Results 2025 iGEM · SYPHU-CHINA
Complete ATRA biosynthetic pathway in E. coli: from gene cloning to functional validation.
Executive Summary
- Full genetic stack: all key genes amplified and verified; upstream β-carotene and downstream retinal→ATRA modules constructed and co-expressed.
- Strong chassis performance: high transformation efficiency; robust enzyme expression.
- Functional outputs: β-carotene (upstream) and ATRA (combined strain) detectable.
- Smart control: lactate-responsive induction with wide dynamic range in a physiologically relevant window.
- Operability: plasmid retention >90% over 20 generations under our conditions.
Gene Amplification and Verification
Goal. Obtain clean, size-correct PCR products to maximize downstream assembly efficiency.
Figure A. Gene amplification analysis (gene-amplification-analysis). Representative gels and QC summaries for the four pathway genes.
Interpretation: Band sizes match theoretical lengths within tolerance; A260/A280 indicates high nucleic-acid purity suitable for cloning.
Analytical summary of PCR products
| Gene | Theoretical size | Experimental size | Concentration (ng/μL) | Purity (A260/A280) | Amplification efficiency (%) |
|---|---|---|---|---|---|
| blhSR | 1,812 bp | 1,809 ± 7 | 45.2 ± 3.1 | 1.87 ± 0.03 | 94.3 ± 2.1 |
| raldhHS | 1,521 bp | 1,518 ± 5 | 38.7 ± 2.8 | 1.85 ± 0.02 | 96.1 ± 1.8 |
| IIdR | 897 bp | 894 ± 4 | 52.1 ± 4.2 | 1.89 ± 0.04 | 97.8 ± 1.2 |
| crtEBIY | 5,214 bp | 5,198 ± 12 | 28.3 ± 2.5 | 1.83 ± 0.05 | 85.4 ± 3.5 |
Plasmid Construction and Verification
Strategy. Homologous recombination for upstream (crtEBIY) and downstream (blhSR–raldhHS) modules; validated by colony PCR and sequencing.
Figure B. Plasmid quality analysis (plasmid-quality-analysis). Concentration, purity indices and functional colony screens.
Interpretation: High A260/A230 & A260/A280 and >108 CFU/μg transformation support plasmid integrity; positive-clone rate >88% confirms efficient assembly.
| Plasmid construct | Conc. (ng/μL) | A260/A280 | A260/A230 | Transform. efficiency (CFU/μg) | Positive clones (%) |
|---|---|---|---|---|---|
| Downstream pathway | 68.5 ± 5.2 | 1.86 ± 0.02 | 2.15 ± 0.05 | (2.8 ± 0.3)×10⁸ | 92 ± 3 |
| Upstream pathway | 72.3 ± 4.8 | 1.84 ± 0.03 | 2.12 ± 0.07 | (2.5 ± 0.2)×10⁸ | 88 ± 4 |
| Acceptance criteria | >50 | 1.8–2.0 | >2.0 | >1×10⁸ | >80 |
Protein Expression and Functional Characterization
Result. SDS-PAGE confirmed robust expression at expected MW. Induction at 25–30 °C balanced total expression and solubility.
Figure C. Temperature optimization (temperature-optimization-analysis). Solubility vs. total expression across induction temperatures.
Interpretation: 25–30 °C yields the best compromise for active enzymes supporting downstream flux.
Expression & activity metrics
| Enzyme | Theor. MW (kDa) | Expression | Solubility | Specific activity (U/mg) | Note |
|---|---|---|---|---|---|
| BlhSR | 67.8 | Strong | 68 ± 5% soluble | 12.3 ± 1.2 | Drives retinal formation |
| RALDH | 55.1 | Strong | 75 ± 4% soluble | 8.7 ± 0.9 | Terminal oxidation to ATRA |
| CrtE/B/I/Y | 20–44 | Moderate | Membrane-associated | N/A | β-carotene confirmed |
Metabolic Pathway Functionality
Outcome. Flux from β-carotene to retinal and ATRA was confirmed using phenotype and LC–MS quantification.
Figure D. Static metabolic pathway (static-metabolic-pathway). Upstream carotenoid & downstream retinoid modules overview.
Context: The schematic maps measured intermediates to specific enzymatic steps.
Figure E. Metabolite production (metabolite-production-analysis). Module-wise outputs vs full pathway.
Interpretation: Retinal >> ATRA implies RALDH oxidation is rate-limiting → priority for enzyme/cofactor engineering.
Figure F. Production kinetics (production-kinetics-analysis). Growth-associated β-carotene formation across 24 h.
Interpretation: Peak volumetric productivity near transition to stationary phase → informs induction timing for fed-batch.
