iGEM 2025 — Team squirrel-beijing

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
{% block page\_content %}

Content

* [Cycle 1 – Bacterial Cellulose (BC) Production System (06/16–07/08)](#section-1)
  + [Daily Experiment Schedule](#subsection-2)
* [Cycle 2 – Reusable Whole-Cell Biocatalyst System (07/10–07/30)](#section-3)
  + [Daily Experiment Schedule](#subsection-4)
* [Cycle 3 – Curcumin Biosynthetic Pathway System (08/01–08/23)](#section-5)
  + [Daily Experiment Schedule](#subsection-6)
* [Cycle 4 – System Integration and Suture Prototyping (08/25–09/17)](#section-7)
  + [Daily Experiment Schedule](#subsection-8)

# Cycle 1 – Bacterial Cellulose (BC) Production System (06/16–07/08)

From June 16 to July 8, 2025, we successfully established and characterized a system for producing the structural biopolymer, bacterial cellulose (BC), in *E. coli* BL21(DE3). We constructed the core production system by introducing a dual-gene cassette containing codon-optimized*bcsA* and *bcsB* from *Gluconacetobacter xylinus* into the pSB1A3 plasmid, driven by a strong constitutive promoter. Comparative analysis against a control strain (carrying an empty vector) confirmed the system's efficacy; the engineered strain demonstrated significantly higher BC yield (p < 0.05) via dry weight analysis and superior per-cell BC production as validated by a Congo Red binding assay (p < 0.0001). A comprehensive 60-hour time-course experiment was conducted to characterize production kinetics, revealing that BC accumulation enters a plateau phase at approximately 48 hours. This study established 48 hours as the optimal fermentation period and achieved a peak yield of approximately 970 mg/L. The successful construction of the genetic circuit was verified by colony PCR and agarose gel electrophoresis. All data were analyzed to validate the effectiveness of the design strategy. This robust "version 1.0" BC production system provides a critical performance baseline and serves as the foundational structural backbone for the ReGenStitch project.

## Daily Experiment Schedule

| Date | Experiment Title | Experiment Objective | Key Steps | Main Results | Notes |
| --- | --- | --- | --- | --- | --- |
| 2025-06-16 | Cycle 1 — Kickoff & Safety SOPs | Align DBTL plan and confirm BSL-1 practices | Team briefing; waste/sterility SOPs; assign roles; reagent inventory | Project plan approved; safety roles assigned | Final product is cell-free biomaterial; BSL-1 rules logged. |
| 2025-06-18 | Cycle 1 — bcsAB Design Freeze | Lock design for *E. coli* bcsA/bcsB expression | Confirm RFC10 sites; J23100+B0034 parts; primer list finalized | Design pack completed | Cycle-1 goal: BC production in *E. coli* BL21(DE3). |
| 2025-06-20 | Cycle 1 — Media & Competent Cells | Prepare plates/media and cells for cloning | Pour LB-Amp plates (invert, 4 °C); prep DH5α/BL21(DE3) competent cells | Plates ready; aliquoted competent cells | Media/antibiotic usage per cloning steps. |
| 2025-06-22 | Cycle 1 — PCR of bcsAB Inserts | Obtain clean bcsAB fragments | Gradient PCR; gel; gel extraction; quantify | Clear bands at expected size | Store inserts on ice → −20 °C; minimize UV time. |
| 2025-06-24 | Cycle 1 — pSB1A3 Digest & Ligation (bcsAB) | Assemble bcsAB into pSB1A3 | Double digest pSB1A3; T4 ligation (insert:vector ratios); include vector-only control | Ligation reactions complete | Standard BioBrick/RFC10-style build. |
| 2025-06-26 | Cycle 1 — DH5α Transformation (bcsAB) | Introduce ligation products for plasmid amplification | Heat-shock TF; SOC recovery; plate on LB-Amp | Colonies on ligation plates | Negative control plate clean (good). |
| 2025-06-28 | Cycle 1 — Colony PCR Screen (bcsAB) | Identify correct recombinant clones | Pick 12 colonies; colony PCR across junctions; choose positives | 8/12 positive clones | Start overnight cultures for positives. |
| 2025-06-30 | Cycle 1 — Miniprep & Sanger Submit (bcsAB) | Purify plasmids and submit for sequencing | Miniprep; NanoDrop; prepare VF2/VR + internal primers; ship | 8 plasmids (60–180 ng/µL) submitted | Sequence verification per plan. |
| 2025-07-02 | Cycle 1 — Wait: Sequencing in Progress | Hold wet work; pre-plan expression tests | Draft BC culture conditions; label BL21 plates; update DBTL tracker | — | DBTL loop documented for Cycle-1. |
| 2025-07-04 | Cycle 1 — Sequence Review & BL21(DE3) TF | Select error-free bcsAB plasmid(s) and move to host | Review chromatograms; transform BL21(DE3); make glycerol stocks | 2 perfect clones; robust BL21 colonies | BL21 (DE3) chosen as Cycle-1 chassis. |
| 2025-07-06 | Cycle 1 — BC Pilot Production (Static Culture) | Validate bacterial cellulose production | Inoculate glucose-rich static bottles (28–30 °C) with bcsAB strain + EV control | Pellicles visible in 24–48 h | Congo red binding planned as identity check. |
| 2025-07-08 | Cycle 1 — BC Harvest & Basic QC | Harvest and quantify BC | Alkali wash; neutralize; dry mass (mg/L); image pellicles | Measurable BC yield; consistent morphology | Record bottle fill volume and area for normalization. |

