WET LAB

Objective: Assemble the complete HepaGuard genetic circuit: pSIP411‑P_efp‑P_fad‑P_nfkb‑P_lux‑TyrI‑ThyB‑FcFGF21, integrating three sensing promoters, AND‑gate controlled LuxI, dual‑drug operon (TyrI‑ThyB for T2, Fc‑FGF21), and safety modules.

Materials:

• Backbone: pSIP411 (chloramphenicol resistance, suitable for Lactobacillus).
• Gene fragments: TyrI (synthesized from Laminaria digitata), ThyB (from B. subtilis), Fc‑FGF21 (gene synthesis with signal peptide, Fc domain, linker, His‑tag).
• Promoters: P_efp, P_fad, P_nfkb (iGEM Registry or synthesis).
• Enzymes: BsaI (Golden Gate), T4 DNA ligase.
• Competent cells: E. coli DH5α (cloning), L. plantarum LP‑115 (expression).

Expected Results:

• Positive clone rate >50% (colony PCR).
• Sequencing accuracy >90% (no mutations in promoters/coding frames).
• Transformation efficiency in L. plantarum >10⁴ CFU/μg.

Validation: Colony PCR + sequencing report.

Objective: Confirm the three‑key AND gate activates only when multiple NASH signals exceed threshold, preventing false activation by single signals.

Materials:

• Reporter strain: LP‑115/pSIP411‑AND‑gfp (complete three‑input circuit).
• Inducer combinations: CDCA (0/50 μM), oleic acid (0/5 mM), TNF‑α (0/100 pg/mL).
• Detection: flow cytometry (single‑cell) or microplate reader (bulk).

Expected Results:

• Triple induction → fluorescence >20‑fold over background.
• Single inducers → <2‑fold increase.
• Pairwise combinations → intermediate responses depending on promoter weights.
• Hill coefficient >2, digital‑like threshold.
• Truth table matches computational predictions (P_nfkb=1.0, P_fad=0.5, P_efp=0.2, total >1.0 activates).

Validation: Fluorescence bar graph for 8 combinations, truth table, dose‑response curves.

Objective: Quantify T2 and Fc‑FGF21 production in activated HepaGuard, verifying optimal 2:1 synergistic ratio.

Materials:

• Engineered strain: LP‑115/pSIP411‑complete circuit.
• Induction: CDCA 50 μM + oleic acid 5 mM + TNF‑α 100 pg/mL in MRS + 5 mM NaI (for T2 synthesis).
• Instrumentation: LC‑MS/MS (triple quadrupole) for T2, His‑tag capture ELISA for FGF21.
• Time points: 12, 24, 48, 72 h.

Expected Results:

• Total T2 yield: 8–12 mg/L (peak at 48 h).
• FGF21 yield: 4–6 mg/L.
• Stable T2:FGF21 ratio ≈ 1.5–2.5:1 (matching 2:1 synergy).
• Combined therapeutic protein >10 mg/L, maintained 48–72 h.

Validation: Standard curves, chromatograms, ELISA calibration, ratio time‑course.

Objective: Verify that OMVs encapsulate therapeutic cargo, protect from degradation, and target liver naturally.

Materials:

• Engineered bacteria induced 24 h.
• Differential centrifugation: 10,000×g (remove cells) → 100,000×g (pellet OMVs).
• Characterization: TEM (morphology), DLS (size, PDI), Western blot (OmpA marker).
• Encapsulation efficiency: drug concentration in purified OMVs vs. total production.

Expected Results:

• Spherical vesicles, 50–100 nm diameter (TEM).
• Narrow size distribution (PDI <0.3).
• OmpA signal confirmed (Western blot).
• Encapsulation: T2 >60%, FGF21 >50%.
• Yield >10¹² particles/mL.

Validation: TEM images, DLS histograms, Western blot bands, encapsulation efficiency data.

Objective: Demonstrate OMV internalization by hepatocytes in a NASH‑mimetic model.

Materials:

• HepG2 cells cultured in DMEM + 10% FBS.
• NASH model: 0.5 mM palmitic acid‑BSA for 48 h.
• OMVs labeled with DiO or PKH67 (10–100 μg/mL, 1–4 h).
• Detection: flow cytometry (% DiO⁺, MFI) and confocal microscopy.

Expected Results:

• Dose‑dependent uptake: >40% positive cells at 10 μg/mL, 4 h.
• Rapid internalization within 1 h, plateau at 4 h.
• Punctate cytoplasmic fluorescence (endosomes), not surface binding.
• Enhanced uptake in steatotic cells.
• No cytotoxicity (LDH assay).

Validation: Flow scatter plots, confocal images, LDH release data.

Objective: Validate therapeutic efficacy of OMV‑encapsulated drugs in a physiologically relevant 3D liver model.

Materials:

• HepG2 spheroids formed in ultra‑low attachment plates (72 h, 200–300 μm diameter).
• NASH induction: 1 mM FFA (oleate:palmitate 2:1) + 50 ng/mL TNF‑α for 48 h.
• Treatment groups: control, NASH, NASH + empty OMVs, NASH + T2‑OMVs, NASH + FGF21‑OMVs, NASH + dual‑drug OMVs (2:1).
• Endpoints: Oil Red O (lipid quantification), ELISA (TNF‑α, IL‑6), live/dead staining.

Expected Results:

• NASH spheroids show >3‑fold lipid accumulation vs. control.
• Dual‑drug OMVs reduce lipid area >40% compared to NASH.
• Inflammatory cytokines decrease >50%.
• Viability >80% in all groups.
• Single‑drug treatments show intermediate efficacy (synergy confirmed).

Validation: Oil Red O quantification, cytokine ELISA, viability staining.

Objective: Confirm that redundant safety systems prevent horizontal gene transfer and maintain genetic stability over therapeutic duration.

Materials:

• Horizontal transfer: co‑culture HepaGuard (donor, Cmᴿ) with E. coli MG1655 (recipient, Cmˢ) on solid medium 24 h; select transconjugants on Cm plates, confirm by PCR.
• Stability: continuous passage in MRS + DAP for 20 generations (72 h); sample every 4 generations; re‑induce AND gate; sequence key genes (LuxI, TyrI, FGF21, safety modules).

Expected Results:

• Transfer frequency <10⁻⁶ per donor (ΔsrtA + narrow‑host‑range pWV01 effective).
• After 20 generations, AND gate induction efficiency decrease <5%.
• No mutations in critical regions (sequencing).
• Plasmid retention >95% (mok/sok TA system).

Validation: Conjugation plates, PCR, sequencing data, antibiotic sensitivity tests.