▶ Time-course (24 h) table
| Time (h) | OD600 | β-Carotene (mg/L) | Vol. productivity (mg·L⁻¹·h⁻¹) | Specific yield (mg/OD) |
|---|---|---|---|---|
| 0 | 0.65 ± 0.05 | 0.05 ± 0.01 | – | 0.08 |
| 6 | 1.82 ± 0.08 | 0.38 ± 0.06 | 0.055 | 0.21 |
| 12 | 2.95 ± 0.12 | 1.12 ± 0.15 | 0.123 | 0.38 |
| 18 | 3.42 ± 0.15 | 1.89 ± 0.22 | 0.128 | 0.55 |
| 24 | 3.68 ± 0.18 | 2.35 ± 0.32 | 0.077 | 0.64 |
Lactate-Responsive Regulation
Relevance. Tumor-like lactate levels (1–10 mM) trigger a strong, selective induction with acceptable viability.
Figure G. Lactate induction (lactate-induction-analysis). Dose–response across a physiological window.
Interpretation: Response window overlaps tumor microenvironment; supports in situ activation concepts.
▶ Temporal activation after induction
| Time (min) | Protein (%) | mRNA (rel.) | Apparent translation | State |
|---|---|---|---|---|
| 0 | 100 | 100 | 1.00 | Baseline |
| 15 | 185 ± 12 | 220 ± 15 | 0.84 | Initial |
| 30 | 420 ± 25 | 580 ± 32 | 0.72 | Accumulation |
| 60 | 720 ± 35 | 850 ± 42 | 0.85 | Near-max |
| 120 | 870 ± 28 | 920 ± 38 | 0.95 | Steady |
Genetic Stability
Finding. High plasmid retention and functionality across serial passages; suitable for batch and short continuous runs.
| Generation | Plasmid retention (%) | Functional expression (%) | Segregational loss (%/gen) | Specific productivity (rel.) |
|---|---|---|---|---|
| 0 | 100 | 100 | – | 100 |
| 5 | 98 ± 1 | 97 ± 2 | 0.40 | 97 |
| 10 | 96 ± 2 | 94 ± 3 | 0.41 | 94 |
| 15 | 94 ± 2 | 91 ± 3 | 0.43 | 91 |
| 20 | 92 ± 3 | 88 ± 4 | 0.45 | 88 |
Interpretation: For very long continuous processes, consider genomic integration or addiction systems to further reduce segregational loss.
Key Performance Indicators
Visualization set. Bubble chart for milestone impact, 3D response surface for interaction effects, parallel coordinates for multi-objective trade-offs.
Figure H. Performance metrics bubble chart (static-bubble-chart). Compact view of milestones vs relative impact.
Interpretation: Bubble size/position highlight key contributors (e.g., cloning efficiency, inducible control range).
Figure I. 3D response surface (3d-response-surface). Output predicted across two interacting factors under fixed constraints.
Interpretation: A ridge emerges at moderate induction and mid-temperature, consistent with wet-lab screens.
Figure J. Parallel-coordinates optimization (parallel-coordinates-optimization). Feasible regions satisfying yield, stability and viability constraints.
Interpretation: Guides DBTL cycles by exposing trade-offs and Pareto-like fronts.
▶ KPI summary table
| Metric | Target | Achieved | Rating | Significance |
|---|---|---|---|---|
| Gene cloning | 100% | 100% | Excellent | Foundation for DBTL cycles |
| Plasmid construction | >80% | 90% | Excellent | Faster iteration |
| Transformation | >10⁸ CFU/μg | 2.8×10⁸ | Exceeded | Library readiness |
| Protein expression | Detectable | Strong | Excellent | Functional capacity |
| β-Carotene | Detectable | 2.35 mg/L | Achieved | Upstream validated |
| ATRA | Detectable | 28.4 nM | Achieved | Full pathway confirmed |
| Induction range | >5-fold | 8.7-fold | Excellent | Precise control |
| Stability (20 gen) | >80% | 92% | Exceeded | Scale-up feasibility |
Analysis and Discussion
▶ Technical achievements
- Complete pathway integration with detectable ATRA output.
- High-efficiency cloning & recombination framework (>90% positive clones).
- Lactate-responsive circuit with large dynamic range.
- Kinetic profiling informs temperature and induction set-points.
▶ System insights
- 25–30 °C balances solubility and expression for active yield.
- RALDH oxidation appears rate-limiting (retinal accumulation).
- Low segregational loss suggests manageable burden across passages.
▶ Translational potential
- Sustainable ATRA bioproduction complementing chemical synthesis.
- Environment-responsive control for tumor-localized activation concepts.
- Platform extensibility to other retinoids.