To enhance immunostimulatory capacity and the tumor-specific colonization of engineered bacteria, we designed and constructed a hypoxia-responsive c-di-GMP expression plasmid, pUCP20-GmR-P_fnrs-yedQ. This system utilizes the oxygen-sensitive promoter P_fnrs, derived from the fnrS gene, to drive the expression of the diguanylate cyclase gene yedQ (dgcQ), leading to the intracellular production of the secondary messenger c-di-GMP specifically under low-oxygen conditions such as the tumor microenvironment (TME). c-di-GMP is a potent bacterial signaling molecule that not only promotes biofilm formation—improving bacterial retention in tumors—but also acts as a strong STING pathway agonist in eukaryotic cells. STING activation triggers type I interferon and pro-inflammatory cytokine production, polarizing macrophages toward an antitumor M1 phenotype. The YedQ enzyme used here is a constitutively active, cytosolic diguanylate cyclase optimized for high-yield c-di-GMP production. The plasmid was constructed by cloning the P_fnrs-yedQ expression cassette into a high-copy-number pUCP20 backbone for elevated c-di-GMP expression after several attempts. The design ensures high gene dosage and robust c-di-GMP output under hypoxia, eliciting a target anti-tumor response.

Figure A exhibits the plasmid map and sequencing results of pUCP20-GmR-P_fnrs-yedQ; Figure B displays an agarose gel confirming successful ligation of the pUCP20-GmR backbone and P_fnrs-yedQ insert following colony PCR; Figure C presents ELISA results demonstrating significantly elevated c-di-GMP production under anaerobic and microaerobic conditions in selected clones (e.g., 1223, 2112, 3222), validating the hypoxia-responsive activity of the P_fnrs promoter partially and functional expression of DgcQ.

To ensure the delivering efficiency of endogenous c-di-GMP and also the biosafety of our engineered bacterial therapy, we designed and validated a "self-lysis" module based on the quorum-sensing system, designated pTBR2iB. This system utilizes externally added AHL signal molecules to bind the luxR protein, thereby activating the PluxI promoter and initiating the expression of its downstream lysis gene, phlA. This leads to the self-lysis of the engineered bacteria, ensuring the controllability of bacterial clearance. This design not only provides a reliable "switch" for the lysis of engineered bacteria at the tumor site and the subsequent bulk release of c-di-GMP molecules, but also offers a strategy for the safe clearance of bacteria post-treatment.

Figure A displays the consistent design map and sequencing results of pTBR2iB; The growth curve in Figure B shows that the strain transformed with pTBR2iB exhibits growth inhibition under specific AHL induction; Figure C, using a spot plate assay, shows a significant reduction in the number of surviving colonies for the strain after AHL treatment compared to the control group; Figure D demonstrates that after the phlA gene was knocked out from pTBR2iB (thus named pTBR2iB-deletion), the corresponding strain exhibits attenuated sensitivity to AHL treatment, resulting in a higher colony count compared to the pTBR2iB group. This corroborates the role of the phlA gene in mediating membrane lysis and the self-cleaning function.

This study used Escherichia coli Nissle 1917 as the host for double-plasmid co-transformation to build strain resources for subsequent work on engineered E. coli–induced polarization of RAW264.7 cells. The target combination was the suicide plasmid ptb (pTBR2iB, AmpR) and the c-di-GMP synthesis plasmid 2112 (pUCP20-GmR-P_fnrS-yedQ), alongside three parallel controls (ptb+pUCP20, pUC18+2112, pUC18+pUCP20-GmR). Following transformation of chemically competent cells by heat shock, colonies were grown for 2 days on Amp-Gm double-selection plates and identified by colony PCR and agarose gel electrophoresis. Strains showing the expected bands (AmpR 822 bp, GmR 564 bp) were cryopreserved.

The first transformation yielded cryostocks for the three control strains. For the ptb+2112 dual-function plasmid strain, initial transformants were few and liquid cultures lacked turbidity; after extended incubation and a repeat transformation, three new positive clones were obtained, overnight cultures became turbid, and cryopreservation was completed. Upon replating and rechecking cryostocks, certain clones (e.g., ptb+2112 no.1 and 2, and pUC18+pUCP20 no.3) failed PCR and were discarded. Ultimately, all four classes of double-plasmid strains were successfully constructed, verified, and stably cryopreserved. In summary, the experiment achieved acquisition and quality control of the target double-plasmid engineered strains, providing reliable resources for subsequent cell experiments.

The core finding of this report is the clear demonstration of oxygen-dependent regulation mediated by the P_fnrS promoter in the pGL3-P_fnrS luciferase reporter system.

Functional analysis was conducted by measuring the normalized luciferase activity (Relative Light Units per OD₆₀₀, RLU/OD₆₀₀) in E. coli DH5α harboring the recombinant plasmid under two distinct incubation conditions: aerobic (shaking) and anaerobic (static within an anaerobic pack). The quantitative data are graphically summarized in the box plot. Statistical evaluation was performed using an unpaired, two-tailed Student's t-test.

