In modern surgery, surgical sutures, as one of the most fundamental medical devices, have a performance that directly impacts the quality of a patient's postoperative recovery. However, sutures currently used in clinical practice commonly suffer from inherent drawbacks, such as being monofunctional, prone to causing infections and inflammatory responses, and sometimes requiring a second procedure for removal. These issues not only cause significant patient suffering but also increase the burden on the healthcare system. Facing this long-standing clinical challenge, our team envisioned a transformative question: Can we upgrade the surgical suture from a passive "physical connector" into an active "bio-intelligent wound management platform"?

Based on this vision, we initiated the ReGenStitch project. The core engineering goal of this project is to leverage the modular design principles of synthetic biology to develop a new generation of bioactive sutures that integrate three major functions: high-strength structural support, broad-spectrum antimicrobial and rapid hemostatic properties, and precise anti-inflammatory and scar-inhibiting capabilities.

To systematically achieve this ambitious goal, we rigorously followed the Design-Build-Test-Learn (DBTL) engineering cycle championed by iGEM. We deconstructed the entire project into three independent, parallel-developable functional modules. Each module underwent at least one complete DBTL cycle to ensure its functional robustness and design validity. This modular engineering strategy enabled us to tackle technical challenges one by one, mitigate overall project risks, and lay a solid foundation for final system integration.

Our engineering practice was divided into four core cycles:

Cycle 1: Engineering of the Bacterial Cellulose (BC) Production System (Structural Backbone Module)

Engineering Challenge: To achieve efficient and controllable production of bacterial cellulose in a non-native, safe, and reliable chassis cell.

Core Strategy: To construct a basic production unit by heterologously co-expressing the key cellulose synthesis genes, bcsA and bcsB, from Gluconacetobacter xylinus in Escherichia coli BL21(DE3).

Cycle 2: Engineering of the Chitosanase (CHI) Surface Display System (Antimicrobial & Healing Module)

Engineering Challenge: To overcome the cost and efficiency bottlenecks of traditional intracellular enzyme extraction processes by developing a reusable "whole-cell biocatalyst" that does not require cell disruption.

Core Strategy: To utilize Ice Nucleation Protein (INP) as a molecular anchor to stably display chitosanase (CHI1) on the outer membrane of E. coli, enabling efficient catalysis of chitosan.

Cycle 3: Engineering of the Curcumin Biosynthetic Pathway (Anti-inflammatory Module)

Engineering Challenge: To successfully reconstruct a complex plant secondary metabolic pathway in a prokaryotic chassis for the de novo biosynthesis of the anti-inflammatory molecule, curcumin.

Core Strategy: To co-express two key synthesis enzyme genes from turmeric, DCS and CURS1, in Escherichia coli BL21(DE3).

Cycle 4: System Integration and Manufacturing of the Suture Prototype (Suture Prototype Integration & Manufacturing)

Engineering Challenge: To organically composite the distinct bioactive components produced in the first three cycles using materials engineering methods, forming a macroscopic thread-like material prototype that possesses both sufficient mechanical strength (for suturing) and flexibility (for knotting).

Core Strategy: To employ a solution blending, casting, and slitting fabrication process to composite bacterial cellulose (BC) as the structural backbone with chitosan/chitooligosaccharides (COS) and curcumin as the functional matrix. Concurrently, to explore and optimize the physical properties and handling characteristics of the final suture prototype by adding glycerol as a plasticizer and systematically testing different concentrations of thermoplastic starch (TPS) as a performance modulator.

Through these four interconnected engineering cycles, we not only validated the effectiveness of each functional module but also deepened our understanding of the system with each iteration, obtaining critical performance parameters and directions for optimization. This series of rigorous engineering practices collectively forms the solid stepping stones for ReGenStitch's journey from concept to reality.

Figure 0. The modular engineering design approach for the ReGenStitch project.

Design

Engineering Goal: To construct the core structural backbone of ReGenStitch. We selected Bacterial Cellulose (BC) as the ideal foundational material due to its purity, which far surpasses that of plant cellulose, its unique three-dimensional nanofiber network structure, and its outstanding mechanical strength and biocompatibility. The primary task of this cycle is to establish a functional, high-yield BC synthesis system in a chassis cell that is easy to manipulate and has high expression efficiency.

Design Rationale and Chassis Selection: The synthesis of bacterial cellulose is a complex process regulated by the bcs operon. Literature research indicates that bcsA (encoding the catalytic subunit of cellulose synthase) and bcsB (encoding the regulatory subunit that binds to the allosteric activator c-di-GMP) are the two absolute core components of the entire synthesis pathway. Therefore, our core design strategy is to enhance and drive the entire cellulose synthesis pathway by overexpressing these two key proteins in a chassis cell. We chose Escherichia coli BL21(DE3) as the chassis for this proof-of-concept cycle because it grows rapidly, has a well-defined genetic background, and possesses strong protein expression capabilities, making it the "gold standard" for validating new pathways in synthetic biology.

