diff --git a/docs/WET_LAB_HANDOFF.md b/docs/WET_LAB_HANDOFF.md index 662fd1dc..d8938f87 100644 --- a/docs/WET_LAB_HANDOFF.md +++ b/docs/WET_LAB_HANDOFF.md @@ -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 @@ -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) @@ -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 diff --git a/tests/test_reference_set.py b/tests/test_reference_set.py index e152442e..ea32abe7 100644 --- a/tests/test_reference_set.py +++ b/tests/test_reference_set.py @@ -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