Staphylococcus xylosus ATCC 29971 R-M System

To overcome the restriction–modification (R-M) systems of Staphylococcus xylosus ATCC 29971, we selected Escherichia coli JM110 as the plasmid amplification host. The plasmid pLI50 was first introduced into JM110 for amplification and extraction, and subsequently transformed into Staphylococcus xylosus ATCC 29971. Correct transformants were obtained, demonstrating that the R-M system of Staphylococcus xylosus ATCC 29971 was effectively bypassed and that plasmid-containing strains were successfully constructed.

According to the competent cell preparation method we adopted, plasmids could be successfully introduced into Staphylococcus xylosus ATCC 29971 using this approach (glycine wash), but the transformation efficiency could be markedly low. After transformation, only a few colonies appeared on the plates, which was consistent with the expected outcome.

Staphylococcus epidermidis ATCC 14990 R-M System

To ensure the successful expression of the series of plasmids pLI50 in Staphylococcus epidermidis ATCC 14990, we consider tackling this problem from its R-M system.

Strain Construction and Validation

We tried to replace dcm from the genome of Escherichia coli DH5α with our constructed linear fragment, yedJ-J23100-RBS-kanR-T2-J23104-RBS-hsdM-hsdS-vsr.

1. Amplify fragment hsdM-hsdS from Staphylococcus epidermidis ATCC 14990, and kanR from the iGEM Distribution Kit, while at the same time add important units like RBS, promoter, or terminator at the 5’ or 3’ site of each sub-fragment.

For this linear fragment used to conduct λRed homologous recombination, it bears yedJ (left homologous arm) and vsr (right homologous arm), with functional parts (kanR and hsdM-hsdS) in between and important gene-expression-related parts (RBSs, terminators, promoters, and intentionally added A/T repeats to ensure the smooth work of ribosome translation).

2. Connect kanR and hsdM-hsdS, and add homologous arms of the genome of Escherichia coli DH5α at the 5′ or 3′ site.

Use overlap PCR, run the first 6 cycles with the homologous templates (sub-fragments) as primers themselves, without the primers we designed (binding to left homologous arm, the end of hsdM-hsdS and adding the right homologous arm vsr) for the full-length amplification. While adding the designed primers at the last 36 cycles to accomplish the full-length amplification.

Although the fragment was expected to be 3806 bp, the gel image quality was compromised because the E-gel Imager in the collaborating lab was not functioning properly. As a result, the fragment size could only be approximately assessed, and subsequent experiments confirmed that the assembly had failed.

However, the promoters’ sequences of kanR and hsdM-hsdS are way too similar, which leads to primer specificity loss, thus making it much easier for the 5′ end primer to bind at the middle of the linear fragment and resulting in the inefficiency of overlap PCR — the full length will not be amplified, only half of it is attained. So, we tried to add the left homologous arm yedJ (60 bp) first (at the 5′ site) to avoid the problem.

3. Transform pKD46 into E. coli DH5α.

DH5α competent cells were prepared using CaCl₂ washing, followed by incubation on ice for 30 min, heat-shock at 42 °C for 90 s, and recovery on ice for 3–5 min. Cells were shaken at 37 °C, 185 rpm for 1 h before being plated on LB agar containing 100 ng/mL ampicillin.

4. Incubate the bacteria at 37 °C overnight with shaking. The next day, dilute the culture at a ratio of 1:100 into 5 ml of fresh medium and shake for 1.5 h. Then add 60 μl of pre-mixed L-arabinose and continue shaking for another 1 h. When the OD₆₀₀ reaches around 0.5, collect the bacteria to prepare electroporation-competent cells. Once the competent cells are prepared, perform electroporation immediately to transform the constructed linear fragment. After the procedure, plate the bacteria on LB agar containing 100 ng/ml ampicillin and 100 ng/ml kanamycin.

5. Pick one colony and incubate at 37 °C, and conduct colony PCR.

6. Shake the bacteria overnight at 40 °C in medium containing 100 ng/ml kanamycin but no ampicillin to eliminate pKD46. Then prepare the bacteria as competent cells using CaCl₂ (aq) washing and transform pLI50[P_SarA1-sfgfp].

Validation of Staphylococcus xylosus ATCC 29971 strain construction

To enhance the delivery efficiency of the system, we need to improve the invasion capacity of the engineered bacteria. We plan to construct Sinon α (pLI50[P_SarA1-fnbA-RBS-sfgfp]) and Sinon β (pLI50[P_cap-fnbA-RBS-sfgfp]), employing the FnBP protein to achieve efficient invasion.

