We designed 20 basic parts in total this year, integral to our whole cycle. You can click on the part to see details.

ID	Name	Type
BBa_25I6XUO4	CTT1	Coding
BBa_2573VF26	P2A	Coding
BBa_25TP2U5B	NCW2	Coding
BBa_25H7TVQ3	K1 killer preprotoxin	Coding
BBa_25US78QJ	Mature K1 toxin, αβ	Coding
BBa_25AHJ8GK	Aga2	Coding
BBa_250TUPOE	HFBI	Coding
BBa_25S7V4WC	Agasp	Coding
BBa_25HGKJYB	Fast-PETase	Coding
BBa_25MM547K	MHETase_W397A	Coding
BBa_256ZL14D	Ade2	Coding
BBa_25BPYSZW	dCas9	Coding
BBa_25ZA5KX4	scRNA	Coding
BBa_25RAOO82	TFF3	Coding
BBa_25SZAZHQ	MCP-MXI1	Coding
BBa_25QQBIJL	nucA	Coding
BBa_25JDFJTZ	pKlPDC1	Promotor
BBa_25276CLP	pGPX2	Promotor
BBa_25DEG8JB	pHSP26	Promotor

This year we designed a total of 13 composite components, each assembled from different Basic Parts, forming the core of the circuit system. Click on a component to view detailed information.

ID	Name	Type
BBa_25FTUMM2	pGPX2-NCW2-SOD1-P2A-NCW2-TFF3-T2A-NCW2-CTT1	Device
BBa_25CH63HC	Pgal1-Agasp-HFBI-(GGGGS)₃-Aga2-GGGGS-V5Tag	Device
BBa_25JZF6MI	Pgal1-Agasp-HFBI-(GGGGS)8-Aga2-GGGGS-V5Tag	Device
BBa_25GGWA1K	lox66-ADE2-lox71	Device
BBa_2505D14H	pHXT1-dCas9-ADH1t	Device
BBa_25CW3NA4	pCUP1-scRNA-ADH1t	Device
BBa_257VLKZS	pKlPDC1-MCP_MXI-ADH1tI	Device
BBa_25TG86BB	pFUS1-α_factor-FastPETase	Device
BBa_2578DUB6	pFUS1-α_factor-MHETase_W397A	Device
BBa_258Y1FP1	pHSP26-tetR-ptetR-Cre Recombinase	Device
BBa_25PVXDIE	pHSP26-tetR-ptetR-nucA	Device
BBa_25WZWETT	pNOTAra-K1 killer preprotoxin	Device
BBa_254EJU32	pAra-mature K1 toxin (αβ)	Device

(1) Degradation Module
The enzymatic degradation module enables optimized design, precise expression, and quorum-sensing-based regulation of microplastic-degrading enzymes, ensuring efficient degradation under intestinal conditions while minimizing metabolic burden.
a. Enzyme Optimization: We introduced two enzymes, Fast-PETase and the modified MHETase_W397A, both exhibiting high degradation activity toward microplastics at low temperatures and within the gut environment. Each enzyme is secreted via an α-factor signal peptide to prevent intracellular accumulation, thereby enhancing degradation efficiency and system stability. Related parts: BBa_25HGKJYB (Fast-PETase), BBa_25MM547K (MHETase_W397A), BBa_25TYTW3T (α-factor signal peptide).
b. Conditional Expression Control: To ensure activation only under specific intestinal conditions, we employed glucose-sensitive promoter pHXT1, copper-ion-sensitive promoter pCUP1, and hypoxia-sensitive promoter pKlPDC1 to drive expression of dCas9, scRNA, and MXI1, respectively. These parts together form a ternary CRISPR/dCas9 system responsive to glucose, copper ions, and hypoxia. Only when all three conditions are met is BAR1 repressed, thereby activating the downstream quorum-sensing pathway. Related parts: BBa_2505D14H (pHXT1-dCas9), BBa_25CW3NA4 (pCUP1-scRNA), BBa_257VLKZS (pKlPDC1-MCP_MXI1).

c. Quorum-Sensing Regulation: Upon BAR1 repression, α-factor accumulates, activating the FUS1 promoter via quorum-sensing signaling and driving enzyme expression. This design couples enzyme production with cell density, ensuring degradation only after population stabilization and achieving temporal and energetic efficiency. Related parts: BBa_25TG86BB (pFUS1-α_factor-FastPETase), BBa_2578DUB6 (pFUS1-α_factor-MHETase_W397A).

