Week 2 HW: DNA Read, Write, Edit — SOD1 Molecular Journey

🧬 Week 2 Documentation

DNA Read → DNA Write → DNA Edit

A Molecular Design Journey

This week was not just a technical exercise. It was an exploration — from abstract sequence to physical plasmid, from conceptual art to molecular execution. Below is the full documentation of my process, including failures, iterations, and insights gained.

🧪 Part 0: Basics of Gel Electrophoresis

Lectures + Recitation

I attended/watched all required lecture and recitation materials.

Conceptual Understanding

Gel electrophoresis separates DNA fragments based on size using:

  • Negatively charged DNA backbone
  • Electric field
  • Agarose matrix
  • Size-dependent migration

Smaller fragments travel further.

🎨 Part 1: Benchling & In-silico Gel Art

Step 1: Benchling Account + Lambda DNA Import

  • Created Benchling account
  • Imported Lambda DNA reference sequence

Step 2: Simulated Restriction Digestion

Enzymes used:

  • EcoRI
  • HindIII
  • BamHI
  • KpnI
  • EcoRV
  • SacI
  • SalI

Initial Failure

My first digestion simulation produced fragmented bands that were too similar in size. The pattern looked visually indistinct.

Iteration Strategy

  • Tested different single and double digests
  • Compared fragment size outputs
  • Adjusted enzyme combinations

Eventually, I selected combinations that produced strong band separation.

Kindly find attach all the simulations carried out for the same task:

The following image represents setting up the Benchling account and loading lambda sequence, ultimately I was able to visualize as shown here- Figure1 Figure1

The following image shows the end result after carrying out the digestion process, I worked on a pattern design of “H Letter”, reason being my startup company’s first letter is H! Although, I must say I struggled alot and I intend to re run all of these simulations and tasks at least 5-6 times! Figure2 Figure2

🧪 In-Silico Gel Art

I did try to work out on gel art, but yet again this part of the homework was something I really struggled.

In Silico Gel In Silico Gel

Insight

Never had I imagined that biological mechanisms could generate such striking and beautiful art forms. As someone who once dreamed of becoming an artist but ultimately pursued engineering, I find this intersection deeply exciting. Working with gel patterns and molecular design has rekindled a childhood aspiration I once held close — the dream of opening an art studio.

🧬 Part 3: DNA Design Challenge

3.1 Choose Your Protein

Selected Protein: Human Superoxide Dismutase 1 (SOD1)

UniProt ID: P00441

sp|P00441|SODC_HUMAN
Superoxide dismutase [Cu-Zn]
OS=Homo sapiens OX=9606 PE=1 SV=2

Why SOD1?

SOD1 converts:

O₂⁻ → O₂ + H₂O₂

It protects against oxidative stress and is implicated in ALS.

It also integrates mechanistically with my LungLite platform — serving as a biochemical actuator.

Kindly find attached an image of the protein sequence:
protein protein

Amino Acid Sequence

MVKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTA
GCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDH
CIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ

3.2 Reverse Translation

Using online reverse translation tools, I generated a nucleotide sequence.

Failure

Reverse translation produced multiple valid sequences due to codon degeneracy.

There is no single “correct” DNA sequence for a protein.

Resolution

I selected one biologically valid version as a starting template.

Pre-optimization DNA:

atggtgaaagcggtgtgcgtgctgaaaggcgatggcccggtgcagggcattattaacttt...
Kindly find attached images showing conversion of amino acid sequences to dna sequence (extremely interesting!):
dna dna

dna dna

3.3 Codon Optimization

Why Optimize?

Different organisms prefer specific codons due to tRNA abundance.

