Three Layers of Proprietary IP
From metabolic chassis to optogenetic valve to physical scaffold — each independently defensible, all synergistic.
Layer 1 — Chassis
Metabolically Orthogonal N. crassa
The iJDZ836 genome-scale metabolic model (836 genes, ~1,200 reactions) describes a metabolically capable organism. Flux-balance analysis confirms xylose as a preferred carbon source and the valid chassis feed for the chow reservoir.
The carbon source screen found 24 of 78 tested sources support growth. Gene essentiality analysis flagged ~130 of 836 genes (~15.5%) as essential — the hard boundary for knockout targeting.
We screened 10,440 double-knockout pairs from a 145-gene pool of transporter and central carbon genes, ranking each by Composite Orthogonality Score. Sixteen strains clear the chip-integration threshold (RBS ≥ 0.868) — the recommended chassis candidates, with enough residual xylose growth for robust chip colonization.
Phenotype Tiers
| Tier | Ranks | Characteristics |
|---|---|---|
| 1 — Best | 1–16 | Best sugar blocking, ~30% WT xylose growth |
| 2 — Balanced | 17–32 | More aggressive blocking, marginal xylose |
| 3 — Fitness | 33–50 | Best xylose fitness, weaker sugar blocking |
Model
iJDZ836
KO Pairs Screened
10,440
Chip-Eligible Strains
16
Metabolic Orthogonality
IP Coverage
Layer 2 — Valve
Optogenetic Septal Pore Valve
Blue light (450 nm) triggers pore closure. 100× dynamic range between dark and lit states. Fail-safe open logic — no light means no plug, flow is unrestricted.
Component A — LOV2-HEX1 Plug
LOV2 + Jα — blue-light photosensor from Avena sativa phototropin 1. Uses ubiquitous FMN as its chromophore — no foreign cofactor required. N-terminal placement keeps the HEX1 self-assembly contacts free. pLDDT = 91 (ColabFold).
HEX1 core (150 aa) — N. crassa Woronin-body protein, naturally evolved to plug septal pores under stress. Fused C-terminal via a (GGGGS)×3 linker; freely cytoplasmic until light-tethered. pLDDT = 88.
Component B — SPA1-ePDZ Anchor
SPA1 (aa 1–146) — N. crassa septal-pore-rim localizing domain. Constitutively anchored at the pore rim; no light dependence in localization.
ePDZ (engineered PDZ, iLID system) — light-gated affinity switch: ~130 nM in the lit state (Jα exposed) vs. >10 µM dark (Jα docked, epitope hidden). pLDDT = 88.
Thermal reversion: LOV2 dark recovery (seconds–minutes) automatically reopens the valve without any input signal. True fail-safe open behavior.
Valve Mechanism
100×
Dynamic range
450 nm
Activation wavelength
IP Coverage
Layer 3 — Scaffold
H-Tree Scaffold & Integration
The H-tree topology is the critical design choice. Every one of the 16 terminal reagent ports is exactly the same path length from the central chow reservoir — ensuring simultaneous, pressure-balanced reagent delivery.
Channel Sizing Rationale
| Level | Width | Hyphae | Function |
|---|---|---|---|
| Root–L1 | 20 µm | 4–8 | Main trunk — flow redundancy |
| L2–L3 | 13–17 µm | 2–3 | Valve control zone |
| Depth (all) | 15 µm | — | Matches hyphal diameter |
Chip Size
10×10 mm
Ports
16 reagent + 1 chow
Levels
4 branching levels
Compatibility
SU-8 / PDMS / Print
IP Coverage
H-Tree scaffold — 10×10 mm, 4 levels, 16 ports · channel width 13–20 µm
Five patent families — provisional ready to file
| # | Family | Description | Strength |
|---|---|---|---|
| 01 | Metabolically orthogonal chassis | Specific genotype + six-gene KO screening method | HIGH |
| 02 | Optogenetic septal pore valve | LOV2-Jα-HEX1 + SPA1-ePDZ constructs & flow control method | HIGH |
| 03 | Living microfluidic device | Chip + organism + scaffold as integrated system | HIGH |
| 04 | H-tree scaffold, fungal dimensions | Fungal-specific channel geometry (13–20 µm) novel over PDMS art | MED |
| 05 | Six-gene chassis (Δcre-1 + Δwc-1) | Requirement for 450 nm optogenetic activation in orthogonal host | HIGH |