Plasmid Amplification and Reconstruction

The plasmid backbone used in this study is pLI50. To achieve both efficient invasion and convenient visualization, we constructed two plasmids by inserting P_SarA1-fnbA-RBS-sfgfp and P_cap-fnbA-RBS-sfgfp fragments into pLI50, thereby generating Sinon α (pLI50[P_SarA1-fnbA-RBS-sfgfp]) and Sinon β (pLI50[P_cap-fnbA-RBS-sfgfp]). In this design, P_SarA1 and P_cap serve as promoters, FnBPA facilitates the invasion of engineered bacteria into cancer cells, and sfGFP provides fluorescence, enabling subsequent monitoring of bacterial entry into host cells.

The purified gene fragment was ligated into the plasmid backbone pLI50 using the One Step Cloning method, resulting in the plasmid construct shown in the figure. The recombinant plasmid was then introduced into Escherichia coli JM110 for amplification and demethylation, after which the plasmid was extracted.

Strain Construction and Validation

Colony PCR verification

We have constructed the Sinon α and Sinon β plasmids and subsequently introduced them into Staphylococcus xylosus ATCC 29971. The following colony PCR results confirm the successful construction of the strains TrojanHorseβ (S. xy[P_SarA1-fnbA-RBS-sfgfp]) and TrojanHorseβ (S. xy[P_cap-fnbA-RBS-sfgfp]).

Fluorescent microscopic examination

Since a bicistronic structure was employed in plasmid construction, FnBPA expression could be directly indicated by fluorescence. To rapidly verify its expression, we used confocal microscopy to directly observe the bacterial suspension.

The detection of sfGFP expression implies successful expression of FnBPA.

Growth Curve of the Constructed Staphylococcus xylosus ATCC 29971

To evaluate the growth status of the strain required for co-culture, we determined the growth curve of Staphylococcus xylosus ATCC 29971 carrying the fnbA and sfgfp. The engineered strain was inoculated into tryptic soy broth (TSB) at a 1:100 inoculum-to-medium volume ratio and cultured in 15 ml centrifuge tubes at 37 °C with shaking at 199 r/min. Samples were collected every 30–60 min, vortexed, and the turbidity was measured at 600 nm using a microplate reader. The resulting data points were fitted to generate the growth curve.

The results showed that S.xylosus ATCC 29971 remained in the lag phase during 0–100 min, followed by a rapid logarithmic growth phase between 100–300 min. After ~300 min, the growth rate declined, and the culture entered the stationary phase, during which OD₆₀₀ values remained between 1.3 and 1.6, with minor fluctuations possibly attributable to experimental error or population heterogeneity.

In summary, for the co-culture experiments we used bacterial suspensions cultured for more than 20 h, ensuring that the cells had reached the late stage of the growth curve and thereby providing an optimal state for co-culture.

Co-culture

To validate and quantify the function of the exogenous protein FnBPA, we conducted a series of co-culture experiments between engineered bacteria and mammalian cells, followed by flow cytometry and fluorescence microscopy analyses.

The engineered strains were derived from Staphylococcus xylosus ATCC 29971 (TrojanHorseβ), which exhibited different phenotypes depending on the introduced plasmids: pLI50[P_SarA1-sfgfp] (Reporter α) and pLI50[P_SarA1-fnbA-RBS-sfgfp] (Sinon α). In this project, the resulting bacterial strains are referred to as TrojanHorseβ[Reporter α] and TrojanHorseβ[Sinon α], respectively. The cell lines used included two human-derived and two mouse-derived lines. The human-derived lines were MDA-MB-231 (a TNBC cell line) and HBL-100 (a normal mammary epithelial cell line). The mouse-derived lines were 4T1 (a TNBC cell line) and HC11 (a normal mammary epithelial cell line).

The invasion rate was defined as the proportion of host cells exhibiting a positive fluorescence signal indicative of bacterial internalization. Flow cytometric analysis was performed by gating the main cell population on the forward scatter (FSC) versus side scatter (SSC) plot to exclude debris and doublets. The invasion-positive gate was determined based on the fluorescence intensity of the negative control, with the upper 1% threshold used to define positive events.

Under these experimental conditions, the comparative analysis of different bacterial and cellular combinations allowed a comprehensive characterization of the functional role of the FnBPA protein.