The results reveal a substantial and statistically significant difference in promoter activity between the two oxygen regimes. Under aerobic conditions, the P_fnrS promoter exhibited a low level of basal activity, with a mean normalized luminescence of 2.82 ± 1.14 RLU/OD₆₀₀. In stark contrast, cultivation under anaerobic conditions triggered a pronounced activation of the promoter, leading to a mean normalized luminescence of 7.83 ± 2.97 RLU/OD₆₀₀.

This difference corresponds to a 2.78-fold induction of promoter activity in the absence of oxygen. The box plot effectively illustrates this disparity, not only in the median values but also in the overall distribution of the data points between the two groups. The statistical analysis confirmed that this observed enhancement was highly significant, with a p-value of less than 0.01, allowing us to reject the null hypothesis that oxygen conditions have no effect on P_fnrS activity.

In conclusion, the data visualized in the box plot provide robust empirical evidence that the P_fnrS promoter functions as a potent oxygen-responsive genetic element. Its significantly elevated activity under anaerobic conditions validates its potential for use in synthetic biology applications where precise, hypoxia-triggered gene expression is desired, such as in targeted therapeutic interventions within hypoxic environments like tumors.

The experiments confirm that the plasmid's P_fnrs promoter functions anaerobically to drive YedQ-mediated c-di-GMP synthesis, enhancing biofilm formation under tumor-like hypoxic conditions. Biofilm production was quantified by crystal violet staining, normalized as OD₅₇₀/OD₆₀₀ (corrected against LB blank controls) shown in the figure. This validates "ptb+2112" as a promising candidate for tumor-targeted therapy, with its oxygen-responsive biofilm formation ensuring localized retention and therapeutic efficacy.

Flow cytometry results showed that, compared with wild-type E. coli, ptb+2112 under AHL induction drove macrophages toward an M1 (CD86⁺) rather than an M2 (CD206⁺) phenotype, with stronger effects at 22 h than at 4 h, indicating time dependence. Anaerobic pre-culture (which enhances c-di-GMP production) yielded stronger responses than aerobic conditions, and the extent of c-di-GMP release broadly correlated with polarization strength. Exogenous c-di-GMP or LPS could induce M1 but less robustly than co-culture with the engineered strain. The ptb+2112 group without AHL also showed a mild M1 increase, likely due to bacterial immunogenicity and low-level basal expression. AHL exhibited a partial dose-response with a plateau. Despite limitations—such as incomplete inclusion of empty-vector controls in flow cytometry and effect sizes somewhat smaller than literature reports—the data collectively show that, under AHL and hypoxia, the engineered bacteria activate innate immune pathways via c-di-GMP release and stably drive RAW264.7 toward an M1 phenotype, supporting potential application in tumor microenvironment immunotherapy.

ELISA showed that AHL-induced dual-plasmid bacteria (ptb+2112) drove the highest cytokine release, significantly elevating IL-6 and IFN-β versus all controls (p < 0.05), with anaerobic preculture further enhancing responses. The c-di-GMP–producing single-control (pUC18+2112) yielded intermediate induction, while the double-empty (pUC18+pUCP20), Normal, and non-induced ptb+2112 (Without AHL) remained near baseline. Responses were time-dependent (22 h > 4 h) and showed a partial AHL dose effect with plateauing at higher concentrations. An independent c-di-GMP ELISA confirmed greater release under AHL and anaerobic conditions, broadly correlating with cytokine levels, supporting that inducible autolysis–mediated c-di-GMP delivery underlies the observed macrophage activation.

Our team successfully constructed a ΔthyA Escherichia coli strain using λ-RED homologous recombination, wherein the thyA gene was stably replaced with an FRT-flanked chloramphenicol resistance cassette. Sequencing verification confirmed precise gene knockout without off-target mutations. This thyA deficiency directly addresses the core safety objective of confining bacterial survival to the tumor microenvironment (TME).

As thyA encodes thymidylate synthase—a key enzyme in de novo thymidine biosynthesis—the ΔthyA strain exhibits strict thymidine auxotrophy. The TME, characterized by aberrant nucleotide metabolism, contains elevated thymidine levels (10–100μM) that support bacterial proliferation. In contrast, normal tissues maintain thymidine concentrations below 1 μM, which are insufficient to sustain ΔthyA cell growth.

Notably, even with the retention of the chloramphenicol resistance cassette, the strain's dependence on exogenous thymidine prevents uncontrolled proliferation outside the TME, thereby fulfilling the "tumor-restricted survival" design. Additionally, the resistance cassette provides a practical advantage for subsequent experiments: supplementing chloramphenicol in culture media enables specific selection of the ΔthyA strain, minimizing contamination by wild-type or non-target microorganisms and enhancing experimental reproducibility.