Genetic Circuit Design: To maximally test the function of these two genes, we designed a dual-gene co-expression genetic circuit. We selected classic parts from the iGEM Registry: the strong constitutive promoter J23100 (BBa_J23100) and the strong ribosome binding site (RBS) B0034 (BBa_B0034) to drive the expression of bcsA and bcsB, respectively. The use of a constitutive promoter allows for continuous and efficient expression of the target proteins without the need for an inducer, enabling us to focus on evaluating the efficiency of the synthesis pathway itself. The entire genetic circuit was designed to be cloned into the high-copy plasmid pSB1A3, with the goal of achieving a higher gene dosage effect.

Figure 1. Genetic circuit diagram of pSB1A3-bcsAB.

Figure 2. Plasmid map of the recombinant plasmid pSB1A3-bcsAB.

Build

Gene Synthesis and Plasmid Construction: We first performed codon optimization on the bcsA and bcsB gene sequences from Gluconacetobacter xylinus to adapt them to the translation system of E. coli, thereby avoiding low expression efficiency due to codon rarity. The optimized gene sequences were synthesized by Genewiz. Subsequently, we used the high-efficiency Golden Gate assembly method to seamlessly ligate the two expression cassettes and clone them into the pSB1A3 vector.

Strain Transformation and Verification: The successfully constructed recombinant plasmid was transformed into E. coli BL21(DE3) competent cells and screened on LB agar plates containing ampicillin. We picked single colonies for colony PCR verification. As shown in Figure 3, the agarose gel electrophoresis results clearly displayed target bands of approximately 2262 bp (bcsA) and 2406 bp (bcsB) in the lanes of positive clones, which perfectly matched the expected sizes. This result conclusively demonstrates that the recombinant plasmid pSB1A3-bcsAB was successfully constructed and transformed into the engineered strain.

Figure 3. Agarose gel electrophoresis analysis of colony PCR products for the recombinant plasmid pSB1A3-bcsAB.

Test

To quantitatively assess the cellulose production capability of our engineered strain and to confirm the chemical nature of the product, we designed two complementary tests:

Dry Weight Yield Analysis of Cellulose: We cultured the engineered strain BL21-bcsAB and a control group, BL21 carrying an empty vector, in shake flasks. After cultivation, impurities such as cell proteins and nucleic acids were removed by alkali treatment (4M NaOH), and the cellulose product was purified, dried, and finally weighed on an analytical balance. The results (Figure 4) showed that the cellulose yield of the engineered strain BL21-bcsAB was significantly higher than that of the control group, which produced almost no cellulose (p < 0.05). This direct evidence indicates that our genetic circuit successfully conferred and greatly enhanced the cellulose synthesis ability of E. coli.

Figure 4. Analysis of cellulose yield from the engineered strain and the control strain.

Congo Red Specific Binding Assay: To provide further chemical evidence that the product is indeed cellulose and to rule out the influence of cell growth differences on total yield, we employed a Congo Red binding assay. Congo Red is a dye that specifically binds to β-1,4-glucan chains. We normalized the cell density of both strains to an OD₆₀₀ of 1 and then incubated them with a quantified amount of Congo Red dye. The results (Figure 5) showed that, compared to the control group, the supernatant of the engineered strain BL21-bcsAB culture had a significantly lower concentration of residual Congo Red (absorbance difference p < 0.0001). This implies that, on a per-cell basis, our engineered strain bound more Congo Red. This not only reconfirms that the product is cellulose but also verifies, at a microscopic level, that the cellulose synthesis capability of individual engineered cells was qualitatively improved.

Figure 5. Comparison of Congo Red adsorption capacity by the bacteria, reflecting cellulose binding.

Production Kinetics Characterization: A successful engineered system must be understood not only in terms of "if" and "how much" but also "when." To investigate the time-course dynamics of cellulose synthesis and to determine optimal process parameters for future scale-up, we conducted a kinetics study. After inoculating the engineered strain, we collected samples at different time points (e.g., 0h, 12h, 24h, 36h, 48h, 60h) and measured the cumulative cellulose yield at each point. By plotting yield against time (Figure 6), we obtained a typical "S"-shaped growth/production curve. The curve shows that production growth was relatively slow in the early phase, rapidly increased during the logarithmic phase, and began to enter a plateau phase at approximately 48 hours, reaching a peak yield of about 970 mg/L, after which the growth rate slowed significantly.