Without optimization:

  • Ribosome stalling
  • Low yield
  • Translation inefficiency

Host Chosen: Escherichia coli

Reasons:

  • Fast growth
  • High recombinant yield
  • Standard lab organism

Final Codon Optimized Sequence

ATGGTTAAAGCGGTATGCGTGCTGAAAGGCGATGGCCCGGTGCAGGGCATTATTAACTTT
GAACAGAAAGAATCAAACGGCCCGGTGAAAGTGTGGGGCAGCATTAAAGGCCTGACCGA
AGGTCTGCACGGCTTTCACGTGCATGAATTTGGCGATAACACCGCGGGCTGCACCAGCG
CCGGCCCGCATTTTAACCCGCTGAGCCGCAAACATGGCGGCCCGAAAGATGAAGAACGCC
ATGTGGGCGATCTGGGCAATGTGACCGCGGATAAAGATGGCGTGGCCGATGTGAGCATT
GAAGATAGCGTGATTAGCCTGAGCGGCGATCATTGCATTATTGGCCGCACCCTGGTTGT
TCATGAAAAAGCAGATGATCTGGGCAAAGGCGGCAACGAAGAAAGCACCAAAACCGGCA
ATGCGGGGAGCCGCCTGGCGTGCGGCGTGATTGGCATCGCCCAG
codon codon
Loading the above sequence directly on benchling platform and visualizing it:
dna dna

3.4 From DNA to Protein

Expression Methods:

Cell-Dependent

  1. Transform plasmid into E. coli
  2. Antibiotic selection
  3. Transcription
  4. Translation
  5. His-tag purification

Cell-Free Option

  • TX-TL system
  • Direct protein production without cells
Building the expression cassette:
dna dna
Create a digital diagram of above cassette:
dna dna

3.5 Central Dogma Alignment

DNA:

ATG GTT AAA GCG

RNA:

AUG GUU AAA GCG

Protein:

Met Val Lys Ala

Each 3 nucleotides = 1 amino acid
T → U during transcription

🧬 Part 4: Prepare a Twist DNA Synthesis Order

4.1 Accounts

  • Created Twist account
  • Created Benchling account

4.2 Build Expression Cassette

Structure:

Promoter
RBS
ATG
SOD1 Coding Sequence
7x His Tag
TAA
Terminator

Failure

Initially forgot to annotate regions in Benchling.

Fix

Annotated:

  • Promoter
  • RBS
  • CDS
  • His Tag
  • Terminator

Verified via Linear Map view.

Final Insert Sequence

>SOD1_LungLite_Expression_Cassette
TTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGCCATTAAAGAGGAGAAAGGTACCATG
GTTAAAGCGGTATGCGTGCTGAAAGGCGATGGCCCGGTGCAGGGCATTATTAACTTTGA
ACAGAAAGAATCAAACGGCCCGGTGAAAGTGTGGGGCAGCATTAAAGGCCTGACCGAAGG
TCTGCACGGCTTTCACGTGCATGAATTTGGCGATAACACCGCGGGCTGCACCAGCGCCG
GCCCGCATTTTAACCCGCTGAGCCGCAAACATGGCGGCCCGAAAGATGAAGAACGCCAT
GTGGGCGATCTGGGCAATGTGACCGCGGATAAAGATGGCGTGGCCGATGTGAGCATTGA
AGATAGCGTGATTAGCCTGAGCGGCGATCATTGCATTATTGGCCGCACCCTGGTTGTTC
ATGAAAAAGCAGATGATCTGGGCAAAGGCGGCAACGAAGAAAGCACCAAAACCGGCAAT
GCGGGGAGCCGCCTGGCGTGCGGCGTGATTGGCATCGCCCAGCATCACCATCACCATC
ATCACTAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTT
TTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGT
GGGCCTTTCTGCGTTTATA

4.3–4.6 Twist Order

Selected:

  • Genes → Clonal Genes
  • Vector: pTwist Amp High Copy

Imported GenBank file back into Benchling to confirm construct.

I built my first plasmid.

The images document the workflow: exporting a FASTA file from Benchling, creating a Twist Bioscience account, (hypothetically) placing an order by selecting Clonal Gene, downloading the resulting gene construct file (a .gb / GenBank file) from the Twist platform, and then uploading that same file back into Benchling.
final finalfinal finalfinal final

🧬 Part 5: DNA Read / Write / Edit

5.1 DNA Read

What Would I Sequence?

The SOD1 gene sequence to understand its structure, variants, and oxidative stress relevance in lung epithelial biology.