(2) Adsorption Module
The adsorption module utilizes a yeast surface display system to enable active microplastic adsorption, increasing degradation efficiency and achieving orthogonal temporal regulation relative to the degradation module.
a. Surface Display System Design: Using the structural components of the α-agglutinin system (Aga1/Aga2) and hydrophobin HFBI, we constructed a Saccharomyces cerevisiae-based surface display system. Aga2 is linked to Aga1 via disulfide bonds, and the HFBI fusion anchors to the cell wall, conferring hydrophobicity and strong PET-binding capacity. This design enhances substrate availability for downstream degradation. Related parts: BBa_25CH63HC (Pgal1-Agasp-HFBI-(GGGGS)₃-Aga2-GGGGS-V5Tag), BBa_25JZF6MI (Pgal1-Agasp-HFBI-(GGGGS)₈-Aga2-GGGGS-V5Tag).
b. Orthogonal Regulation Strategy: Leveraging the carbon-source-responsive GAL1 promoter (Pgal1), the adsorption module activates under glucose depletion, while the degradation module functions under glucose abundance. This reverse regulation ensures temporal separation between adsorption and degradation, minimizing energy waste. The system adsorbs microplastics during fasting and degrades them post-feeding, optimizing energy use and maintaining cell homeostasis.

(3) Anti-inflammatory and Antioxidant Module
The therapeutic module provides triple protection via coordinated expression of antioxidant, mucosal repair, and reactive oxygen species (ROS) scavenging genes, mitigating inflammation and oxidative damage caused by microplastics in the gut.
a. Multigene Co-expression Design: The oxidative stress-inducible GPX2 promoter drives a polycistronic therapeutic circuit comprising SOD1, TFF3, and CTT1, responsible for superoxide removal, mucosal repair, and hydrogen peroxide decomposition, respectively. The genes are connected via 2A peptide sequences to ensure efficient co-translation and stable stoichiometry. Related part: BBa_25FTUMM2 (pGPX2-NCW2-SOD1-P2A-NCW2-TFF3-T2A-NCW2-CTT1).
b. Secretion and Functional Implementation: To enable extracellular activity, each gene includes an N-terminal NCW2 signal peptide for secretion. SOD1 and CTT1 form a cascade reaction to eliminate ROS and H₂O₂, while TFF3 promotes mucosal healing, collectively realizing an integrated “response–repair–clearance” mechanism. This establishes a gut-friendly therapeutic system that enhances the engineered strain’s anti-inflammatory and antioxidant capacity. Related parts: BBa_25TP2U5B (NCW2 signal peptide), BBa_2573VF26 (P2A), BBa_25FTUMM2.

(4) Safety Module
The safety module ensures biocontainment and controllability of engineered yeast both in vivo and ex vivo through a dual-layered safeguard system that allows survival and function only under designated conditions.
a. In Vivo Control: We employed the native yeast K1 killer toxin system to design an arabinose-dependent suicide switch. The system includes two components: K1 killer preprotoxin and mature K1 toxin. In the absence of arabinose (gut conditions), cells express the precursor to neutralize the toxin; in the presence of arabinose, mature toxin accumulates and the precursor is depleted, leading to cell death. This prevents uncontrolled proliferation or environmental escape. Related parts: BBa_25WZWETT (pNOTAra-K1 killer preprotoxin), BBa_254EJU32 (pAra-mature K1 toxin (αβ)).

