccc ataaatttgg 61 caattacaat aaagaagcta aaattgtggt caaactcaca aacattttta ttatatacat 121 tttagtagct gatgcttata aaagcaatat ttaaatcgta aacaacaaat aaaataaaat 181 ttaaacgatg tgattaagag ccaaaggtcc tctagaaaaa ggtatttaag caacggaatt 241 cctttgtgtt acattcttga atgtcgctcg cagtgacatt agcattccgg tactgttggt 301 aaaatggaag acgccaaaaa cataaagaaa ggcccggcgc cattctatcc tctagaggat 361 ggaaccgctg gagagcaact gcataaggct atgaagagat acgccctggt tcctggaaca 421 attgcttttg tgagtatttc tgtctgattt ctttcgagtt aacgaaatgt tcttatgttt 481 ctttagacag atgcacatat cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc 541 gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta 601 tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt 661 gcagttgcgc ccgcgaacga catttataat gaacgtaagc accctcgcca tcagaccaaa 721 gggaatgacg tatttaattt ttaaggtgaa ttgctcaaca gtatgaacat ttcgcagcct 781 accgtagtgt ttgtttccaa aaaggggttg caaaaaattt tgaacgtgca aaaaaaatta 841 ccaataatcc agaaaattat tatcatggat tctaaaacgg attaccaggg atttcagtcg 901 atgtacacgt tcgtcacatc tcatctacct cccggtttta atgaatacga ttttgtacca 961 gagtcctttg atcgtgacaa aacaattgca ctgataatga attcctctgg atctactggg 1021 ttacctaagg gtgtggccct tccgcataga actgcctgcg tcagattctc gcatgccagg 1081 tatgtcgtat aacaagagat taagtaatgt tgctacacac attgtagaga tcctattttt 1141 ggcaatcaaa tcattccgga tactgcgatt ttaagtgttg ttccattcca tcacggtttt 1201 ggaatgttta ctacactcgg atatttgata tgtggatttc gagtcgtctt aatgtataga 1261 tttgaagaag agctgttttt acgatccctt caggattaca aaattcaaag tgcgttgcta 1321 gtaccaaccc tattttcatt cttcgccaaa agcactctga ttgacaaata cgatttatct 1381 aatttacacg aaattgcttc tgggggcgca cctctttcga aagaagtcgg ggaagcggtt 1441 gcaaaacggt gagttaagcg cattgctagt atttcaaggc tctaaaacgg cgcgtagctt 1501 ccatcttcca gggatacgac aaggatatgg gctcactgag actacatcag ctattctgat 1561 tacacccgag ggggatgata aaccgggcgc ggtcggtaaa gttgttccat tttttgaagc 1621 gaaggttgtg gatctggata ccgggaaaac gctgggcgtt aatcagagag gcgaattatg 1681 tgtcagagga cctatgatta tgtccggtta tgtaaacaat ccggaagcga ccaacgcctt 1741 gattgacaag gatggatggc tacattctgg agacatagct tactgggacg aagacgaaca 1801 cttcttcata gttgaccgct tgaagtcttt aattaaatac aaaggatatc aggtaatgaa 1861 gatttttaca tgcacacacg ctacaatacc tgtaggtggc ccccgctgaa ttggaatcga 1921 tattgttaca acaccccaac atcttcgacg cgggcgtggc aggtcttccc gacgatgacg 1981 ccggtgaact tcccgccgcc gttgttgttt tggagcacgg aaagacgatg acggaaaaag 2041 agatcgtgga ttacgtcgcc agtaaatgaa ttcgttttac gttactcgta ctacaattct 2101 tttcataggt caagtaacaa ccgcgaaaaa gttgcgcgga ggagttgtgt ttgtggacga 2161 agtaccgaaa ggtcttaccg gaaaactcga cgcaagaaaa atcagagaga tcctcataaa 2221 ggccaagaag ggcggaaagt ccaaattgta aaatgtaact gtattcagcg atgacgaaat 2281 tcttagctat tgtaatatta tatgcaaatt gatgaatggt aattttgtaa ttgtgggtca 2341 ctgtactatt ttaacgaata ataaaatcag gtataggtaa ctaaaaa

In practice, I would likely use a commercially optimized luciferase variant.

Potential Extensions

The project could evolve beyond a simple art piece into a collaborative science platform sensing perimenopause.