Figure 6. Time-course curve of cellulose production by the BL21-bcsAB strain.

Learn

Conclusion of this Cycle: Our first DBTL engineering cycle was a complete success. Through rational genetic circuit design, rigorous experimental construction, and multi-dimensional performance testing, we demonstrated that heterologous co-expression of the bcsA and bcsB genes in E. coli BL21 is a feasible and effective strategy for constructing an efficient bacterial cellulose production system. We not only created a functional production strain but, more importantly, we quantitatively confirmed its superior production performance and obtained key process parameters.

Key Learnings & Insights:

Design Validation: The success of this cycle strongly validated our initial design hypothesis—that bcsA and bcsB are the core engines driving cellulose synthesis. It also confirmed that our chosen promoter, RBS, and other regulatory elements could effectively drive the expression of these two genes.

Establishment of a Baseline: We have obtained a "version 1.0" BC production strain with well-defined performance. This strain and its yield data will serve as a critical baseline for all subsequent optimizations in our project. Any future improvements (such as changing promoters, optimizing culture media, etc.) will be compared against it.

Identification of Key Process Parameters: The production kinetics study provided us with a crucial process parameter—an optimal fermentation period of 48 hours. This data is essential knowledge for transitioning any engineering project from the lab to industrial production, as it directly guides how to obtain the target product with maximum efficiency.

Transition to the Next Cycle: Now that the "structural backbone" of ReGenStitch has been successfully constructed and preliminarily characterized, our engineering focus logically and naturally shifts to endowing it with its "soul"—active biological functions. A suture with only physical strength is incomplete. Therefore, we move forward with confidence into the second engineering cycle, dedicated to developing an innovative biological module that can provide the suture with broad-spectrum antimicrobial and active healing-promoting capabilities. This will be the key feature that truly distinguishes ReGenStitch from traditional sutures.

Design

Engineering Goal: After successfully constructing the suture's structural backbone, the goal of this cycle is to endow it with active biological functions—specifically, broad-spectrum antimicrobial and active healing-promoting capabilities. We selected chitosan and its hydrolysis products, Chitosan Oligosaccharides (COS), as the core functional molecules. Chitosan itself possesses excellent film-forming, hemostatic, and moderate antimicrobial properties. Its degradation products, COS (especially those with 2-6 sugar units), have higher water solubility, permeability, and biological activity, enabling them to more effectively inhibit bacterial growth and stimulate tissue regeneration.

Core Challenge and Engineering Strategy: The primary commercial method for producing COS is the hydrolysis of chitosan using chitosanase. However, traditional bioprocesses typically involve intracellular expression in engineered bacteria (like E. coli), which makes enzyme acquisition extremely cumbersome. The process requires large-scale bacterial cultivation, cell harvesting, cell disruption via high pressure or sonication, centrifugation to remove cell debris, and finally, multi-step chromatography to purify the target enzyme. This series of steps not only leads to long production cycles and high costs but also results in significant loss of enzyme activity during purification. Furthermore, the purified enzyme is a single-use consumable that cannot be easily recovered and reused.

To completely disrupt this inefficient traditional model, we proposed an innovative engineering solution: constructing a "Whole-Cell Biocatalyst" based on Cell Surface Display technology. Our core design idea is to stop "imprisoning" the enzyme inside the cell and instead "display" it on the cell surface. This transforms the entire living engineered bacterium into a micro-scale catalytic reactor that is ready to use without disruption, easily recyclable, and even capable of self-replication.

Design Rationale and Component Selection:

The Engine (Functional Enzyme): We chose chitosanase CHI1 from Bacillus thuringiensis. This enzyme has been reported to have high catalytic activity, capable of precisely hydrolyzing long-chain chitosan into small-molecule oligosaccharides.

The Anchor (Molecular Anchor): To firmly anchor CHI1 to the outer membrane of E. coli, we selected the classic Ice Nucleation Protein (INP). The N-terminal domain of INP can effectively display any "passenger protein" fused to it on the cell surface, making it a well-validated and highly efficient display tool.

Genetic Circuit Design: We designed a genetic circuit for the fusion expression of the INP gene and the CHI1 gene (pSB1A3-INP-CHI1). To rigorously prove the superiority of the surface display strategy, we also designed a critical control group: a genetic circuit that expresses CHI1 only intracellularly (without the INP anchor) (pSB1A3-CHI1). Both circuits use the same strong constitutive promoter J23100 (BBa_J23100) and strong RBS B0034 (BBa_B0034) as in Cycle 1. This ensures that the driving force for expression is consistent, allowing any performance differences to be directly attributed to the core variable of "surface display versus no surface display."

