This page compiles all the reusable components we created throughout our project — including engineered genetic parts, functional modules, computational models, and educational frameworks. Each resource is well-documented, standardized, and validated, making it easy for future iGEM teams and researchers to adopt, adapt, and build upon our work.

By openly sharing these tools, we aim to reduce repetitive efforts, accelerate design and experimentation, improve safety and reliability, and inspire new innovations in synthetic biology and cancer therapy.

Experimental Contribution

Triple-negative breast cancer (TNBC) represents the most aggressive subtype of breast cancer, marked by rapid progression, poor prognosis, and the absence of effective targeted therapies. These challenges underscore the urgent need for innovative treatment strategies. The inspiration for the TroGen project came from recent studies on intratumoral commensal microbiota. Researchers have detected the presence of bacteria within triple-negative breast cancer (TNBC) cells, which persist as intratumoral commensals and influence tumor development, immune responses, and therapeutic outcomes. This discovery captured our interest — if these bacteria can naturally “recognize” and remain within tumor regions, we might be able to harness this inherent selectivity to develop a controllable tumor-targeting delivery platform. To explore this idea, we surveyed literature and microbiome databases to identify bacterial species enriched in tumor environments. Among them, the genus Staphylococcus stood out due to its highest relative abundance. In our preliminary screening, we selected two representative non-pathogenic species — Staphylococcus xylosus and Staphylococcus epidermidis — as chassis organisms for our engineering design.

Building on this foundation, the TroGen project aims to construct a versatile bacterial platform that integrates three key innovations: receptor-independent TNBC targeting, an intrinsic capacity for cancer cell invasion, and the use of non-pathogenic commensal bacteria as a safe and adaptable chassis. In addition, we developed an invasion-potentiation module and a hypoxia-responsive module, which not only enable the core functions of our project but also expand the genetic toolkit available for Staphylococcus. Using our constructs, bacteria can enter host cells and exhibit hypoxia-responsive expression of downstream proteins. We have also created two novel regulatory units: an RBS optimized for Staphylococcus spp., and a short scar sequence that minimizes the impact of terminator-induced secondary structures, thereby preserving promoter functionality and ensuring efficient protein expression. Collectively, these advances enrich synthetic biology resources and open new avenues for safer and more effective cancer treatment.

Three Innovative and Defining Platform Features

a) Receptor-Independent TNBC-Targeting Potential

Triple-negative breast cancer (TNBC), characterized by the absence of estrogen receptor, progesterone receptor, and HER2, does not benefit from conventional hormonal or targeted therapies. Chemotherapy, although still the mainstay of treatment, is limited by modest efficacy and severe toxicity, underscoring an urgent clinical need. Recent studies have revealed a remarkable selective enrichment of Staphylococcus commensals within TNBC cells, suggesting an inherent tumor-targeting potential shaped by the tumor microenvironment. Building upon this, TroGen pioneers the use of commensal bacteria in TNBC therapy, establishing a receptor-independent platform for targeted drug delivery. This innovation not only introduces a new therapeutic paradigm for TNBC but also enables efficient and precise delivery of diverse therapeutic agents.

b) Intrinsic Capacity for Cancer Cell Invasion

Building on the capacity of engineered bacteria to establish intracellular commensalism within triple-negative breast cancer (TNBC) cells, our delivery platform is particularly suited for therapeutics that require intracellular release. For example, small-molecule agents such as prodrugs, as well as cytoplasm-targeting proteins including Apoptin, Granzyme B, and Cas9, require cytosolic delivery to exert their function. Likewise, nucleic acid therapeutics such as siRNA and CRISPR systems, together with DNA-targeting agents, require nuclear delivery for biological activity. By combining the intrinsic cytoplasmic invasion ability of engineered bacteria with genetically programmed secretion of heterologous proteins, our platform enables precise intracellular drug release in TNBC cells, thereby enhancing the potency of specific therapeutics and improving overall treatment efficacy.

c) Use of non-Pathogenic Commensal Bacteria as the Chassis

In current bacteria-mediated cancer therapies, the chassis is typically derived from attenuated pathogenic strains, such as VNP20009 (from Salmonella enterica), Novyi-NT (from Clostridium novyi), SYNB1891, and Listeria-¹⁸⁸Re (both from Listeria monocytogenes). These engineered strains are generated by deleting major virulence factors, thereby reducing toxicity while retaining certain biological functions to improve therapeutic applicability. However, compared with natural commensal bacteria, attenuated pathogens still present inherent risks in terms of safety, genetic stability, and immunogenicity. In immunocompromised patients, residual toxicity can remain clinically hazardous, whereas commensal bacteria have coexisted with the host over evolutionary timescales and thus exhibit a higher intrinsic safety profile. Moreover, attenuated strains are prone to reversion mutations that may restore virulence and undermine the long-term stability of engineered modifications, while commensals generally possess more stable genomes and naturally lack major virulence determinants, offering a broader safety margin. Finally, attenuated pathogens tend to elicit strong immune responses that trigger inflammation, accelerate bacterial clearance, and shorten the therapeutic window, whereas commensals usually induce weaker immune stimulation, allowing prolonged persistence within the host and enabling sustained release of therapeutic payloads.
Against this backdrop, TroGen introduces a novel chassis class based on commensal Staphylococcus. Unlike attenuated pathogens, our strains combine receptor-independent tumor-targeting capacity with an intrinsic ability for intracellular invasion, while their evolutionary adaptation to the host provides natural safeguards of safety and stability. This design not only addresses the longstanding limitations of pathogen-derived chassis but also establishes a new paradigm and generalizable resource for researchers developing bacteria-mediated cancer therapies.

