Global Regulation Module

We designed and validated a quorum sensing (QS)-coupled feedback inhibition element to achieve autonomous density-dependent control of gene expression in engineered bacteria. The system was constructed by integrating a LuxI/LuxR-based QS module with a TetR-mediated repression module, creating a closed-loop circuit where bacterial population density directly regulates downstream gene expression.

Experimental validation using an AmCyan fluorescent reporter demonstrated the system's functionality: fluorescence intensity showed a characteristic activation-repression dynamic, peaking and then stabilizing at a lower baseline, as shown in Figure 1. Quantitative analysis confirmed a strong correlation (R² = 0.9078) between experimental results and ODE model simulations.

This proof of concept establishes that QS-coupled feedback inhibition can effectively translate population density signals into precise transcriptional control, providing a robust foundation for implementing autonomous regulation circuits in synthetic biology applications.

BioPROTAC

HemorrEaser incorporates a novel bioPROTAC(VHH-VHL) designed to mediate ubiquitin-dependent degradation of non-hydroxylated HIF-1α under hypoxic conditions, thereby inhibiting angiogenesis in hemorrhoidal tissues. The VHH-VHL construct consists of four components: a nuclear localization signal (NLS), an HIF-1α-binding nanobody (VHH212), an E3 ubiquitin ligase recruiter (pVHL) and linker peptides.

Functional validation was performed through integrated dry-lab and wet-lab approaches. Computational assessments included molecular docking (HADDOCK/HDOCK) of the HIF-1α-bioPROTAC-ubiquitin complex, Amber molecular dynamics simulations, and Gaussian 16 transition-state analysis. Results indicated a feasible ubiquitination energy barrier of approximately 8.20 kcal/mol, supporting reaction viability.

Experimental validations confirmed that the VHH-VHL construct maintains stable binding to HIF-1α, as demonstrated by yeast two-hybrid assays and protein co-purification assay. Furthermore, in vitro ubiquitination assays confirmed specific polyubiquitination of HIF-1α in the presence of bioPROTAC, collectively verifying its dual binding and degradation functionality.

Figure 3. Wet-lab validation results.

(A) Yeast two-hybrid assay; (B) Protein co-purification assay; (C) In vitro ubiquitination assay.

Collectively, these findings validate the rational design of VHH-VHL as an effective degrader of HIF-1α under hypoxia, providing a mechanistic foundation for its anti-angiogenic function in hemorrhoid therapy. You can read more at Results

OMV Delivery Module

To verify that Escherichia coli outer membrane vesicles (OMVs) can effectively load therapeutic molecules and achieve targeted transport, we first constructed a physical model to determine whether OMVs can be spontaneously internalized by small intestinal villus epithelial cells.

The core of the model lies in comparing the total energy difference of OMVs in two states—outside the cell membrane and inside the membrane—by evaluating the relative magnitudes of interface energy and membrane bending elastic energy to determine whether the process satisfies the thermodynamic spontaneous condition (ΔG < 0).

Specifically, the model divides the system into the initial state and the final state: in the initial state, the OMV is partially inserted into the membrane, forming a hemispherical protrusion that involves three types of interface energies — OMV-external fluid, OMV-membrane, and membrane-cytosol. In the final state, the OMV is completely wrapped to a spherical vesicle, with energy contributions from OMV-membrane interface energy, membrane-cytosol interface energy, and membrane bending elastic energy (based on Helfrich theory). By establishing a geometric relationship between the two states through volume conservation, the expression for ΔG was derived, yielding a dimensionless criterion: if a combined parameter (including interface tension, OMV radius, and membrane bending modulus) > 1, endocytosis proceeds spontaneously.

After substituting physiological parameters (R₂=50×10^-9 m, σ₁=10^-3 N/m, σ₂=0.8×10^-3 N/m, σ₃=0.5×10^-3 N/m, k=10^-19 J), the ratio reached 15.3—far greater than 1—indicating that OMV transmembrane endocytosis is thermodynamically favorable. Robustness verification further showed that even with smaller OMV size or higher membrane rigidity, spontaneous uptake conditions are still satisfied. This model thus provides theoretical support for the feasibility of OMVs as drug delivery carriers at the physical mechanism level.

To experimentally validate OMV function, gene sequences containing ompA-CAR, ompA-amCyan, and srp-TagRFP were constructed on the pET-28b(+) vector. Two plasmid combinations (ompA-amCyan + srp-TagRFP) ompA-CAR + srp-TagRFP) were co-transformed into E. coli Nissle 1917 (EcN) strains.