# Cycle 2 – Reusable Whole-Cell Biocatalyst System (07/10–07/30)

From July 10 to July 30, 2025, we established and characterized a reusable whole-cell biocatalyst for the production of antimicrobial Chitosan Oligosaccharides (COS). We engineered a cell surface display system in *E. coli* by fusing the chitosanase *CHI1* to the N-terminus of the Ice Nucleation Protein (INP) anchor. A head-to-head performance test demonstrated that our whole-cell biocatalyst was significantly more efficient (p < 0.01) in both COS production and enzyme activity compared to traditional crude enzyme lysate. Further characterization identified the optimal operating conditions as 50°C and pH 7.0. To validate its real-world applicability, we demonstrated an end-to-end sustainable process: first, we achieved a high chitin yield of ~14.5% from shrimp shell waste using a co-fermentation of *B. subtilis* and *Acetobacter sp.*; second, our whole-cell catalyst showed superior hydrolysis efficiency on this crude, sustainably-sourced substrate. The successful construction of the *INP*-*CHI1* fusion gene was verified by colony PCR. This cycle successfully developed a highly efficient, low-cost, and reusable biocatalyst, providing the core antimicrobial and healing-promoting module for the ReGenStitch project.

## Daily Experiment Schedule

| Date | Experiment Title | Experiment Objective | Key Steps | Main Results | Notes |
| --- | --- | --- | --- | --- | --- |
| 2025-07-10 | Cycle 2 — INP-CHI Plan & Primer Check | Prepare surface-display chitosanase build | Confirm INP anchor fusion design; finalize primers and linkers | Design validated | Cycle-2 goal: reusable whole-cell catalyst. |
| 2025-07-12 | Cycle 2 — PCR CHI1 & Backbone Prep | Generate CHI1 insert and prepare vector | PCR CHI1; gel-purify; digest pSB1A3-INP backbone | Clean insert and vector on hand | Proceed to ligation next day. |
| 2025-07-14 | Cycle 2 — Ligation & DH5α TF (INP-CHI) | Clone INP-CHI into plasmid backbone | T4 ligation; transform DH5α; plate on LB-Amp; include no-insert control | Colonies obtained | Track insert:vector molar ratio. |
| 2025-07-16 | Cycle 2 — Colony PCR & Overnights (INP-CHI) | Identify correct assembly clones | Screen 12 colonies; select positives; start ON cultures | 9/12 positives | Prepare glycerols of positives. |
| 2025-07-18 | Cycle 2 — Miniprep & Sanger Submit (INP-CHI) | Verify INP-CHI sequence | Miniprep; quantify; submit VF2/VR + internal | Samples submitted | Maintain both display and intracellular CHI variants. |
| 2025-07-20 | Cycle 2 — Wait: Sequencing/Logistics | Bridge time for reads & consumables | Order standardized chitosan substrate; prepare buffers | — | Plate reader and baseline blanks prepared. |
| 2025-07-22 | Cycle 2 — BL21(DE3) TF (INP-CHI) | Create expression strain(s) | Transform BL21(DE3); select colonies; prepare glycerol stocks | BL21-INP-CHI established | Keep EV and intracellular-CHI controls. |
| 2025-07-24 | Cycle 2 — Expression Induction & Whole-Cell Prep | Confirm surface display and prepare catalysts | Induce at mid-log; gentle handling; prepare washed whole-cells | Good biomass; intact prep | Avoid harsh lysis to preserve display. |
| 2025-07-26 | Cycle 2 — CHI Activity & Benchmark vs Crude | Demonstrate catalytic activity vs crude-enzyme approach | Incubate with chitosan substrate; measure released products vs controls | Displayed CHI > crude enzyme | Matches reported superiority of whole-cell catalyst. |
| 2025-07-28 | Cycle 2 — pH/Temperature Optimization | Identify optimal operating conditions | Test pH 6–8 and 30–55 °C; fixed time; replicate | Best activity near pH 7, ~50 °C | Conditions align with prior results. |
| 2025-07-30 | Cycle 2 — Reusability (Rounds 1–3) | Evaluate recyclability of whole-cell catalyst | Reuse same biomass across runs; wash between cycles | Activity retained over cycles | Confirms “reusable” catalyst concept. |