Future versions might include:

  • Genetic circuits that modulate brightness based on hormonal markers -Cell-free expression systems
  • Multi-color bioluminescent systems using luciferases from marine organisms
  • CRISPR-regulated expression patterns synchronized with hormonal cycles

What technology or technologies would you use to perform this DNA synthesis and why?

The primary technology I would use is phosphoramidite DNA synthesis, which is currently the standard industrial method used by DNA synthesis companies such as Twist Bioscience. I would use this technology because: it is highly precise and commercially accessible, it allows custom-designed DNA sequences, it supports codon optimization and it is scalable for synthetic biology applications.

Essential Steps of the DNA Synthesis Process:

  1. Digital Sequence Design
  2. Oligonucleotide Synthesis : the DNA synthesis machine chemically adds nucleotides sequentially using phosphoramidite chemistry. Because this process is most reliable for short sequences, the construct is synthesized as many smaller oligonucleotides.
  3. DNA Assembly. the short DNA fragments are assembled into a larger construct using methods such as Gibson or Golden Gate Assembly
  4. Sequence Verification: after assembly, the construct would be verified using DNA sequencing to confirm that the synthesized sequence matches the intended design.

Although modern DNA synthesis is powerful, there are several limitations:

  • Sequence Length : chemical synthesis becomes less accurate with longer DNA strands because each nucleotide addition introduces a small probability of error.
  • Error Rates : errors such as deletions, insertions, or substitutions can occur during synthesis and assembly.
  • Speed: although synthesis is much faster than older cloning methods, complex constructs can still require several days to weeks for commercial synthesis, as well as additional time for verification and troubleshooting.
  • Cost: longer or more complex DNA constructs can become expensive.

3. DNA Edit

I want to edit the firefly luciferase gene from Photinus pyralis in order to alter the color of its bioluminescence from the natural yellow-green wavelength toward red emission. I am interested in editing this DNA because red bioluminescence would create a more visceral and blood-like visual effect for my bioart installation involving menstrual blood. Conceptually, the color shift would reinforce themes of embodiment, menstruation, and intimacy.

The exact color of the emitted light depends on the structure of the luciferase protein and the chemical environment of the reaction. I would engineer mutations associated with red-shifted luminescence. Previous studies have shown that certain amino acid substitutions in luciferase can shift emission wavelengths significantly toward orange and red light.

What Technology Would I Use to Perform These DNA Edits and Why?

To edit the luciferase gene, I would use CRISPR-Cas9 combined with site-directed mutagenesis and synthetic DNA assembly techniques.

These technologies are appropriate because they allow precise modification of specific nucleotides within a gene, making it possible to engineer targeted amino acid substitutions.

Essential steps:

  1. Guide RNA Design: a guide RNA is designed to target a specific region of the luciferase gene near the amino acid residues to be edited.
  2. Cas9 DNA Cleavage: the Cas9 protein binds the guide RNA and cuts the DNA at the target location.
  3. DNA Repair with Desired Mutation.
  4. Verification: the edited DNA is sequenced to confirm the mutations were introduced correctly.

Site-Directed Mutagenesis

Because luciferase is relatively small, another efficient strategy is PCR-based site-directed mutagenesis. This method uses specially designed primers containing the desired mutations, amplifies the plasmid DNA, and creates a new edited version of the gene. This approach is commonly used in protein engineering because it is fast, inexpensive, and highly precise for small edits.

Preparation

  1. Identify Target Mutations to shift luciferase emission toward red wavelengths.
  2. Computational Sequence Design: the edited DNA sequence need to be designed digitally to maintain protein stability, preserve enzymatic activity, and optimize mammalian codon usage.
  3. Guide RNA or Primer Design

Although CRISPR and mutagenesis are powerful technologies, they have several limitations:

  1. Off-Target Effects: CRISPR-Cas9 can sometimes cut unintended DNA sequences if the guide RNA partially matches other regions.
  2. Editing Efficiency: Precise edits using HDR are often less efficient than simple gene disruption.
  3. Protein Stability Tradeoffs: Mutations that shift light color can reduce enzyme brightness, thermal stability, or overall activity.
  4. Scalability: editing a few mutations is relatively straightforward, but engineering complex genetic circuits becomes increasingly difficult due to cloning complexity, regulatory interactions, and sequence optimization challenges.
  5. Biological Context Dependence: Luciferase color can also depend on pH, temperature, co-factors, and intracellular environment. This means the same edited luciferase may produce different colors in different systems.

Due to time limitation, this part of the weekly assignments has been generated by ChatGPT without further research work.