Figure 7. Genetic circuit diagram of the surface display system.

Build

Gene Synthesis and Plasmid Construction: We performed codon optimization for the INP and CHI1 genes, which were then synthesized by a commercial company. Using fusion PCR, we joined the INP and CHI1 fragments to create a complete INP-CHI1 fusion gene. Subsequently, using EcoRI and XbaI restriction sites, we cloned the INP-CHI1 fusion gene into the pSB1A3 vector, successfully constructing the experimental plasmid pSB1A3-INP-CHI1.

Strain Transformation and Verification: The plasmid was transformed into E. coli BL21(DE3) cells. Transformants were verified by colony PCR. As shown in Figure 8, agarose gel electrophoresis results clearly showed bands corresponding to the expected sizes of INP (537 bp) and CHI1 (2028 bp) in the positive clones, proving that our plasmid was successfully constructed and ready for subsequent performance comparison tests.

Figure 8. Agarose gel electrophoresis analysis of colony PCR products for the recombinant plasmid pSB1A3-INP-CHI1.

Figure 9. Plasmid map of the recombinant plasmid pSB1A3-INP-CHI1.

Test

The core of this cycle's testing was to answer a key engineering question: Is the performance of our designed whole-cell catalyst truly superior to the traditional crude enzyme lysate method? To this end, we conducted a series of rigorous performance evaluations.

Head-to-Head Performance Test:

Experimental Setup: We established two core comparison groups:

Experimental Group (Surface Display): Directly used the intact, living BL21-INP-CHI1 engineered bacterial suspension as the catalyst.

Control Group (Crude Enzyme): Used the CHI1 strain, which was sonicated to disrupt the cells, followed by centrifugation. The resulting supernatant (i.e., crude enzyme lysate) was used as the catalyst, simulating the traditional enzyme preparation process.

Reaction and Detection: Equal amounts of the two catalysts were reacted with a 1% commercial chitosan solution under identical conditions. The production of COS was then measured using a reducing sugar assay kit (based on the DNS method).

Results: The experimental results clearly demonstrated that both in terms of the absolute amount of product (chitooligosaccharides) generated and the calculated enzyme activity units (U/mL), the catalytic efficiency of the BL21-INP-CHI1 whole-cell catalyst was significantly higher than that of the traditional crude enzyme lysate (p < 0.01). This result provides powerful evidence that our surface display strategy offers a decisive advantage in catalytic performance.

Figure 10. Assessment of chitooligosaccharide production levels by BL21-INP-CHI1 and CHI1 crude enzyme lysate.

Figure 11. Comparison of enzyme activity between BL21-INP-CHI1 and CHI1 crude enzyme lysate.

Catalyst Characterization: After proving its superiority, we further characterized its key operating parameters, treating it like an industrial catalyst, to determine its optimal application conditions.

Optimal Temperature: We tested the activity of the whole-cell catalyst over a range of 25°C to 65°C and found that it exhibited the highest activity at 50°C (Figure 12).

Optimal pH: We conducted tests over a pH range of 4 to 8 and determined its optimal reaction pH to be 7.0 (Figure 13).

These data provide invaluable guidance for process control in future large-scale applications.

Figure 12. Comparison of enzyme activity of BL21-INP-CHI1 at different incubation temperatures.

Figure 13. Comparison of enzyme activity of BL21-INP-CHI1 under different pH conditions.

End-to-End Sustainable Process Validation:

Engineering Question: Can our system be integrated into a complete, environmentally friendly biomanufacturing process that converts waste into high-value products?

Upstream Raw Material Preparation: First, we designed and tested an eco-friendly microbial fermentation method to extract chitin from shrimp shell waste collected from restaurants. We used B. subtilis for deproteinization and Acetobacter sp. for demineralization. The experimental results (Figure 14) showed that when the two bacteria were used in co-fermentation, the chitin yield increased dramatically to approximately 14.5%, far exceeding the ~1% yield from single-strain treatments, demonstrating a significant synergistic effect. This successfully valorized the biomass waste.

Downstream Catalysis Test: Next, we converted the chitin produced via this sustainable method into crude chitosan and used it as a "real-world" substrate to test the catalytic performance of our engineered bacteria. The results (Figure 15) once again proved that even when processing this complex, low-purity crude substrate, the hydrolysis efficiency of the BL21-INP-CHI1 whole-cell catalyst was significantly superior to the traditional crude enzyme lysate at all time points.

Figure 14. Chitin yield from different bacterial combinations.

Figure 15. Hydrolysis efficiency of BL21-INP-CHI1 and CHI1 crude enzyme lysate on the prepared crude chitosan at different time points.