b. Ex Vivo Control: A temperature-dependent safeguard ensures automatic inactivation outside the host. The HSP26 promoter drives a tetR-regulated kill switch: at ≥37 °C, downstream expression is repressed; at lower temperatures, repression is lifted, activating the lethal module. The module includes nucA, a nuclease that degrades intracellular DNA, and an ADE2-deficient strain design that prevents growth in adenine-limited environments. This dual containment guarantees activity only within target conditions. Related parts: BBa_258Y1FP1 (pHSP26-tetR-ptetR-Cre Recombinase), BBa_25PVXDIE (pHSP26-tetR-ptetR-nucA), BBa_256ZL14D (lox66-ADE2-lox71).

In terms of contributions, we experimentally validated and functionally simulated several parts, including BBa_25TP2U5B, BBa_25MM547K, BBa_25DEG8JB, BBa_25US78QJ, etc. And we also verified several composite parts that incorporate basic parts developed by other teams, such as BBa_25FTUMM2, BBa_2578DUB6, BBa_258Y1FP1, BBa_254EJU32, etc. All experimental data and documentation have been uploaded to the corresponding Biological Parts pages. These results may provide valuable references for other teams.

We have organically integrated a series of parts to build an “intelligent” system for intestinal microplastic degradation and therapy. The surface display parts enable yeast to actively “capture” plastic particles, while quorum-sensing parts ensure that degradation enzymes are precisely expressed at the optimal time, achieving efficient degradation. By leveraging the properties of different promoter parts, the adsorption and degradation modules operate in a staggered manner, maximizing the efficiency of energy utilization. In addition, the therapeutic parts allow engineered yeast to sense intestinal oxidative stress and repair damaged intestinal mucosa. Last but not least, the safety parts establish a dual-layer “self-destruction switch” for both internal and external environments, ensuring robust biosafety. The recorded data will be of great value to future teams and contribute meaningfully to intestinal microplastic treatment.

This year, the iGEM Registry underwent a comprehensive reconstruction. Our parts have been standardized and uploaded according to the new requirements, with experimental data and documentation attached to their respective Biological Parts pages, providing validated content for the updated Registry platform. Furthermore, as Saccharomyces cerevisiae was newly added to the whitelist this year, our uploaded parts offer multiple functional components for this chassis, providing valuable resources and references for future synthetic biology research in yeast.

This year, to promote the clinical application of engineered bacteria in treating microplastic hazards in the human intestinal tract, our team has not only simulated the drug delivery kinetics in the gastrointestinal tract from a macroscopic perspective but also constructed multi-dimensional models from a microscopic angle, including signal pathway regulation, metabolic toxicity assessment, and core target prediction. By integrating numerical simulation, kinetic modeling, metabolic flux analysis, and network toxicology, we have established the following key models and research:

(1) Numerical Study on the Convection-Diffusion Wall Adsorption of Intestinal Products under Peristalsis-Segmentation Synergy
With the continuous development of oral drug delivery technology, the movement, mixing, and adsorption behavior of engineered bacteria in the intestinal tract have become key factors affecting therapeutic efficacy. Numerous studies have shown that gastrointestinal peristalsis, pyloric gating, and intestinal segmentation collectively determine the spatial distribution and action time of drugs. However, there is currently a lack of systematic simulation tools that integrate multi-scale dynamic processes.

Taking this opportunity, we have built an integrated numerical model of the stomach, pylorus, duodenum, and jejunum based on COMSOL 6.3, coupling dynamic mesh, laminar flow, dilute substance transport, and boundary ODE/DAE techniques. This model enables the full-process simulation of the engineered bacteria suspension under the synergy of peristalsis and segmentation. The model reveals the dual regulatory mechanisms of pyloric gating determining the migration "jump distance" and segmentation movement dominating the near-wall mixing. The wall adsorption module quantifies the coupling relationship of "concentration-shear-retention".