Preliminary Experiments

The preliminary experiments aimed to assess the biological function of FnBPA and to characterize the relationship between invasion efficiency and the multiplicity of infection (MOI). In addition, the experimental protocol was iteratively optimized during this process to ensure reproducibility and to establish reliable conditions for the subsequent main experiments.

TrojanHorseβ[Reporter α] and TrojanHorseβ[Sinon α] were co-cultured with MDA-MB-231 cells for 6 hours under hypoxic conditions (5% O₂). Fluorescence signals derived from the expression of sfGFP were collected in the FITC channel (excitation at 488 nm).

Preliminary Experiment A and B:

TrojanHorseβ[Reporter α] (Staphylococcus xylosus ATCC 29971 harboring pLI50[P_SarA1-sfgfp]) were co-cultured with MDA-MB-231 cells for 6 hours under hypoxic conditions (5% O₂).

Trojanβ[Reporter α] exhibited consistently low invasion rates, fluctuating between approximately 1%-4%. Moreover, the overall invasion efficiency and fluorescence level remained lower than anticipated, likely due to the low fluorescence intensity of sfGFP.

Preliminary Experiment C and D:

TrojanHorseβ[Sinon α] (Staphylococcus xylosus ATCC 29971 harboring pLI50[P_SarA1-fnbA-RBS-sfgfp]) were co-cultured with MDA-MB-231 cells for 6 hours under hypoxic conditions (5% O₂).

TrojanHorseβ[Sinon α] exhibited markedly enhanced invasion capacity compared to Trojanβ[Reporter α], with an invasion rate of up to 50.4%. The invasion rate displayed a bell-shaped dependence on MOI, reaching its maximum at an MOI of approximately 10. However, this observation requires further validation due to the limited dataset.

The sfGFP fluorescence intensity, reflecting both signal strength and the percentage of positive cells, exhibited stable intra-experimental patterns but inconsistent absolute levels across experiments. This variation is presumed to result from unknown environmental factors regulating sfGFP expression.

Labeling performance validation

Considering the relatively low and unstable fluorescence intensity of sfGFP, we employed the cell proliferation dye CellTrace™ Far Red to label Trojanβ[Reporter α], ensuring that fluorescence data were obtained in a paired manner for direct comparison. Bacterial smears were prepared and imaged using an Olympus spinning disk confocal microscope under identical conditions. Twelve fields of view were randomly selected, and identical regions of interest (ROIs) corresponding to the same group of cells were analyzed in both the far-red (640 nm) and green (488 nm) channels using ImageJ. Mean fluorescence intensity (MFI) was quantified for paired comparison.

The results showed that the CellTrace Far Red signal was significantly stronger than the sfGFP fluorescence under identical imaging conditions, demonstrating the superior labeling efficiency and stability of the dye.

Formal Experiments

The main experiments were performed following validation of the assay’s reliability in accurately reflecting the true invasion efficiency. These experiments aimed to explore the invasion capacity and selectivity of the engineered bacterium.

TrojanHorseβ[Reporter α] and TrojanHorseβ[Sinon α] were labeled with CellTrace™ Far Red and performed repeated (n = 3) co-culture experiments with MDA-MB-231, HBL-100, 4T1, and HC11 cells for 6 hours under hypoxic conditions (5% O₂). Flow cytometry was then used to collect fluorescence signals from both sfGFP and CellTrace™ Far Red. The sfGFP signal (excitation at 488 nm) was used solely for visualization and reference, whereas the CellTrace Far Red fluorescence, detected in the APC-Cy7 channel (excitation at 640 nm), served as the exclusive indicator of bacterial invasion efficiency.

Co-culture of TrojanHorseβ[Sinon α] with human cell lines

TrojanHorseβ[Sinon α] was co-cultured under identical conditions with the human cell lines MDA-MB-231 (TNBC cells) and HBL-100 (normal mammary epithelial cells) to quantitatively assess invasion efficiency and to compare cell-line selectivity.