Learn

Conclusion of this Cycle: This DBTL cycle achieved a breakthrough success. We not only successfully introduced the antimicrobial and healing-promoting functional module into the suture but, more importantly, through cell surface display technology, we simplified a complex biochemical process (enzyme extraction and purification) into a simple physical one (centrifugation and recovery of cells). We created and validated a highly efficient, low-cost, and reusable whole-cell biocatalyst system that is far superior to traditional methods.

Key Learnings & Insights:

Power of Engineering Design: This cycle perfectly illustrates how synthetic biology can solve real-world process bottlenecks through clever engineering design. The success of the surface display strategy proves that "changing the location of the enzyme" can sometimes lead to more significant process advancements than "optimizing the enzyme itself."

Importance of Characterization: We not only proved that the system "works" but also, through detailed parameter characterization, answered "how it works best" (50°C, pH 7.0). This is a necessary step in moving from scientific research to engineering application.

From Ideal to Real: The successful test on a real-world waste substrate greatly enhances the practical significance and sustainability value of our project, demonstrating its potential to close the loop in a bio-based economy.

Transition to the Next Cycle: At this point, our ReGenStitch possesses a sturdy "skeleton" (BC) and a powerful "immune shield" (COS). However, a perfect wound healing process requires not only resistance to external bacterial invasion but also delicate regulation of the body's own inflammatory response to prevent excessive inflammation from causing tissue damage and unsightly scars. This is a critical aspect overlooked by traditional sutures. Therefore, our engineering journey enters its final and most challenging phase: constructing an intelligent module capable of actively modulating inflammation. In Cycle 3, we will take on the challenge of reconstructing a complex plant metabolic pathway in a prokaryotic organism to produce the potent natural anti-inflammatory agent—curcumin.

Design

Engineering Goal: After endowing the suture with a robust "structure" and a powerful "antimicrobial" function, the goal of this cycle is to introduce its third and most sophisticated core function: active anti-inflammatory and scar-regulating capabilities. Wound healing is a dynamic process where an excessive inflammatory response not only delays healing but is also a key culprit behind postoperative pain and the formation of unsightly scars. Therefore, we selected the potent, naturally-derived anti-inflammatory molecule—curcumin—as our functional "drug."

Core Challenge and Engineering Strategy: Curcumin is a structurally complex polyphenol compound, a secondary metabolite formed by plants (turmeric) over a long evolutionary history. Synthesizing it chemically is not only a multi-step, costly process but also prone to causing environmental pollution. Therefore, our core engineering challenge is to "transplant" and reconstruct its core biosynthetic pathway across species within a simple, easily culturable prokaryotic chassis (E. coli). This requires us to precisely identify the key enzymes in the pathway and enable them to work synergistically in the new host.

Design Rationale and Component Selection:

Pathway Analysis: Through in-depth literature research, we identified the minimal enzymatic unit required for the synthesis of curcumin from the primary metabolites feruloyl-CoA and malonyl-CoA. This pathway relies on two key enzymes:

DCS (Diketide-CoA Synthase): A type III polyketide synthase responsible for the "carbon chain extension" reaction, using feruloyl-CoA as a starter unit and malonyl-CoA as an extender unit to construct the core intermediate skeleton of curcumin.

CURS1 (Curcumin Synthase 1): Responsible for the final "assembly" step, catalyzing the intermediate molecules to generate the final curcumin product.

Genetic Circuit Design: We designed a dual-enzyme co-expression system. To achieve precise control over this heterologous, high-metabolic-load pathway, we chose the classic pET expression system. We constructed the genes encoding DCS and CURS1 in a polycistronic structure and placed them under the control of a potent, IPTG-inducible T7 promoter. This inducible design is a critical engineering consideration: it allows us to separate the cell's "growth phase" from the product's "production phase." We can allow the cells to reach a high density before "switching on" curcumin synthesis by adding IPTG, thereby minimizing the toxicity or metabolic burden of the heterologous pathway on normal cell growth, with the aim of achieving a higher final yield.

Figure 16. Genetic circuit diagram of pET28a(m)-DCS-CURS.

Figure 17. Plasmid map of the recombinant plasmid pET28a(m)-DCS-CURS.

Build

Gene Synthesis and Plasmid Construction: To ensure that these two plant-derived genes could be efficiently translated in a prokaryotic host, we first performed comprehensive codon optimization on the DCS and CURS1 gene sequences to match their codon usage bias with that of E. coli. The optimized genes were synthesized by a commercial company. Subsequently, using the NdeI and XhoI restriction sites, we precisely cloned the DCS-CURS1 expression cassette into the pET28a(m) vector, completing the construction of the recombinant plasmid.