OMV precipitates were obtained through differential centrifugation (DC). Subsequently, the OMV marker OmpA (≈38.2 kDa) was used as a key indicator in a 12% SDS-PAGE assay. A distinct OmpA band was clearly visible in the gel electrophoresis results, confirming the successful extraction and enrichment of OMVs.

In future work, fluorescence confocal microscopy will be used to confirm the co-localization of amCyan and TagRFP, verifying the successful loading of engineered OMVs. Finally, a cell scratch assay will be conducted to evaluate the role of CAR in promoting cell migration, validating the targeting ability of engineered OMVs.

Anti-VEGF Nanobody

To inhibit hemorrhoidal vascular proliferation by specifically binding vascular endothelial growth factor (VEGF) and blocking its interaction with the vascular endothelial growth factor receptor (VEGFR), we designed an anti-VEGF nanobody modified with a P17 peptide at the N-terminus and fused to a shielding peptide at the C-terminus can be cleaved by matrix metalloproteinase-3 in the hemorrhoidal microenvironment.

First, we sought to validate the successful expression of P17-Nanobody, which was initially verified in Escherichia coli SHuffle. The P17-nanobody, tagged with 6×His, was constructed into the pET-28b(+) plasmid containing a lactose operon. The IPTG-induced P17-Nanobody was purified using a Ni²⁺-NTA affinity chromatography column and analyzed by SDS-PAGE and Western blot. The SDS-PAGE and Western blot (Figure 6.A, Figure 6.B) results demonstrated that P17-Nanobody was successfully expressed in Escherichia coli SHuffle. We next evaluated the capacity of the purified P17-nanobody to inhibit proliferation of VEGFR2-positive cells and block VEGF signaling. In a cell proliferation assay using VEGFR2 positive HUVECs and VEGFR2 negative HEK293 cells, the nanobody significantly inhibited HUVEC growth in a dose-dependent manner after 24 hours, with inhibition rates of 40-50% (P < 0.05, Figure 6.C). In contrast, no significant inhibition was observed in HEK293 cells at any concentration tested.

The P17 peptide significantly enhances the stability and solubility of anti-VEGF nanobodies. We used Protein-sol to predict the solubility of the P17-fused nanobody. The results showed that the P17 peptide improved the solubility both during and after expression(Figure 7.). Specifically, solubility increased by 27.8% during the expression phase, which facilitates robust protein production.

To validate the feasibility of the masking peptide, we analyzed all docking complexes with the Anti-VEGF antibody using Rosetta InterfaceAnalyzer, and identified several sequences with relatively weak interface energies. The representative complex was further examined using PRODIGY, yielding a binding free energy of -9.3 kcal·mol⁻¹ and a dissociation constant of K_d ≈ 2.6 × 10⁻⁷ M (260 nM), indicative of moderate affinity. The binding interface was dominated by hydrophobic (apolar-apolar, 16) and polar-apolar (10) contacts, while charged-charged (5) and polar-polar (1) interactions contributed less. The nonpolar interface area (41.0%) exceeded the charged area (23.1%), suggesting that binding is mainly driven by a hydrophobic core rather than electrostatic interactions.

To assess the dissociation process, steered molecular dynamics (SMD) simulations were performed with GROMACS to generate the unbinding pathway, followed by umbrella sampling and WHAM analysis to compute the potential of mean force (PMF).

The resulting binding free energy (plateau-minimum) was -9.8 kcal·mol⁻¹, corresponding to K_d ≈ 1.22 × 10⁻⁷ M at 310 K. These results indicate that the masking peptide binds with moderate affinity and reversible stability, consistent with its expected functional behavior.

Medicine-Food Collaboration

The suicide system comprises a riboswitch regulating the toxin gene ccdB, suppressing CcdB expression upon ligand binding and activating bacterial clearance in its absence. Using a TPP riboswitch-controlled vector and modeled optimal TPP concentration, toxicity assays in 96-well plates showed that at the IC₅₀ dose, the system strongly suppressed bacterial growth (Figure 9.), confirming effective riboswitch-controlled bactericidal activity by CcdB.

Figure 10. OD₆₀₀ measurement under 7 mM TPP induction.

Considering the differences between topical and oral administration scenarios, we further integrated a expiry-date circuit to meet the practical requirements of oral application. The simulation results are presented below.