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48 changes: 47 additions & 1 deletion docs/WET_LAB_HANDOFF.md
Original file line number Diff line number Diff line change
Expand Up @@ -67,6 +67,7 @@ before ordering. The report flags:
| `DEAMIDATION_RISK` | N→D conversion at pH > 7.5 | Use pH 7.0 buffer; avoid long storage at room temp |
| `LOW_CHARGE` | Reduced membrane affinity | Lower expected potency; may need higher test concentrations |
| `LONG_PEPTIDE (>30aa)` | SPPS yield risk | Expect lower crude purity; order extra crude for purification |
| `C_AMIDATION_RECOMMENDED` | C-terminal COOH reduces charge and serum stability | **Request "C-terminal amide (CONH₂)"** in synthesis order form — adds ~+0.7 charge, improves stability at zero extra cost |

### Synthesis Difficulty Rating

Expand All @@ -82,7 +83,24 @@ before ordering. The report flags:

### Initial Screen (MIC Assay)

- **Organism priority:** *E. coli* ATCC 25922 (Gram-negative standard), *S. aureus* ATCC 29213 (Gram-positive standard)
**Reference strain panel (Wave 1 minimum):**

| Organism | ATCC strain | Gram | Rationale |
|----------|-------------|------|-----------|
| *E. coli* | ATCC 25922 | − | Gram-negative standard; reference for all AMP comparisons |
| *S. aureus* | ATCC 29213 | + | Gram-positive standard; reference for all AMP comparisons |

**MDR expansion (Wave 1 recommended — adds publication significance):**

| Organism | Strain | Resistance phenotype | Why include |
|----------|--------|----------------------|-------------|
| *S. aureus* | USA300 | MRSA (methicillin-resistant) | Clinically prevalent; any hit is immediately publishable |
| *E. coli* | ST131 (ATCC BAA-2469) | MDR (CTX-M ESBL) | Most prevalent MDR Gram-negative globally |
| *K. pneumoniae* | ATCC BAA-1705 | KPC (carbapenem-resistant) | Highest-priority WHO critical pathogen |

Including MDR strains adds only ~3× material cost but increases publication impact substantially.
Any candidate with MIC ≤ 8 μg/mL against MRSA or KPC-KP qualifies as clinically significant.

- **Growth medium:** MHB (Mueller-Hinton Broth), cation-adjusted
- **Inoculum:** ~5×10⁵ CFU/mL (CLSI standard)
- **Concentration range:** 1–128 μg/mL (2-fold serial dilutions)
Expand All @@ -106,6 +124,34 @@ Hemolysis > 10% at MIC is a fail.

---

## SEED-006 (Mastoparan-X) Special Notes

SEED-006 variants are derived from **Mastoparan-X** (INWKGIAAMAKKLL, Yoshida *et al.* 1990),
a wasp (*Vespa xanthoptera*) venom calmodulin-binding helical peptide. This is structurally
distinct from the other 5 seeds (magainin-like, cecropin-like, tachyplesin-like).

**Mechanism:** Mastoparan-X inserts into bacterial membranes via a predominantly
**hydrophobic helix insertion** mechanism rather than electrostatic carpet disruption.
It also activates G-proteins and has calmodulin-binding properties.

**Expected activity profile:**
- Gram-positive coverage likely > Gram-negative (small outer membrane)
- May show cooperative ("all-or-nothing") dose-response curves
- Hemolytic risk: moderate (assay at MIC/3 per QC guidance)

**Synthesis guidance:**
- No Cys, no Met → standard Fmoc SPPS, standard storage
- **C-terminal amidation strongly recommended**: SEED-006_VAR_xxx candidates end in LL (hydrophobic),
neutral C-terminus. Request `CONH₂` to add +0.7 charge and improve serum stability
- SEED-006 variants have 2–3 interior Lys → serum t½ estimated ~2–4 h

**Assay interpretation:**
- If SEED-006 variants are active but SEED-001–005 are not: supports calmodulin-binding mechanism;
consider calmodulin competition assay to confirm novel mechanism
- High publication value if MIC < 4 μg/mL against MRSA (see MDR panel above)

---

## Troubleshooting

### Too Few Candidates Pass the Gates
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2 changes: 0 additions & 2 deletions tests/test_reference_set.py
Original file line number Diff line number Diff line change
Expand Up @@ -8,8 +8,6 @@
import csv
from pathlib import Path

import pytest

REF_CSV = Path("examples/known_reference/amp_curated_references.csv")
CANONICAL_AA = set("ACDEFGHIKLMNPQRSTVWY")
MIN_LEN = 6
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