Why This Matters

Superoxide Dismutase 1 (SOD1) is a cytosolic antioxidant enzyme that catalyzes the conversion of superoxide radicals (O₂⁻) into oxygen and hydrogen peroxide. Because oxidative stress is central to airway inflammation, SOD1 represents the molecular boundary between resilience and pathology in lung tissue. Mutations in SOD1 are linked to Amyotrophic Lateral Sclerosis (ALS), and its structure and function are well-characterized, making it ideal for recombinant engineering and diagnostic integration.

Technology Chosen: Oxford Nanopore

Generation: Third-generation sequencing

Input:

  • Extracted DNA containing SOD1
  • Adapter ligation

Mechanism:

  • DNA passes through nanopores
  • Ionic current changes → base calling

Output:

  • FASTQ long reads of SOD1 sequence

Why Nanopore?

  • Long reads allow full-length SOD1 sequencing
  • Detects structural variants and potential regulatory regions
  • Portable and scalable

Limitations:

  • Higher error rate than Illumina
  • Correctable with sequencing depth and consensus alignment

5.2 DNA Write

What Would I Synthesize?

A codon-optimized SOD1 expression cassette and ROS-responsive genetic circuits for LungLite.

Rationale

To integrate SOD1 into LungLite, the gene must be optimized for expression in bacterial or cell-free systems. This enables recombinant production and functional embedding into oxidative stress detection circuits.

Technology

  • Phosphoramidite oligo synthesis
  • PCR assembly
  • Clonal gene insertion into expression vector
  • 7×His tag for purification

Application in LungLite

  1. Biological Amplifier Strategy

    • ROS activates redox-sensitive promoter
    • Induces SOD1 expression in freeze-dried TX–TL system
    • SOD1 converts superoxide → H₂O₂
    • Coupled colorimetric/fluorescent reaction produces smartphone-readable signal

  2. Calibration Standard Strategy

    • Purified recombinant SOD1 embedded in microfluidic wells
    • Known concentrations normalize ROS dye response
    • Enables quantitative oxidative stress scoring

Limitations

  • Length constraints in synthesis
  • Synthesis errors
  • Cost scaling for large constructs

5.3 DNA Edit

What Would I Edit?

Upregulate antioxidant pathways — including SOD1 expression — in lung epithelial cells.

Technology: CRISPR-Cas9

Steps

  1. gRNA design targeting regulatory region
  2. Cas9-induced double-strand break
  3. HDR-mediated repair with enhanced promoter template

Input:

  • gRNA plasmid
  • Cas9
  • Donor DNA template
  • Target lung epithelial cells

Goal

Increase endogenous SOD1 buffering capacity to restore redox balance in oxidative stress conditions.

Limitations

  • Off-target effects
  • Variable editing efficiency
  • Delivery challenges in airway epithelium

🌬 Final Reflection

What began as:

Lambda DNA
→ Restriction digest
→ Gel electrophoresis

Evolved into:

DNA Read → Sequencing SOD1
DNA Write → Engineering ROS-responsive SOD1 circuits
DNA Express → Recombinant protein production
DNA Integrate → Embedding SOD1 into LungLite microfluidic diagnostics

SOD1 is not merely a recombinant protein in this project. It becomes a functional biochemical actuator — translating environmental oxidative exposure into measurable signal output.

Growing up in Delhi, where severe air pollution makes oxidative stress a daily lived experience, reframes SOD1 from an abstract enzyme to a molecular proxy for environmental exposure. LungLite transforms this molecular logic into a portable, AI-integrated, noninvasive public health device.

The DNA Design Challenge is no longer just molecular cloning — it becomes the foundation for a programmable redox-sensing health platform.

I acknowledge that I used artificial intelligence tools, including ChatGPT-5.0, for language refinement, structural organization, and improvement of clarity in this documentation.

All scientific concepts, experimental designs, sequence selections, analytical reasoning, and technical interpretations presented in this work reflect my own understanding and independent effort. The AI tool was used solely to enhance readability, coherence, grammar, and overall presentation quality.

The prompts primarily included instructions such as: “Rewrite the text and correct grammatical errors.”