Strain Transformation and Verification: The successfully constructed recombinant plasmid was transformed into E. coli BL21(DE3) competent cells, which express T7 polymerase. After screening for positive transformants on LB plates containing kanamycin, we verified them using colony PCR. As shown in Figure 18, the agarose gel electrophoresis results clearly displayed target bands of approximately 1161 bp (DCS) and 1170 bp (CURS1) in the lanes of positive clones, perfectly matching the expected sizes. This result conclusively proved that our curcumin synthesis plasmid was successfully constructed, laying the foundation for subsequent functional testing.

Figure 18. Agarose gel electrophoresis analysis of colony PCR products for the recombinant plasmid pET28a(m)-DCS-CURS.

Test

The core of this cycle's testing was to answer a fundamental question: Can these two enzymes we "borrowed" from a plant successfully work together in the "foreign land" of E. coli to synthesize curcumin from scratch?

1. In Vitro Enzymatic Assay & Yield Quantification:

Engineering Strategy: To bypass the complexities of detecting metabolites within living cells, we adopted a more direct and purer testing strategy—an in vitro enzymatic reaction. This method isolates the function of our constructed enzymes from their complex intracellular environment, allowing for a direct and clear validation of whether the pathway itself is "functional."

Experimental Setup: We first induced the engineered strain BL21-DCS-CURS with IPTG to express large quantities of the DCS and CURS1 enzymes. We then prepared a crude enzyme lysate by sonicating the cells. This lysate was mixed with quantified substrates (feruloyl-CoA and malonyl-CoA) in a buffer solution to react. After the reaction, the product was extracted with ethyl acetate and precisely quantified by measuring its characteristic absorption peak at 420 nm, using a curcumin standard curve.

Results: The results (Figure 19) were exciting. Our engineered strain's crude enzyme lysate successfully catalyzed the substrates to synthesize curcumin, with a yield reaching approximately 13-14 μM. In stark contrast, the crude lysate prepared from the wild-type BL21 strain showed a product concentration almost indistinguishable from the background. The yield from the engineered strain was over 26 times higher than the background level, a difference of extremely high statistical significance (p < 0.001). This irrefutably proves that the DCS-CURS dual-enzyme co-expression system we constructed is functional and has successfully achieved the de novo biosynthesis of curcumin.

Figure 19. Curcumin production test of the engineered strain co-expressing DCS and CURS.

2. Enzyme Kinetics Characterization:

Engineering Question: After proving the pathway "works," we wanted to further understand "how well it works." The catalytic efficiency of a system is a key indicator of its future industrial potential.

Experimental Setup: While keeping the enzyme concentration constant, we set up a series of reaction systems with varying substrate concentrations and measured the initial reaction rate for each concentration.

Results: By plotting the reaction rate against the substrate concentration (Figure 20), we obtained a very typical Michaelis-Menten kinetics curve. Through non-linear regression fitting, we calculated the two key kinetic parameters for this dual-enzyme system: the maximum reaction rate (Vmax) was approximately 345.6 nM/min, and the Michaelis constant (Km) was approximately 114.6 μM. This result indicates that our constructed system is not only functional but also exhibits good catalytic performance and moderate substrate affinity.

Figure 20. Reaction kinetics curve for curcumin synthesis by DCS-CURS.

Learn

Conclusion of this Cycle: This DBTL cycle was a major success. Through rational pathway design and rigorous experimental validation, we successfully reconstructed a functional plant secondary metabolic pathway in E. coli, achieving the de novo biosynthesis of the anti-inflammatory molecule curcumin. This marks the validation of ReGenStitch's final core functional module—"anti-inflammatory and scar regulation."

Key Learnings & Insights:

Feasibility of Interspecies Engineering: The success of this cycle is a powerful testament to the concepts of "gene part standardization" and "cross-species engineering" in synthetic biology. It demonstrates that functional modules from even evolutionarily distant species (plants and bacteria) can be "transplanted" and successfully activated through engineering techniques like codon optimization.

Value of a Stepwise Testing Strategy: Adopting the "in vitro crude lysate test" strategy allowed us to quickly and clearly validate the function of the core enzymes. This avoided the interference and uncertainty that could have arisen from direct, complex analysis of metabolic flux in living cells, proving to be a highly efficient method for validating pathway feasibility in the early stages of a project.

Leap from Qualitative to Quantitative: The enzyme kinetics study elevated our understanding of the system from a qualitative "yes/no" level to a quantitative "how fast/how well" level. The obtained Vmax and Km values provide us with precise evaluation benchmarks and clear directional guidance for future optimization efforts (e.g., by increasing substrate supply or by improving Vmax/lowering Km through protein engineering of the enzymes themselves).

