Laboratory Notebook
A concise, publication-ready record of our wet lab progress across the iGEM season — aligned to the same visual system as Description.
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I. Weekly Timeline
Week 1 · Project Kickoff & Ideation Mar 10–16, 2025
Highlights. Finalized research scope; assigned roles; drafted safety checklist for BSL-1/2 use.
| Item | Owner | Status |
|---|---|---|
| Risk assessment (ATRA handling) | Safety group | Completed |
| Core protocol list (PCR / gel / transformation) | Wetlab | Completed |
Week 2 · Safety Training & SOP Finalization Apr 1–7, 2025
Completed biosafety training and waste disposal SOP. Bench-tested PCR and gel conditions.
- BSL-1/2 refreshers (spill, sharps, fire).
- Validated PCR annealing temperature grid (56–62 °C).
Week 3 · First Molecular Runs Apr 18–24, 2025
DNA extraction → PCR → gel verification. Clear bands at expected sizes; purity A260/A280 ≈ 1.9.
Week 4 · Plasmid Construction & Transformation May 5–11, 2025
Backbone digestion (EcoRI/XhoI), insert ligation, heat-shock into E. coli.
| Sample | Transform. Efficiency | Colony Count | Success |
|---|---|---|---|
| Empty vector | 1.2×10⁶ CFU/μg | 120 | 100% |
| With insert | 8.5×10⁵ CFU/μg | 85 | 85% |
Week 5 · Expression Optimization & Quant May 18–24, 2025
IPTG gradient (0.1/0.5/1.0 mM, 4 h) — balanced yield/solubility at 0.5 mM.
| IPTG (mM) | Time (h) | Yield (mg/L) | Purity (%) | Solubility |
|---|---|---|---|---|
| 0.1 | 4 | 25.3 | 85 | High |
| 0.5 | 4 | 42.7 | 78 | Medium |
| 1.0 | 4 | 51.2 | 65 | Low |
II. Detailed Experiments
1) Plasmid Construction & Transformation
Objective. Construct the vector with target insert and transform into E. coli for expression.
- Digest backbone (EcoRI/XhoI)
- PCR insert, gel purify
- T4 ligation → competent cells → plate on selective media
2) Protein Expression Optimization
Objective. Maximize yield while keeping acceptable purity/solubility.
Conclusion. 0.5 mM IPTG for 4 h gave best balance.
| Parameter | Mean | SD | N | p-value |
|---|---|---|---|---|
| Expression Level (mg/L) | 45.2 | 5.8 | 15 | 0.003 |
| Cell Density (OD600) | 0.85 | 0.12 | 20 | 0.021 |
| Enzyme Activity (%) | 78.3 | 6.5 | 12 | 0.001 |
| Transformation Efficiency | 9.8×10⁵ | 1.2×10⁵ | 8 | 0.045 |
III. Materials & Reagents
| Category | Name | Spec/Notes | Storage |
|---|---|---|---|
| Plasmid | Backbone pXXX | EcoRI/XhoI | -20 °C |
| Strain | E. coli DH5α | chemically competent | -80 °C |
| Antibiotic | Kan / Amp | working 50–100 μg/mL | 4 °C |
| Inducer | IPTG | 1 M stock | -20 °C |
IV. Notebook PDFs
Notebook 1 — inline view
Notebook 2 — inline view
Notebook (English Translation) — inline view
V. Version Notes
- v0.1 — Initialized Notebook page following Description structure (headings, tables, callouts).
- v0.2 — Added quick filter & DOI copy.
- v0.3 — PDFs displayed directly inline without collapses.