Engineered functional modules

a) Invasion-Potentiation Module (IPM)

We constructed an Invasion-Potentiation Module (IPM) to enhance the ability of engineered Staphylococcus strains to invade triple-negative breast cancer (TNBC) cells. The central element of this module is the fnbA gene from the Staphylococcus aureus genome, which encodes fibronectin-binding protein A (FnBPA). By promoting integrin-mediated endocytosis, FnBPA markedly increases bacterial internalization into host cells. To integrate functional expression with monitoring, the IPM was designed as a bicistronic construct in which a single constitutive promoter (P_SarA1) drives both fnbA and sfgfp. In this configuration, sfGFP fluorescence provides a direct proxy for FnBPA expression, offering a built-in reporter system while avoiding additional verification steps for protein production and localization. Beyond its immediate utility in our project, the IPM establishes a standardized invasion-enhancement cassette that can be readily adopted by future iGEM teams and researchers to study bacterial–host interactions, improve intracellular delivery platforms, and expand the synthetic biology toolkit available for commensal Staphylococcus engineering.

b) Hypoxia-Responsive Module (HRM)

We constructed a Hypoxia-Responsive Module (HRM) to enable engineered Staphylococcus strains to sense the hypoxic tumor microenvironment (TME). The HRM consists of the endogenously conserved nreABC operon and its downstream promoter P_narT. This three-component regulatory system activates P_narT under hypoxic conditions and reaches maximal induction in the presence of nitrate, thereby providing graded control of safety modules in response to oxygen and nitrate inputs. In general, this HRM can serve as a basic TME-sensing genetic element in Staphylococcus species. Beyond serving as a fundamental TME-sensing element in Staphylococcus species, we have already harnessed this module to build two functional applications — a suicide switch and a controlled drug release system — demonstrating both its immediate utility and its broader potential as a reusable biosafety component. In the future, the HRM can also be extended to drive other environment-responsive circuits, thereby enriching the synthetic biology toolkit for tumor therapy and microbial engineering.

Novel Genetic Parts

a) Hypoxia-Inducible Promoter: nreABC-P_narT (BBa_25OWXPOU / BBa_25CNB3FP)

The nreABC operon and the P_narT promoter were obtained from the genomes of Staphylococcus sylosus ATCC 29971 and Staphylococcus epidermidis ATCC 14990. The operon functions as a sensor of low oxygen levels and drives the expression of downstream genes, whereas the promoter is activated under hypoxic conditions in the presence of nitrate or nitrite. Consequently, this part can be positioned upstream of any coding sequence (with an RBS) to achieve hypoxia-responsive expression of the target gene.

b) Invasion Protein: fnbA (BBa_25L8LT5I)

The modified fnbA gene (codon-optimized for Staphylococcus) encodes a member of the Fibronectin-Binding Protein (FnBP) family, which mediates host cell invasion. FnBPs form a high-affinity complex upon binding to extracellular fibronectin (Fn), ultimately promoting progressive membrane extension and bacterial engulfment. When expressed under standard regulatory elements for single ORF, fnbA efficiently facilitates entry into multiple host cells.

c) NNB001 (BBa_25YLO08T)

NNB001 is a novel regulatory element that we originally designed for use in Staphylococcus chassis strains. It is composed of repeated adenine and thymine residues, designed to serve as a neutral spacer between transcription units. This design mitigates the formation of terminator-induced secondary structures and provides insulation between upstream terminators and downstream promoters.

In our experiments, we verified that inserting NNB001 between an upstream terminator and a downstream promoter did not interfere with downstream gene expression, confirming its compatibility in Staphylococcus chassis strains. Although we did not conduct a systematic quantitative characterization of its regulatory effects, this validation indicates that NNB001 can serve as a simple and non-disruptive tool for modular design. By submitting this part, we contribute a novel regulatory resource to the synthetic biology toolkit, specifically expanding the options available for engineering Staphylococcus.

d) NNB002 (BBa_25R9JV89)

NNB002 is a novel regulatory element originally designed and validated by our team in Staphylococcus. It is intended to be positioned between a promoter and a start codon with the following arrangement: promoter-[short spacer]-upstream linker (5 bp)-AGGAGG (RBS motif)-downstream linker (8 bp)-start codon. The short spacer (typically 5–10 bp), which may consist of a restriction site or auxiliary fragment, is recommended for proper function, as our validation was performed under this configuration; the outcome of omitting it has not been systematically tested.

This modular design balances flexibility and stability. The spacer provides freedom for user-defined sequence additions, while the streamlined RBS core minimizes secondary structure formation that could interfere with ribosome binding. As a result, NNB002 supports broad compatibility, reliable ribosome accessibility, and efficient translation initiation across diverse sequence contexts.