Project Summary & Transition to Final Integration:

At this point, through three rigorous and independent DBTL engineering cycles, we have successfully constructed and validated the three core biological functional modules required for ReGenStitch:

1. A high-yield production system for the structural backbone (bacterial cellulose).
2. An efficient, recyclable preparation system for antimicrobial repair (chitooligosaccharides).
3. A functional biosynthetic system for anti-inflammatory regulation (curcumin).

The next stop on our engineering journey, Cycle 4, and our final challenge, will be System Integration. The future focus will shift to: how to organically composite these three bioactive materials obtained through biomanufacturing—bacterial cellulose, chitooligosaccharides, and curcumin—in the optimal ratio and manner to produce the final ReGenStitch suture product. Subsequently, we will conduct a comprehensive performance evaluation of this composite material, including its mechanical properties, drug release kinetics, in vitro antibacterial and anti-inflammatory effects, and ultimately, its biocompatibility and tissue repair efficacy at the cellular and animal levels. Our engineering story will now move from building independent "parts" to assembling a "complete machine" that works in synergy.

Design

Engineering Goal: After the successful validation of ReGenStitch's three core biological modules (structural, antimicrobial, anti-inflammatory) in the preceding cycles, the engineering goal of this cycle is to achieve System Integration. Our task is to start with the separated bioactive "parts" and, through the methods of materials engineering, design and fabricate the first physical prototype—a genuine, suture-ready ReGenStitch multifunctional suture.

Core Challenge and Engineering Strategy: The core challenge lies in how to organically composite biomacromolecules of different properties (the rigid bacterial cellulose and the positively charged chitosan) to form a uniform material that possesses both sufficient mechanical strength to withstand suturing tension and good flexibility for easy knotting, while also serving as a carrier for the active ingredients.

Design Rationale and Formulation Optimization:

Basic Composite Formulation: Our core design is to construct a bacterial cellulose-chitosan (BC-CS) composite system. In this system, the BC obtained from Cycle 1, with its three-dimensional nanofiber network, serves as the suture's "reinforcing skeleton," providing the primary mechanical strength and biocompatible scaffold. The CS, obtained from the upstream process of Cycle 2, acts as a "functional coating/matrix," imparting hemostatic and antimicrobial activity to the material. The curcumin, obtained from Cycle 3, serves as the "anti-inflammatory core," providing the suture's anti-inflammatory function.

Selection of Additives:Plasticizer: To overcome the potential brittleness of pure BC and CS materials, we introduced glycerol as a plasticizer. Small glycerol molecules can insert themselves between the biopolymer chains, increasing chain mobility and thus significantly improving the material's flexibility and extensibility, which is crucial for the suture's knotting performance.

Performance Modulator: To further explore and optimize the composite material's overall properties (such as degradation rate and mechanical strength), we introduced Thermoplastic Starch (TPS) as a biodegradable performance modulator.

Manufacturing Process Design: For the prototype stage, we selected a solution blending, film casting, and slitting manufacturing process. This method is simple to operate and easy to implement under laboratory conditions, making it highly suitable for rapid screening and proof-of-concept validation of different material formulations.

Build

Prototype Manufacturing Process: We systematically fabricated the prototype materials for the ReGenStitch suture in the laboratory, strictly following the designed formulation and process.

1.Raw Material Dissolution: We separately dissolved the bacterial cellulose (BC) and chitosan (CS) produced via our biological methods in a 1% glacial acetic acid solution, obtaining uniform polymer solutions through prolonged stirring.

2.Blending and Addition: While continuously stirring, the CS solution was slowly added to the BC solution to form a BC-CS blend. Subsequently, a quantified amount of glycerol was added as a plasticizer.

3.Formulation Gradient Preparation: TPS and curcumin were added to the blend to prepare the composite material precursor solution.

4.Molding and Drying: The solution for each formulation was poured into a circular plate and dried through controlled evaporation, ultimately forming a composite film of a certain thickness.

5.Suture Preparation: The dried film was cut into thin, long threads using a precision cutting tool, yielding our first-generation ReGenStitch suture prototypes.

Test

In this cycle, our testing focus was on Prototype Validation & Benchmarking. This involved assessing whether our manufactured ReGenStitch prototype possessed the fundamental qualities of a surgical suture and conducting a direct comparison with established products on the market.

Macroscopic Morphology and Basic Performance Evaluation:

1.Test Method: We placed the ReGenStitch prototype alongside two commonly used commercial surgical sutures—an absorbable PGA suture and a non-absorbable polyamide (nylon) suture—for direct visual and tactile comparison.