TrojanHorseβ[Sinon α] achieved full-scale invasion of MDA-MB-231 cells, maintaining >99% infection efficiency across all tested MOIs (5–25). Statistical analysis using a two-way repeated-measures ANOVA revealed significant main effects of MOI (F(5,20)=1124.36, p<0.0001) and cell line (F(1,4)=1328, p<0.0001), as well as a strong MOI × cell line interaction (F(5,20)=441.8, p<0.0001). These results indicate that invasion efficiency was jointly modulated by infection dose and cell type, with a significant interaction between the two factors. Post hoc multiple comparisons (Šídák’s test) demonstrated consistently higher invasion rates in MDA-MB-231 cells relative to their non-malignant counterpart across low to moderate MOIs (p<0.0001 at MOI 5–10; p=0.015 at MOI 15), confirming a pronounced cell-line selectivity and preferential tropism of TrojanHorseβ[Sinon α] for the TNBC phenotype at lower MOIs (5–15). Consistently, no significant difference was observed between cell lines at higher MOIs (adjusted p>0.7 at MOIs 20–25), suggesting a saturation effect that diminishes selectivity at excessive infection doses. The mean difference in invasion efficiency between cell lines, averaged across all tested MOIs, was 2.65 ± 0.07% (95% CI, 2.45–2.85), underscoring the robustness and reproducibility of this cell-line–specific effect.

Co-culture of TrojanHorseβ[Sinon α] with murine cell lines

To extend the analysis of host specificity, TrojanHorseβ[Sinon α] was co-cultured under identical experimental conditions with murine cell lines, including 4T1 (TNBC cells) and HC11 (normal mammary epithelial cells) cells, to evaluate invasion efficiency and cell-line selectivity across species.

TrojanHorseβ[Sinon α] achieved high invasion efficiency in 4T1 cells, with infection rates remaining above 92% even at the lowest tested MOI (20). Statistical analysis using a two-way repeated-measures ANOVA revealed significant main effects of MOI (F(5,20)=5520, p<0.0001) and cell line (F(1,4)=4485, p<0.0001), as well as a strong MOI × cell line interaction (F(5,20)=72.94, p<0.0001). These results indicate that invasion efficiency was jointly influenced by bacterial dose and cell type, with distinct invasion dynamics between the two cell lines. Post hoc multiple comparisons (Šídák’s test) further confirmed that TrojanHorseβ[Sinon α] exhibited markedly higher invasion efficiency in 4T1 cells than in HC11 cells across all tested MOIs (p<0.0001). The difference remained stable at higher infection doses, suggesting a consistent cell-line selectivity within the murine system. The mean difference in invasion efficiency between 4T1 and HC11 cells was 14.90 ± 0.22% (95% CI, 14.28–15.51), underscoring the robustness and reproducibility of this cell-line–specific effect.

Functional assessment of FnBPA across varying MOIs

To provide a molecular evidence for the observed cell-line invasion and selectivity, we next examined the functional role of FnBPA across different infection doses. Specifically, TrojanHorseβ[Reporter α] and TrojanHorseβ[Sinon α] were labeled with CellTrace™ Far Red and performed repeated (n = 3) co-culture experiments with 4T1 cells for 6 hours under hypoxic conditions (5% O₂).

TrojanHorseβ[Sinon α] exhibited markedly higher invasion rates in 4T1 cells than TrojanHorseβ[Reporter α] across all tested MOIs (p < 0.0001 at MOI 5–10, p < 0.0003 at MOI 15–25; two-way RM ANOVA with Šídák’s multiple comparisons). The overall mean difference between strains was 78.99 ± 0.26% (95% CI, 78.26–79.72), suggesting the pronounced invasion-promoting effect of FnBPA and confirming its essential role in facilitating bacterial entry into host cells.

Together, these results provide molecular evidence that FnBPA serves as the principal mediator of the enhanced invasion phenotype. Functional validation confirmed that FnBPA expression substantially increases bacterial entry efficiency, while dose–response analysis demonstrated a strong but saturable invasion-promoting effect. This establishes FnBPA as the molecular basis for the enhanced cellular invasion of TrojanHorseβ[Sinon α].

Previously unreported intrinsic invasion of the commensal strain

To establish the baseline invasion profile of the TrojanHorseβ (commensal strain Staphylococcus xylosus ATCC 29971), the reporter strain TrojanHorseβ[Reporter α] was co-cultured with MDA-MB-231 and HBL-100 cells, across different MOIs. Unexpectedly, the results revealed a previously unreported phenotype of the commensal bacterium Staphylococcus xylosus ATCC 29971: the strain exhibited an intrinsic ability to actively invade TNBC cells, accompanied by marked cell line selectivity.

