Synthetic biology seeks to achieve the predictable behavior, reusability, and standardization of biological systems through modular design, standardized biological components, and engineering methodologies. In our project, we address the challenge of material damage in soft robots during marine exploration by developing a dual-functional soft material system with both self-healing and adhesive capabilities, integrating engineering principles of synthetic biology throughout the design process. Our system is composed of several standardized and modular components, including a self-healing module, an adhesion module, and a tyrosinase catalytic module. Each module is systematically engineered to meet specific functional needs and can be flexibly combined to optimize overall system performance.

Within this self-healing system, the squid ring teeth protein (BBa_K5398001) confers self-healing properties. Its highly repetitive amino acid sequences form stable β-sheet structure, enabling the material to recover its original structure and function via hydrogen bonding when damage occurs. The mussel foot protein (Mfp5) within the fusion protein TRn4-Mfp5 (BBa_K5398030) imparts strong adhesive properties, particularly under wet and dynamic marine environments. To ensure stable adhesion, we integrated tyrosinase (TyrVs, BBa_K5398600), which catalyzes the conversion of tyrosine in Mfp5 into L-DOPA, thereby facilitating effective binding to the soft robot's surface. Furthermore, we co-expressed mussel foot protein 6 (Mfp6, BBa_K5398601) to prevent excessive oxidation of L-DOPA, thus enhancing adhesion stability and robustness.

Fig. 1 | Integrative engineering framework for the three modules.

●Self-Healing Module Subcollection

●Adhesion Module Subcollection

●Tyrosinase Catalysis System Subcollection

All of the biological parts used, whether basic or composite, obey iGEM standards RFC [10] and RFC [1000]. In our experimental procedures, we employed E. coli strain DH5α for routine cloning purposes and BL21(DE3) for protein expression, ensuring efficient implementation of standardized workflows.

Our modular design ensures that each component functions independently while maintaining synergistic interactions that contribute to enhanced system performance. This approach is consistent with the foundational principles of synthetic biology—standardization, reusability, and predictability—enabling our system to achieve self-repair and long-term operational robustness in challenging marine environments.

Table 1 | Part Collection Collection

To impart self-healing functionality to the material, we utilized the squid ring teeth protein (BBa_K5398001), composed of highly repetitive amino acid sequences that form stable β-sheet structures. These structures interact through intermolecular hydrogen bonds, creating robust cohesion that enables the material to self-heal after damage. To facilitate the production of these repetitive proteins, we employed self-splicing ribozymes (BBa_K5398002 and BBa_K5398003), leveraging the cyclization of the T4 phage td gene intron to generate circular mRNA, providing a continuous translation template and using the RBS we constructed (BBa_K5398004 )to achieve efficient expression of these repetitive proteins. This design allows ribosomes to translate the mRNA loop repetitively, generating long polypeptide chains, thereby significantly enhancing self-healing efficiency and demonstrating the advantages of high-level engineered expression.

The rapid self-healing properties of this module, enabled by hydrogen bonding and π-π interactions, allow the material to recover its mechanical strength and function quickly, making it suitable for aerospace materials, flexible electronics, and robotic skin. Furthermore, the circular mRNA system facilitates the efficient production of high-strength composite materials with repetitive sequences, such as spider silk proteins and keratins, expanding the module's applicability to smart sensors and high-strength building materials.

Table 2 | Self-Healing Module Subcollection

To achieve stable attachment of the self-healing material to the soft robots' surface, we developed a fusion protein, TRn4-Mfp5 (BBa_K5398030), which integrates both self-healing and adhesion properties. Tyrosine residues in Mfp5 are enzymatically converted to L-DOPA by tyrosinase (TyrVs, BBa_K5398600), allowing L-DOPA to form non-covalent bonds with the surface of the soft robot, ensuring effective adhesion even in highly humid environments. The incorporation of the TRn4 repeat sequence facilitates robust binding through β-sheet interactions, further enhancing adhesion.

This module's excellent adhesion performance makes it suitable for diverse applications, including wound repair and underwater ecosystem restoration. By providing stable tissue adhesion in biomedical settings and serving as an eco-friendly alternative to chemical adhesives in environmental applications, this module supports both medical and conservation needs.

Table 3 | Adhesion Module Subcollection

To catalyze the conversion of tyrosine residues in Mfp5 to L-DOPA, we integrated tyrosinase TyrVs (BBa_K5398600) into our system. However, TyrVs can also induce over-oxidation of L-DOPA to dopaquinone, compromising adhesion performance. To mitigate this issue, we co-expressed Mfp6 (BBa_K5398601), which contains cysteine residues capable of reducing oxidized dopaquinone back to L-DOPA, thereby maintaining stable adhesive properties and preventing over-oxidation. The integration of the tyrosinase catalytic module with Mfp6 enhances the system's adaptability in humid environments, allowing effective adhesion even under prolonged high-humidity conditions.

This catalytic module provides precise control of L-DOPA production and oxidation, making it highly valuable for developing DOPA-based functional materials. Such control is particularly beneficial in surgical applications, including tissue adhesive gels and coatings for medical implants, enhancing both adhesive stability and functional controllability.

Table 4 | Tyrosinase Catalysis System Subcollection

Our project integrates the self-healing module, adhesion module, and tyrosinase catalytic system to work synergistically. The self-healing module provides resilience through hydrogen bonding and β-sheet structures of the squid ring teeth protein, while the adhesion module offers strong binding in wet environments via mussel foot protein. The tyrosinase module enhances adhesion robustness by regulating L-DOPA production. Together, these modules ensure the material can recover its strength and maintain stable adhesion after damage.

Beyond application in marine exploration soft robots, our modules have significant transferability to various fields, including implantable medical devices, wound adhesives, robotic skin, and flexible electronics. The self-healing properties provide protection, while the adhesion module is biofriendly and well-suited for biomedical adhesion. The tyrosinase system provides stable adhesion, making it crucial for maintaining performance in challenging environments.

Future teams can utilize our modules across different scenarios. The self-healing module supports efficient expression of repetitive protein sequences, making it suitable for developing high-performance materials like spider silk proteins and keratins. The tyrosinase module can be used for precise control of DOPA-based systems by adjusting Mfp6 expression, particularly for applications such as biosensors and biocompatible coatings. This modular design provides a flexible framework for diverse applications in synthetic biology.