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Design

Engineering design of CradleShield

Project Design

From concept to engineered probiotic

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Chassis & Core Mechanism

🦠 Chassis: Bacillus subtilis 168
  • Spores naturally resistant to cephalosporins/amoxicillin.
  • Room‑temperature stable for >12 months.
  • No long‑term gut colonization (cleared in 3‑5 days).
🧪 Functional Molecule: Butyrate
  • Produced on‑site at 0.5‑1.5 mM (therapeutic window).
  • Repairs tight junctions via HDAC8/NF‑κB/Slc26a3 pathway.
  • No colonization lag – acts within 48 h.
🔒 Safety System: dal Auxotrophy
  • dal knockout → D‑alanine dependent growth.
  • Grows only in gut (D‑alanine present), self‑limiting in environment.
  • Replaces antibiotic resistance markers.
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Chassis Innovation

First combination of B. subtilis spore properties with butyrate mechanism.

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Safety Innovation

dal auxotrophy, no antibiotic markers, environmentally self‑limiting.

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Application Innovation

“Antibiotic companion” – co‑administered, no cold chain.

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Engineering Innovation

DBTL cycle: plasmid validation → chromosomal integration.

💡 Dual‑Core Solution

  • Spore chassis + butyrate mechanism
  • Physical & molecular synergy
Dual core

🔬 Core Engineering Steps

Step 1

1️⃣ Δdal Host Strain Construction

  • Knockout of dal gene via pK18mobsacB‑Δdal.
  • Double‑crossover recombination → D‑alanine auxotrophy.
  • Validated by PCR and growth tests (M9 ± D‑alanine).
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Step 2

2️⃣ Plasmid Construction

🧬 pHT43‑butyrate (Golden Gate)

For phase 1 yield validation

🧩 pAX01‑butyrate‑dal (Gibson)

For chromosomal integration

✂️ pK18mobsacB‑Δdal

For dal knockout

3️⃣ Chromosomal Integration

  • Electroporation of pAX01‑butyrate‑dal into Δdal cells.
  • Integration into amyE locus via homologous recombination.
  • Final strain: CradleShield (Δdal::amyE‑P43‑butyrate‑dal)
  • No free plasmids, no antibiotic resistance.
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Step 3
🌱

Spore Preparation & Validation

  • 🧫 Induction in SP medium with Mn²⁺ (48 h, sporulation ≥90%).
  • 🧼 Purification: ethanol treatment (75%, 30 min) kills vegetative cells.
  • 💧 Washed spores resuspended in saline.
  • 🛡️ Resistance: Grow on LB + cephalosporins/amoxicillin.
  • ⏳ Stability: ≥90% germination after 7 days at 25 °C.
🧪🌡️❄️
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Functional Validation

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Biochemical

HPLC butyrate ≥1.5 g/L

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Cellular (Caco‑2)

TEER ↑30%, permeability ↓40%

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Molecular

HDAC8 ↓50%, Slc26a3 ↑2‑fold

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In vivo

Diarrhea index ↓40‑60%

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Triple Biosafety Assurance

🌿 Natural Safety

Spores do not colonize gut; cleared in 3‑5 days.

🔒 Active Safety

dal auxotrophy → no survival in environment.

🧬 Structural Safety

All genes integrated into chromosome; no plasmids, no resistance markers.

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Application Prospects

CradleShield is designed as an “antibiotic companion” for children, addressing the three bottlenecks of current probiotics. It can be co‑administered with antibiotics, requires no cold chain, and acts rapidly with proven safety. This product fills a technological gap in pediatric AAD prevention and holds great potential for clinical use in hospitals and community healthcare centers.