The application of this model provides quantitative basis for optimizing the dosage of engineered bacteria and predicting the action sites. By adjusting parameters such as the pyloric gating period and segmentation frequency, the drug distribution under different physiological states can be predicted, guiding the design of formulations. We expect this simulation platform to provide strong support for the development of oral engineered bacteria formulations and promote the advancement of precision drug delivery technology.

(2) α-Factor Quorum Sensing Kinetic Model
In the design of synthetic biology signal circuits, achieving density-dependent gene expression initiation and amplification is a core challenge. Although the yeast α-factor system has been widely used, there is a lack of kinetic models that quantitatively describe its self-stimulatory amplification and self-limiting steady-state mechanisms.

We have constructed a complete kinetic model of the Saccharomyces cerevisiae α-factor signaling pathway based on the ODE framework, covering receptor activation, MAPK cascade amplification, and autocrine positive feedback. The model reveals the dual mechanism of "self-stimulatory amplification-self-limiting steady-state" in this system: a significant increase in Fus3 concentration achieves signal amplification, while negative regulation at high concentrations effectively avoids non-physiological over-amplification. This provides a kinetic basis for the quantitative design of density-dependent initiation modules in engineered bacteria.

(3) Metabolic Toxicity Assessment Model
The introduction of complex genetic circuits imposes significant metabolic burdens on host cells, but there is currently a lack of quantitative tools for systematically assessing the energy consumption of exogenous gene expression. We used COBRApy to conduct metabolic flux analysis on Saccharomyces cerevisiae with different functional modules introduced, quantifying the energy consumption for the synthesis and secretion of nine exogenous proteins.

Sensitivity analysis shows that protein length is the most sensitive parameter for energy consumption, with long-chain proteins having a significant energy burden. At conventional expression levels, the host growth rate remains above 90% of the baseline, verifying the metabolic compatibility of the circuit. This framework provides a quantitative basis for optimizing engineered bacteria circuits and avoiding toxicity.

(4) FVA-Constrained Model
Metabolic flux exchange in symbiotic systems is crucial for the colonization and functional performance of engineered bacteria. We have constructed a metabolic coupling model of Saccharomyces cerevisiae and Lactobacillus plantarum based on COBRApy, integrating FBA and FVA analysis to simulate the dynamics of dual-bacteria symbiosis.
The model reveals the bidirectional transfer pathways of key metabolites such as lactic acid and amino acids, quantifying the coupling relationship of "symbiotic enhancement-energy relief". The results demonstrated that the symbiotic system significantly enhanced the growth rate of yeast and reduced the ATP maintenance burden, verifying the metabolic feasibility and robustness of the system and providing a theoretical basis for the collaborative design of multiple strains.

(5) Core Target and Pathogenic Mechanism Prediction Model
With the increasing severity of environmental microplastic pollution, the potential impact of PET microplastics on human health has attracted widespread attention. Although some scholars have noted the association between microplastic exposure and IBD, there is a lack of systematic molecular mechanism research to clarify the pathogenic pathways.
We developed a multi-level target prediction and mechanism analysis model, integrating network toxicology, machine learning, molecular docking, and dynamics simulation techniques. Ultimately, we identified three core molecular targets: JAK2, IL2, and TGFBR2, and systematically elucidated the four major mechanisms by which PET affects IBD progression: oxidative stress, immune dysregulation, uncontrolled cell proliferation, and intestinal barrier disruption.
Based on the systematic identification of core targets, this model provides multiple potential application directions for the precise treatment of IBD. From a preventive perspective, this model can also help identify high-risk populations exposed to microplastics, enabling early warning and intervention. We hope that this model not only deepens the understanding of the pathogenic mechanisms of environmental pollutants but also translates into practical clinical applications, opening up new avenues for the prevention and treatment of diseases related to microplastic exposure.
In addition, to assist in the experimental verification of the MXI1-mediated transcriptional inhibition pathway, we systematically analyzed the interaction mechanism of the MXI-Sin3-HDAC complex through bioinformatics methods, providing a theoretical basis for the promoter inhibition mechanism and guiding experimental design and functional verification. (For detailed content, see the Model section.)