Quantitative analysis revealed a clear dependence of bacterial invasion on both MOI and cell type. As the MOI increased, the invasion rates rose correspondingly, demonstrating a dose-responsive invasion pattern. Two-way repeated-measures ANOVA identified significant main effects of MOI (p < 0.0001) and cell line (p = 0.0248), as well as a significant interaction effect between MOI and cell line (p = 0.0018), indicating that the response of each cell type to bacterial load differed significantly. Specifically, Šidák’s multiple comparisons test indicated that invasion rates in MDA-MB-231 cells were consistently higher than those in HBL-100 cells at higher MOIs. The difference became statistically significant at MOI 25 (mean difference = 25.40%, 95% CI = 12.96–37.84, p < 0.0001), while lower MOIs showed no significant difference. This pattern suggests a threshold-like invasion behavior, likely reflecting the requirement of sufficient bacterial density to induce effective interactions or active internalization processes in tumor cells.

Collectively, these results demonstrate that S. xylosus ATCC 29971 exhibits a previously unreported, intrinsic ability to invade tumor cells, with a pronounced preference for TNBC cells (MDA-MB-231) over non-tumorigenic epithelial cells (HBL-100). This unexpected finding prompted us to carefully reconsider whether the cell line selectivity observed in TrojanHorseβ[Sinon α] arises from the introduced FnBPA module, or simply reflects the intrinsic invasion properties of the commensal host strain. The inherent tumor-targeting capacity of S. xylosus thus makes it a promising chassis bacterium for the development of commensal-based TNBC-targeted intracellular delivery platform.

Gentamicin protection assay validation

Since the gentamicin protection assay relies on the antibiotic’s ability to completely eliminate extracellular bacteria, failure in this process could lead to false-positive results. Therefore, we performed a validation experiment to confirm that gentamicin treatment under our co-culture conditions is sufficient to eradicate bacteria remaining in the culture medium. This validation step is essential for ensuring the authenticity and reproducibility of the experimental results.

In this validation assay, bacterial suspensions were co-incubated with gentamicin in DMEM containing 10% FBS, and samples were collected at 5-minute intervals. After centrifugation and resuspension, serial dilutions of each sample were plated for CFU enumeration to assess bacterial survival over time.

Intracellular localization and endosomal escape assay

To determine whether TrojanHorseβ[Sinon α] enables intracellular bacteria to escape from endosomal compartments and enter the host cytoplasm, we examined their intracellular localization by multichannel fluorescence imaging.

Bacterial cytoplasm was labeled with CFSE (green) to allow long-term tracking, while late endosomes and lysosomes were stained with LysoTracker Red (red). Nuclei were marked by Hoechst 44334 (blue), and cellular boundaries were outlined based on bright-field images due to instrument limitations in DID detection. Following co-culture of infected host cells under hypoxic conditions (5% O₂) for approximately 10 hours, confocal fluorescence microscopy revealed distinct cellular distributions of the labeled bacteria. Representative fluorescence micrographs are presented in Figure 26, where nuclei (blue), endosomes/lysosomes (red), and CFSE-labeled bacteria (green) are shown, with white contours delineating individual cell boundaries. The left and right panels correspond to the same microscopic field, displaying different channel combinations: the left panel shows the merged image of all fluorescence channels, whereas the right panel excludes the 488 nm channel to enhance visualization of bacterial spatial distribution.

Fluorescence microscopy revealed that TrojanHorseβ[Sinon α] were predominantly localized within the host cytoplasm rather than confined to endosomal or lysosomal vesicles. Only a minor subset of bacterial signals showed spatial overlap with LysoTracker staining, indicating limited retention within endosomal compartments. This spatial distribution pattern suggests that the majority of intracellular bacteria had successfully escaped from endosomal confinement and gained access to the cytosol.

Collectively, these observations demonstrate that TrojanHorseβ[Sinon α] facilitates efficient endosomal escape, thereby promoting cytoplasmic localization of intracellular bacteria following internalization.

Time-Dependent Bacterial Invasion Assay

To quantify the invasion efficiency of TrojanHorseβ[Sinon α] and provide experimental data for subsequent modeling, a time-course infection assay was performed. Engineered bacteria expressing FnBPA were added to adherent tumor cell cultures, and samples were collected every 2 hours over an 8-hour period. After each time point, extracellular bacteria were removed by gentamicin treatment, followed by washing and cell lysis with saponin. The resulting lysates were serially diluted (10×, 100×, and 1000×), and aliquots (10 μL) of each dilution were plated for colony formation counting after overnight incubation.

