PepperProof of Concept
Scoville Splice · ScovilleSplice.farm
SS-0089 — Hostile Agent
Product Specification
- Product ID
- SS-0089
- Product Name
- Hostile Agent
- Target Organism
- Pepper (C. chinense)
- Engineering Tier
- Pepper
- Series
- Heat Ladder
- Target SHU
- 5,000,000
- Pun1/CS Variant
- Triple
- Gene Count
- 8
- Construct Length
- 13,345 bp
- Construct Type
- T-DNA binary vector
- Selection Marker
- nptII (kanamycin resistance)
- Promoter System
- 2A11
- Codon Optimization
- pepper
- Capsaicinoid Profile
- extreme multi
- Burn Character
- Heat Ladder: Hostile Agent
Gene Cassette Map
LB — [nptII] — [pAMT] — [Pun1/CS Triple] — [BCAT] — [KAS] — [FAR] — [PAL] — [C4H] — [4CL] — RB
| Gene | Function | Role |
|---|---|---|
| pAMT | Putative aminotransferase | Vanillylamine biosynthesis |
| Pun1/CS (Triple) | Capsaicin synthase | Acyl-vanillylamine condensation |
| BCAT | Pathway enzyme | Capsaicinoid pathway |
| KAS | Pathway enzyme | Capsaicinoid pathway |
| FAR | Pathway enzyme | Capsaicinoid pathway |
| PAL | Pathway enzyme | Capsaicinoid pathway |
| C4H | Pathway enzyme | Capsaicinoid pathway |
| 4CL | Pathway enzyme | Capsaicinoid pathway |
Flux Balance Analysis
- Predicted Capsaicin
- 234.375 μmol/g
- Predicted DHC
- 46.875 μmol/g
- Total Flux
- 2.5899 μmol/h
- Vanillylamine Branch
- 0.365
- Acyl Branch
- 0.256
- Condensation Efficiency
- 0.436
- Growth Impact
- 0.531 (fraction of WT)
- Feasibility
- EXPLORATORY
CRISPR Guide RNAs (POX Knockout)
| Target | Spacer (20-mer) | PAM | GC% | Efficiency |
|---|---|---|---|---|
| CaPOX1 | TGGCCCTCGCCGCCGCCGAT | NGG | 80.0% | 0.657 |
| CaPOX2 | AGACTATGCAGCTGAGCGTT | NGG | 50.0% | 0.799 |
| CaPOX3 | GTTTGGAGTGTGGGCTTGTG | NGG | 55.0% | 0.578 |
| CaPOX4 | GATGGCGGGGGCAGCGCGAG | NGG | 80.0% | 0.558 |
| CaPOX5 | TAAATCTATCTTCCACGGCC | NGG | 45.0% | 0.738 |
Transformation Protocol Summary
- Vector
- T-DNA binary vector in Agrobacterium tumefaciens GV3101
- Selection
- 50 mg/L kanamycin
- Confirmation
- PCR with construct-specific primers + HPLC capsaicinoid assay
- Expected Timeline
- 4-6 weeks (callus to T0 plant)