2.Results: As shown in Figure 21, our ReGenStitch prototype exhibited a uniform and smooth appearance. Compared to the commercial sutures, it could be made thinner while maintaining good flexibility and stretchability. In theory, a smaller suture diameter means less tissue trauma and a milder foreign body reaction.

Figure 21. Appearance comparison of the finished surgical suture (bottom) with existing commercial surgical sutures (middle: PGA, top: polyamide).

Functional Testing in a Simulated Use Scenario:

1.Test Method: To simulate the suture's handling performance and mechanical behavior in a real surgical setting, we used fresh, skin-on pork purchased from the market as a tissue model to perform a simulated suturing experiment. This served as a critical stress test for the prototype in an environment closely resembling its intended application.

2.Results: As shown in the video (Figure 22), during the suturing process, the ReGenStitch prototype demonstrated sufficient mechanical strength to penetrate the tough pigskin tissue without breaking. Concurrently, its good flexibility allowed for smooth knotting operations, and the resulting knots were secure, effectively approximating the wound edges. This simulation test successfully proved that our prototype possesses the core functionalities essential for a surgical suture: mechanical integrity and operability.

Figure 22. Simulated use comparison of the finished surgical suture with existing commercial surgical sutures.

Learn

Conclusion of this Cycle: Cycle 4 was a landmark success. We successfully assembled the biological "parts" produced in the first three cycles into a functional macroscopic "device," completing a decisive leap from molecular biology to materials science and engineering. The test results indicate that our designed ReGenStitch prototype is not only conceptually feasible but also demonstrates great potential in its basic physical and handling properties, proving its viability as a novel surgical suture.

Key Learnings & Insights:

Successful Validation of System Integration: The most important outcome of this cycle was the validation that our integration strategy was successful. BC and CS can be effectively composited, and through the modulation of additives (glycerol), can form a thread-like material that balances both strength and flexibility. This proves that our modular design approach is closed-loop at the final integration stage.

Limitations and Value of Qualitative Validation: We are also keenly aware that the testing in this cycle was primarily qualitative and descriptive. Although the simulated suturing experiment proved it is "usable," we cannot yet provide a quantitative conclusion on "how well it works" or the superiority of different formulations. However, the value of this qualitative validation lies in the strong confidence and intuitive evidence it provides, confirming that the project is on the right track and setting a clear direction for the next phase of more refined engineering iterations.

Evolution of the Engineering Problem: Our core engineering question has successfully evolved from "Can we manufacture these components via biological methods?" (Cycles 1-3) to "How do we fabricate these components into a suture with optimal performance?" (Cycle 4 and beyond).

With the successful completion of the fourth engineering cycle, we have brought the engineering exploration phase of the ReGenStitch project to a satisfying conclusion. Looking back at the entire journey, we have systematically constructed and validated a complete technical prototype for a multifunctional bioactive suture through four interconnected DBTL cycles:

In Cycle 1, we successfully built a high-yield production system for the structural backbone (bacterial cellulose), providing the suture with a tough "body."

In Cycle 2, we innovatively developed a highly efficient, recyclable whole-cell catalysis system for antimicrobial repair (chitooligosaccharides) and established a sustainable waste-to-raw-material pathway, dressing the suture in powerful "armor."

In Cycle 3, we precisely reconstructed a functional biosynthetic system for anti-inflammatory regulation (curcumin), implanting the suture with an intelligent "brain."

In Cycle 4, we finally integrated these biological "parts" into a tangible, operable suture prototype and validated its basic functions.

This series of engineering practices fully demonstrates our team's full-stack engineering capability, applying the principles of synthetic biology from gene design, to biomanufacturing, to material forming and prototype testing. We not only created independent biological components but, more importantly, we proved the feasibility of integrating them into a complex, synergistically working system.

Project Outlook

This project has successfully developed ReGenStitch v1.0, a "proof-of-concept" prototype with clear functional validation and immense potential. It lays a solid foundation for the research and development of the next generation of surgical suture materials. Future work will build upon this foundation, focusing on more in-depth, product-oriented optimization and validation, which will primarily include:

Preclinical Biological Evaluation: Comprehensive assessment of the biocompatibility, degradation behavior, in vivo antibacterial and anti-inflammatory effects, and ultimately, the true ability to promote high-quality wound healing of the final optimized ReGenStitch suture, using in vitro cell models and in vivo animal models.

We firmly believe that through continuous engineering iteration and scientific validation, the technical path pioneered by the ReGenStitch project will eventually move from the iGEM competition stage to real-world clinical applications, bringing a safer, more comfortable, and higher-quality postoperative recovery experience to hundreds of millions of surgical patients worldwide.

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