As shown in Figure 27, the intracellular bacterial concentration increased rapidly during the initial 2 hours and gradually reached a plateau at approximately 6 hours, remaining relatively stable thereafter. Quantitative analysis revealed that bacterial concentration rose from approximately 0.8 × 10⁻¹¹ mol/m³ at the onset to around 2.5 × 10⁻¹¹ mol/m³ at 6 hours, with no significant further increase up to 8 hours. This trend suggests that TrojanHorseβ[Sinon α] efficiently invades tumor cells within the first few hours of co-culture, followed by a saturation phase likely limited by cellular uptake capacity. The experimentally measured bacterial concentrations were consistent with the model predictions, differing by less than one order of magnitude.

Bacterial invasion assay in 4T1 tumor spheroids

In this experiment, we performed a 3D spheroid co-culture of 4T1 cells with our TrojanHorseβ [Sinon α]. To monitor bacterial movement, we used an upright fluorescence microscope (Eclipse Ni-U) to capture images at time intervals ranging from 30 minutes to 25.5 hours. Three parallel replicates were included, and for each microscopic observation, samples were randomly selected and imaged.

To simulate the invasion and activity of the engineered bacteria TrojanHorseβ [Sinon α] in a microenvironment more closely resembling in vivo conditions, this study employed a three-dimensional tumor spheroid model as the primary in vitro platform for cell–bacteria co-culture. Within the spheroids, gradients of oxygen and nutrients are established between the center and the periphery, with cellular activity generally decreasing from the outer layers toward the core. Such gradients cannot be observed in conventional plate-based cultures but represent critical factors influencing bacterial behavior and drug diffusion.

In this study, tumor spheroids were cultured in a honeycomb plate, and their fluorescence dynamics were monitored at defined time intervals ranging from 30 minutes to 25.5 hours using microscopy. By recording and analyzing the fluorescence signals over time, the invasion, distribution, and activity of the engineered bacteria within the spheroids were systematically assessed.

The following are the primary results of our experiments. Only potentially viable cells are presented here, while scientifically meaningless data have been excluded:

The experimental results showed that, despite strong background fluorescence and considerable disturbances, bacterial invasion into tumor cells was observed in most samples after 17.5 h of co-culture. This indicates that the engineered bacteria possess a clear ability to invade tumor cells. Compared with previous studies on bacterial invasion of tumor spheroids, the invasion process of this engineered strain was completed over a shorter duration, suggesting higher invasion efficiency and the potential to achieve the desired distribution within a shorter time frame. This characteristic also provides a basis for further optimization of co-culture duration and dosing strategies, facilitating more precise therapeutic control and improving experimental reproducibility.

Validation of E. coli Nissle 1917 strain construction

To validate the general applicability of the strategy of heterologous expression of invasion proteins in engineered bacteria, we plan to express the invasion protein in the probiotic strain Escherichia coli Nissle 1917. Specifically, we will construct the plasmids pUC57[J23100-sfgfp-RBS-invasin] (Sinon γ) and pUC57[J23105-sfgfp-RBS-invasin] (Sinon δ) to evaluate their effectiveness.

Plasmid Amplification and Reconstruction

The plasmid backbone used in this study is pUC57. To achieve both efficient invasion and convenient visualization, we constructed two plasmids by inserting J23100-sfgfp-RBS-invasin and J23105-sfgfp-RBS-invasin fragments into pUC57, thereby generating Sinon γ and Sinon δ. In this design, J23100 and J23105 serve as promoters, Invasin facilitates the invasion of engineered bacteria into cancer cells, and sfGFP provides fluorescence, enabling subsequent monitoring of bacterial entry into host cells.

The purified gene fragment was ligated into the plasmid backbone pUC57 using the One Step Cloning method, resulting in the plasmid construct shown in the figure. The recombinant plasmid was then introduced into Escherichia coli JM110 for amplification and demethylation, after which the plasmid was extracted.

Strain Construction and Colony PCR Verification

We have constructed the Sinon γ and Sinon δ plasmids and subsequently introduced them into Escherichia coli Nissle 1917. The following colony PCR results confirm the successful construction of the strains TrojanHorseγ[Sinon γ] (Nissle 1917[J23100-sfgfp-RBS-invasin]) and TrojanHorseγ[Sinon δ] (Nissle 1917[J23105-sfgfp-RBS-invasin]).