In our project, we placed an OSC linker G and a BamHI-3’ restriction site in the spacer position upstream of NNB002. Experimental validation confirmed that this arrangement enabled stable, reproducible, and high-level gene expression in Staphylococcus. By contributing NNB002, we expand the synthetic biology toolkit with a structurally optimized, context-resilient, standardized RBS element, providing a valuable resource for modular genetic design in Staphylococcus.

Modeling Contribution

We integrated protein engineering and computational modeling to analyze multiple FnBPs, establishing a comprehensive framework for FnBP screening. Additionally, we constructed a diffusion–convection–growth model of bacterial dynamics within tumor cells, providing a reusable and optimizable foundation for future research in synthetic biology and bacteria-based cancer therapy.

Analysis Data of Common FnBPs

We collected all commonly studied FnBPs and conducted molecular-level investigations. A systematic molecular study was carried out on the interactions between representative fibronectin-binding proteins (FnBPs) and fibronectin (Fn). The analyses of RMSD, binding free energy and binding hotspot residues could provide valuable reference data for related research.

Reusable Protein Screening Framework

We established a standardized and transferable computational pipeline for FnBP screening and evaluation.

1. Phylogenetic level:
   Perform 16S rRNA-based phylogenetic analysis between the donor strains carrying FnBPs and the engineered host strains, providing an evolutionary basis for protein selection.
2. Molecular level:
   •Predict protein complex structures using AlphaFold-Multimer.
   •Conduct molecular dynamics (MD) simulations with GROMACS to assess structural stability.
   •Calculate the binding free energy of protein complexes using MM/PBSA, based on the RMSD-stable time window.
   •Perform energy decomposition analysis to identify binding hotspot residues that contribute most to the binding free energy.
3. Integrative analysis Integrate results from both the phylogenetic and molecular levels to identify the most suitable FnBP as an exogenous invasion protein.

This standardized workflow provides a reproducible framework for future research in protein engineering and synthetic biology.

Reference Model for Intracellular Bacterial Diffusion–Convection–Growth

To capture the migration and proliferation of bacteria within tumor tissues, we developed a systematic kinetic model. This model explicitly decomposes flux into diffusion and convection components, couples Monod nutrient-dependent kinetics with a Logistic density-limiting mechanism, and incorporates a yield coefficient to quantitatively describe the relationship between bacterial growth and substrate consumption. Such decomposition and treatment provide a practical reference for future teams constructing similar models. Moreover, this framework may inspire studies of bacterial dynamics following cellular invasion, where the model can be further refined and optimized for related research.

Bacterial equation

$$\frac{\partial b}{\partial t}\;-\;\nabla\!\cdot\!\left(D_{b}\,\nabla b\right)\;+\;\vec{u}\!\cdot\!\nabla b \;=\; \mu_{\max}\,\frac{c}{K_{m}+c}\,\left(1-\frac{b}{K_{B}}\right)\,b$$

Substrate equation

$$\frac{\partial c}{\partial t}\;-\;\nabla\!\cdot\!\left(D_{c}\,\nabla c\right)\;+\;\vec{u}\!\cdot\!\nabla c \;=\; -\,\frac{1}{Y}\,\mu_{\max}\,\frac{c}{K_{m}+c}\,b$$

Human Practices Contribution

In order to make a tangible and valuable contribution to the iGEM community, our team has summarized and systematically constructed an “Integrated All-ages Education Model” file. Guided by the core idea that “education should not be confined to a single group but should cover all stages of life,” this model integrates synthetic biology education with the characteristics of learners at different ages, forming a framework that is both universal and practical.

Within this model, we divided learners into several stages: children, adolescents, university students, and middle-aged/elderly groups. For each stage, we outlined not only the corresponding educational objectives and curriculum design, but also appropriate teaching methods. For example, the elementary school stage focuses on sparking interest and hands-on practice; the middle school stage emphasizes logical thinking and analogy-based understanding; the university stage highlights research literacy and interdisciplinary exploration; while the elderly stage is centered on health awareness, gentle reminders, and supportive care. Through this step-by-step and stage-specific approach, we strive to realize the principle of “education tailored to both age and need.”

Moreover, this model was not built only on theoretical considerations, but has been continuously tested and iterated through our diverse educational practices. We implemented it in elementary classrooms with “clay cell modeling,” in middle schools through “experiments and case-based teaching,” in high schools via “summer schools and logic-oriented lessons,” in universities with “art exhibitions and interdisciplinary discussions,” and in communities for the elderly through “health advocacy and companionship activities.” These practices not only enriched the content of the model but also validated its feasibility and scalability.

We have compiled this model into a structured document and made it available to the community, hoping it can serve as a valuable resource for future iGEM teams. Whether used as a reference framework for designing educational activities, or as a foundation for improvement and expansion, it can help teams save exploration costs, provide systematic guidance, and inspire further innovative approaches. We believe that as more teams adopt and refine this model, it will continue to evolve globally, thereby advancing the outreach and development of synthetic biology education.