Part 5: DNA Read, Write, & Edit
Part 5: DNA Read, Write, & Edit
Answers framed around the BioVolt DIY electroporation pipeline: plasmid amplification → transformation → PCR verification → gel electrophoresis. What DNA would we read, write, and edit to make this frugal pipeline sing?
5.1 DNA Read
(i) What DNA would you want to sequence and why?
In the BioVolt pipeline: After electroporation, we transform E. coli with plasmids (e.g., sfGFP expression cassette). We run post-transformation PCR and gel electrophoresis to infer success—but we don’t know the exact sequence. Sequencing the plasmid (or PCR amplicon) confirms that:
- The insert is correct (no truncations, no wrong gene)
- Electroporation didn’t introduce mutations (high voltage can stress DNA)
- The expression cassette is intact for downstream experiments
Broader applications (aligned with BioVolt’s democratization goals):
- Environmental monitoring — e.g., sewage/wastewater DNA for microbiome analysis in Panama; biodiversity surveys
- Human health — disease-associated genes, pharmacogenomics
- DNA data storage — archival sequences in synthetic DNA
- Biobank validation — verifying stored samples
(ii) What technology would you use and why?
Technology chosen: Oxford Nanopore (MinION) — third-generation sequencing
Why Nanopore for BioVolt / frugal labs:
- Portable — USB-sized device; runs on laptop; fits in a backpack. Ideal for Panama, field sites, or home labs.
- Real-time — base calling as reads stream; no batch wait.
- Long reads — can span full plasmids; fewer assembly gaps.
- Low capital — compared to Illumina, much cheaper to get started.
- No PCR required for some workflows — direct DNA sequencing possible (native DNA).
| Question | Answer |
|---|---|
| Output? | FASTQ files (reads + quality scores); can be base-called in real time to BAM/FASTA. |
| Essential steps & base calling? | (1) DNA passes through a nanopore; (2) each base disrupts ionic current differently; (3) base caller (e.g., Guppy) converts current traces → A/T/G/C; (4) reads assembled/compared to reference. |
| Input & preparation? | Option A (PCR amplicon): PCR product → end-prep → adapter ligation → load onto flow cell. Option B (native): Fragment DNA (e.g., g-TUBE or sonication) → repair ends → adapter ligation → load. Key: adapters enable motor protein to thread DNA through pore. |
| First-, second-, or third-generation? | Third-generation. Single-molecule, real-time; no amplification required for some lib preps; long reads; portable form factor. |
5.2 DNA Write
(i) What DNA would you want to synthesize and why?
For BioVolt: The expression cassettes we electroporate! Specifically:
- sfGFP plasmid — promoter + RBS + sfGFP CDS + terminator (e.g., BBa_J23106, BBa_B0034, sfGFP, BBa_B0015). This is the “make E. coli glow green” construct we build in Part 4.
- Custom reporters — e.g., biosensors that fluoresce in response to environmental cues (pH, metals, toxins) for citizen-science monitoring.
- Validation controls — known sequences for PCR/gel positive controls in the frugal pipeline.
Broader: Therapeutics (mRNA vaccines), genetic circuits, DNA origami, gene clusters for metabolic engineering.
(ii) What technology would you use and why?
Technology: Column-based phosphoramidite synthesis (e.g., Twist Bioscience, IDT) — the industry standard for gene synthesis.
Why: High fidelity, scalable, cost-effective for genes and gene fragments. Twist can deliver clonal genes (circular) ready for transformation—perfect for BioVolt.
| Question | Answer |
|---|---|
| Limitations? | Speed: days to weeks. Accuracy: ~1 error per 1–3 kb; may need sequencing to confirm. Scalability: great for genes; whole genomes get expensive. Length: very long constructs may need assembly. |
| Essential steps? | (1) Design sequence (e.g., codon-optimized); (2) split into overlapping oligos; (3) synthesize oligos (phosphoramidite chemistry, base-by-base); (4) assemble oligos (PCR, Gibson, or enzymatic); (5) clone into vector; (6) sequence to verify. |
5.3 DNA Edit
(i) What DNA would you want to edit and why?
For BioVolt:
- Improve electroporation efficiency — edit E. coli to knock out or modify genes that affect membrane composition, cell wall, or DNA repair (e.g., recA, mutS) to get more transformants per zap.
- Biosensor chassis — edit strains to express reporter circuits (e.g., GFP under metal-responsive promoter) for environmental sensing in the DIY pipeline.
- Safety — auxotrophic markers, kill switches, or containment edits for responsible DIYbio.
Broader: Human therapeutics (e.g., sickle cell), agriculture (nitrogen fixation, disease resistance), conservation (genetic rescue), longevity research.
(ii) What technology would you use and why?
Technology: CRISPR/Cas9 (with HDR for precise edits) — or base editors for single-nucleotide changes without double-strand breaks.
Why: Programmable, precise, widely adopted. gRNA design is straightforward; many tools (Benchling, etc.) support it.
| Question | Answer |
|---|---|
| Limitations? | Efficiency: not 100%; mixed populations. Precision: off-target cuts possible; PAM requirement constrains target sites. Delivery: need to get Cas9 + gRNA into cells (electroporation works!). |
| Preparation & input? | Design: gRNA(s) targeting locus; donor template (ssODN or plasmid) for HDR. Input: DNA template, Cas9 nuclease, gRNA (or plasmid expressing both), cells. Optional: base editor (e.g., ABE, CBE) for point mutations. |
| Essential steps? | (1) Design gRNA (avoid off-targets; check PAM, e.g., NGG for SpCas9); (2) deliver Cas9 + gRNA + donor (electroporation, conjugation, etc.); (3) Cas9 cuts DNA; (4) cell repairs via NHEJ or HDR; (5) screen for edits (PCR, sequencing). |