Growth Curve of Constructed E. coli Nissle 1917

To assess the growth status of bacteria required for co-culture, we measured the growth curve of Escherichia coli Nissle 1917 (EcN) carrying the invasin and sfgfp. In the experiment, the engineered strain was inoculated into LB liquid medium at a 1:100 ratio of culture to medium, placed in 15 mL centrifuge tubes, and incubated on a shaker at 37 °C and 199 r/min. During cultivation, samples were taken every 30–60 minutes, mixed by vortexing, and the optical density at 600 nm (OD₆₀₀) was measured using a microplate reader to generate the growth curve.

The results showed that EcN underwent exponential growth approximately between 0 and 150 minutes, after which it entered the stationary phase relatively early, with OD₆₀₀ stabilizing at around 0.8–1.0.

Validation and Characterization of Invasin (Experimental Planning Only)

We plan to co-culture the engineered strains TrojanHorseγ (Sinon γ) and TrojanHorseδ (Sinon δ) with triple-negative breast cancer cells, and we anticipate observing a pronounced invasion effect.

Hypoxia-Responsive Module Functional Test (Experimental Planning Only)

To evaluate the expression efficiency of the hypoxia-inducible promoter and facilitate the subsequent construction of a suicide switch for anticancer compound expression, we first plan to construct GFP reporter plasmids, Reporter ε (pLI50[nreABC-P_narT-sfgfp]Sep) and Reporter ζ (pLI50[nreABC-P_narT-sfgfp]Sxy), for promoter characterization. The successfully constructed plasmids will then be introduced into Staphylococcus epidermidis ATCC 14990 or Staphylococcus xylosus ATCC 29971. Cultures will be grown overnight under normoxic conditions or 1% O₂, and the results will be examined.

Plasmid Amplification and Reconstruction

First, primers were designed to amplify the target fragment nreABC-P_narT from Staphylococcus xylosus ATCC 29971 and Staphylococcus epidermidis ATCC 14990 (Vazyme: P520-01). Homology arms derived from pLI50[sfgfp] were added to both ends of the fragment to create overlapping regions for subsequent One Step Cloning. In addition, inverse PCR primers were designed to linearize the plasmid pLI50[sfgfp].

We employed the One Step Cloning (OSC) technique to insert the target fragment [nreABC-P_narT] into the plasmid pLI50[sfgfp].

We successfully constructed two reporter plasmids, Reporter ε and Reporter ζ, and introduced them into Escherichia coli JM110 for amplification and extraction. The subsequent plasmid sequencing results confirmed the correctness of the [nreABC-P_narT-sfgfp]Sep and [nreABC-P_narT-sfgfp]Sxy sequences, enabling their use in the next step of Staphylococcus transformation.

Strain Construction and Validation

Colony PCR verification

After plasmid extraction from JM110, we successfully introduced the plasmid into Staphylococcus xylosus ATCC 29971. The colony PCR results (shown below) confirmed the successful construction of the strain S. xy[nreABC-P_narT-sfgfp] (TrojanHorseβ[Reporter ζ]).

Due to the unresolved R-M system issue in Staphylococcus epidermidis ATCC 14990, only the plasmid Reporter ε was constructed, amplified, and sequenced in Escherichia coli JM110, without subsequent transformation into S. epidermidis.

Fluorescent microscopic examination

We employed confocal microscopy to examine the fluorescence of TrojanHorseβ[Reporter ζ] under both normoxic and hypoxic conditions. As controls, TrojanHorseβ[Reporter α] (S. xy[P_SarA1 + sfgfp]) under hypoxia was included as the positive control, while the wild-type Staphylococcus xylosus ATCC 29971 under normoxia served as the negative control. The results are shown in the figure below.

Characterization of Strain Growth Under Aerobic and Anaerobic Conditions

To test whether NO₃⁻ could mimic the bacterial growth state observed under normoxic conditions, we measured the static growth curve of Staphylococcus xylosus ATCC 29971 carrying Reporter α (TrojanHorseβ[Reporter α]). The engineered strain was inoculated into TSB medium at a 1:100 ratio of culture to medium and cultured in 10-cm cell culture dishes. In the experimental group, potassium nitrate was supplemented at a final concentration of 20 mmol, whereas the two control groups were cultured without potassium nitrate, under either normoxic or anoxic conditions, with three replicates each. Because the anaerobic incubator available in our laboratory lacked gas flow, while the bacterial incubator provided a minimum aeration of 40%, some measurement error was unavoidable. All plates were incubated statically at 35 °C, and samples were collected every 2–4 hours, vortexed to ensure uniformity, and measured for optical density at 600 nm (OD₆₀₀) using a microplate reader over a total period of 30 hours. Scatter plot data were subsequently fitted to obtain the growth curves shown below.

