PROJECT

The core innovation of HepaGuard lies in its precise patient‑identification system. We engineered a three‑key AND logic gate that activates therapeutic production only when three NASH‑specific biomarkers simultaneously exceed their thresholds: bile acid (>30 µM via P_efp), free fatty acid (>2 mM via P_fad), and TNF‑α (>50 pg/mL via P_nfkb). These thresholds correspond to bile acid dysmetabolism from liver injury, increased intestinal lipid absorption with barrier dysfunction, and inflammatory markers distinguishing simple steatosis from NASH.

The three promoters drive LuxI expression with weighted contributions: 0.2× (P_efp), 0.5× (P_fad), and 1.0× (P_nfkb). Activation occurs only when the total score exceeds 1.0, requiring either the inflammatory signal P_nfkb together with at least one metabolic signal, or all three simultaneously. This design prevents false activation in healthy individuals—postprandial FFA spikes alone cannot trigger the gate.Pnfkb (TNF-a) must be activated as a master switch, and PEFP (bileacid) and PFAD (FFA-RAct act synergistically as enhancing signals.Three promoters in tandem drive Luxl expression, enabling preciseidentification of Nash patients only. Python simulations based on literature data yield an AUC of 0.947, with 94 % specificity and 89 % sensitivity.

Once activated, the engineered strain initiates a bicistronic operon that simultaneously produces two therapeutic molecules, T2 and Fc‑FGF21, in an optimal 2 : 1 synergistic ratio. T2, a highly selective THR‑β agonist (selectivity >1000‑fold, no cardiac side effects), is synthesised from L‑tyrosine via a two‑step enzymatic pathway using TyrI (a bifunctional iodinase from brown algae) and ThyB (from B. subtilis, responsible for DIT coupling). The final yield reaches 10 mg/L.

Fc‑FGF21 is a fusion protein incorporating a signal peptide for secretion, an Fc fragment to prolong half‑life to 24–48 h, a flexible linker to preserve independent folding, and a His‑tag for easy purification. It improves insulin resistance, suppresses de novo lipogenesis, and protects hepatocytes, with a yield of 5 mg/L. The 2 : 1 ratio is derived from a 2023 Nature Medicine study showing that this combination reduces liver fat by 50 % more than either monotherapy with minimal side effects. Differential RBS strengths and a LuxI/LuxR auto‑calibration system maintain this stable ratio for 48–72 h. Both drugs accumulate in the periplasm and are packaged into outer membrane vesicles (OMVs) induced by TolA/TolB, achieving >60 % encapsulation efficiency, protecting against intestinal proteases, and enabling natural portal‑vein‑to‑liver delivery.

HepaGuard incorporates quadruple redundant safety locks to ensure automatic clearance after completing its task, preventing long‑term colonisation and ecological risks.

• 🔒 ΔdapA auxotrophy: Genomic deletion of dapA renders the strain dependent on exogenous diaminopimelic acid (DAP). Once the 72‑h DAP supply in the formulation is exhausted, or upon leakage outside the gut, cell‑wall synthesis fails and the bacterium dies.
• 🔒 mok‑sok TA system: Constitutive weak expression of toxin combined with inducible antitoxin RNA leads to toxin accumulation after ~20 divisions due to antitoxin dilution, triggering suicide.
• 🔒 pH‑inducible holin‑endolysin: The P_acid promoter activates at pH < 5.5 (e.g., gastric acid or gut leakage), causing lysis and release of drugs before final self‑destruction.
• 🔒 Horizontal transfer blockade: ΔsrtA knockout reduces surface‑protein anchoring, decreasing conjugation efficiency by 90 %; the pWV01 plasmid has a narrow host range, replicating only in Lactobacillus.

Monte Carlo simulations indicate that while individual lock failure probabilities range from 10⁻³ to 10⁻⁶, the combined failure probability is below 10⁻¹⁸, satisfying FDA standards for live biotherapeutic products. Compared with the chemical drug Resmetirom, HepaGuard offers no cardiac side effects, no hepatic burden, no long‑term residue, and a daily cost of only ¥0.5.

The following BioBrick parts are used in our project:

Part Name	ID
LuxI	BBa_C0061
LuxR	BBa_C0062
Plux	BBa_R0062
Strong RBS	BBa_B0034
Medium RBS	BBa_B0032
Terminator	BBa_B0015
GFP	BBa_E0040
dCas9	BBa_K1216001

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