The results showed that under normoxic conditions without nitrate supplementation, bacterial growth was the most rapid, entering the exponential phase at approximately 10–15 hours and reaching a stable phase around 25–30 hours, with the final OD₆₀₀ reaching 2.5–3.0. In contrast, under anoxic conditions with nitrate supplementation, the growth rate was markedly reduced; the growth curve rose gradually and eventually stabilized at an OD₆₀₀ of about 1.5–2.0, indicating that nitrate could partially support bacterial growth in the absence of oxygen. Growth was most limited under anoxic conditions without nitrate, with OD₆₀₀ values remaining low and stabilizing at around 0.8, suggesting that bacterial metabolism was strongly inhibited in the absence of an electron acceptor. These findings demonstrate that nitrate can serve as an alternative electron acceptor under anoxic conditions, significantly improving the growth of S. xylosus ATCC 29971 and enabling higher cell density in the absence of oxygen; however, its growth capacity remained substantially lower compared with normoxic conditions. This result supports the hypothesis that nitrate can, to some extent, mimic the role of an electron acceptor in anoxic environments.

Suicide Switch Functional Test

To ensure that the engineered bacteria survive only within the tumor region, we placed the erythromycin resistance gene (ermC) under the control of the hypoxia-responsive module. By adding erythromycin to the culture medium, we tested whether the engineered bacteria could survive exclusively under specific conditions.

Plasmid Amplification and Reconstruction

For simplicity, we directly used the nreABC-P_narT fragment containing homology arms from pLI50[sfgfp]. Inverse PCR primers were designed for pUC57[ermC], and the same nreABC-P_narT homology arms were added at the insertion site to create overlapping regions for subsequent OSC assembly. For pUC57[ermC], the same plasmid was used for both Staphylococcus strains, and the nreABC-P_narT homology arms were derived from sfgfp, which share identical sequences in both strains.

We used the One Step Cloning (OSC) technique to insert the target fragment nreABC-P_narT into the plasmid pLI50[sfgfp].

We successfully constructed two plasmids, SuicideSwitch α and SuicideSwitch β, and introduced them into Escherichia coli JM110 for amplification and extraction. Plasmid sequencing results confirmed the correctness of the [nreABC-P_narT-ermC]Sep and [nreABC-P_narT-ermC]Sxy sequences, enabling their use in the next step of Staphylococcus transformation.

Strain Construction and Colony PCR Verification

After plasmid extraction from JM110, we successfully introduced the plasmid into Staphylococcus xylosus ATCC 29971. The colony PCR results (shown below) confirmed the successful construction of the strain S. xy[nreABC-P_narT-ermC] (TrojanHorseβ[SuicideSwitch β]).

Validation and Characterization (Experimental Planning Only)

We plan to add erythromycin to the culture medium and incubate the bacteria under different oxygen concentrations. The colony-forming ability under these conditions will be examined to evaluate whether the suicide switch has been successfully constructed.

Anticancer Substance Efficacy Test (Experimental Planning Only)

We plan to construct an engineered bacterial strain capable of secreting Apoptin. The P_narT promoter fragment will be amplified from Staphylococcus epidermidis ATCC 14990 and Staphylococcus xylosus ATCC 29971, and subsequently cloned into an Apoptin expression plasmid. The resulting construct will then be introduced into S. xylosus ATCC 29971 to evaluate the functional expression of Apoptin under hypoxic conditions.

Plasmid Amplification and Reconstruction

Reverse primers were designed to amplify the plasmid pLI50[apoptin], yielding a linearized vector. In parallel, the nreABC–P_narT fragment containing the apoptin homologous region was amplified from Staphylococcus xylosus ATCC 29971 and Staphylococcus epidermidis ATCC 14990. These overlapping regions were constructed to facilitate subsequent OSC assembly.

After constructing the two plasmids, they were transformed into Escherichia coli JM110 for amplification and extraction. Sequencing of the extracted plasmids revealed the absence of the nreABC-P_narT fragment.

Strain Construction and Colony PCR Verification

Upon successful plasmid construction, we plan to extract the plasmids and introduce them into Staphylococcus strains for subsequent evaluation of anticancer substance efficacy.

Validation and Characterization

Based on previous studies, Apoptin has been shown to selectively induce apoptosis in cancer cells while exerting minimal effects on normal cells. Therefore, we hypothesize that the engineered system will exhibit tumor-specific cytotoxicity in the TNBC model.