# Cycle 3 – Curcumin Biosynthetic Pathway System (08/01–08/23)

From August 1 to August 23, 2025, we established and characterized a biosynthetic pathway for the anti-inflammatory agent curcumin in *E. coli*. We successfully reconstructed the plant-derived pathway by co-expressing the codon-optimized genes *DCS* and *CURS1* in *E. coli* BL21(DE3) using an IPTG-inducible pET expression system. The successful construction of the plasmid was verified by colony PCR. An in vitro enzymatic assay using crude enzyme lysate demonstrated the de novo synthesis of curcumin, achieving a yield of approximately 13-14 μM, which was over 26 times higher than the control (p < 0.001). Further enzyme kinetics characterization yielded a Vmax of approximately 345.6 nM/min and a Km of 114.6 μM, confirming the system's catalytic efficiency. This cycle successfully validated the final core functional module—anti-inflammatory and scar regulation—for the ReGenStitch project.

## Daily Experiment Schedule

| Date | Experiment Title | Experiment Objective | Key Steps | Main Results | Notes |
| --- | --- | --- | --- | --- | --- |
| 2025-08-01 | Cycle 3 — DCS/CURS Pathway Plan | Prepare anti-inflammatory module build | Confirm DCS + CURS1 genes and expression design in *E. coli* BL21 | Build plan approved | Cycle-3 goal: curcumin biosynthesis. |
| 2025-08-03 | Cycle 3 — PCR DCS/CURS & DH5α Assembly | Generate inserts and assemble pathway vector | PCR DCS/CURS; gel-purify; ligation into expression backbone; transform DH5α | Colonies observed | Proceed to screening and sequence-check next. |
| 2025-08-05 | Cycle 3 — Colony PCR Screen (DCS/CURS1) | Identify correct DH5α clones | Screen 12 colonies across junctions; select positives for overnight culture | 7/12 positives at expected sizes | Proceed to miniprep & Sanger. |
| 2025-08-07 | Cycle 3 — Miniprep & Sanger Submit | Sequence-verify pathway | Miniprep; quantify; submit VF2/VR + internal primers | Plasmids sent for sequencing | Track any mixed peaks; hold backups. |
| 2025-08-09 | Cycle 3 — Wait: Sequencing in Progress | Hold benchwork pending reads | Prepare BL21(Kan) plates; outline induction conditions and sampling plan | — | DBTL notes updated for Cycle-3. |
| 2025-08-11 | Cycle 3 — Sequence Review & BL21(DE3) Transformation | Select error-free clone(s), move to expression host | Inspect chromatograms; transform BL21(DE3); prepare glycerol stocks | Robust transformants obtained | Keep EV BL21 control. |
| 2025-08-13 | Cycle 3 — Induction Pilot & Lysate Prep | Produce active DCS/CURS1 enzymes | IPTG induction at mid-log; harvest; crude lysate & clarification | Protein-rich lysate prepared | Aliquot & store at −80 °C. |
| 2025-08-15 | Cycle 3 — Curcumin Assay (UV–Vis @420 nm) | Demonstrate pathway activity | Reaction with substrates; extract; measure A420 vs standards | Clear 420 nm peak above controls | Confirms anti-inflammatory module function. |
| 2025-08-17 | Cycle 3 — Kinetics/Optimization | Estimate performance parameters | Substrate gradient; time-course; fit initial rates | Vmax/Km trends consistent with expectations | Choose best induction temperature/time. |
| 2025-08-19 | Cycle 3 — Troubleshooting/Media Tuning | Improve yields if suboptimal | Adjust precursors/cofactors and pH per plan; re-assay | Improved yield vs baseline | Lock selected conditions. |
| 2025-08-21 | Cycle 3 — Module Freeze & Documentation | Finalize constructs and SOPs | Freeze working glycerols; finalize protocols and raw-data sheets | Cycle-3 module closed | Ready for integration. |
| 2025-08-23 | Cycle 3 — Buffer/Contingency | Reserve for repeats or logistics | Use only if sequencing/shipping delays occur; otherwise maintain readiness | — | Maintains schedule flexibility. |

cycle3-notebook.pdf

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[Download](https://static.igem.wiki/teams/5878/notebook/cycle3-notebook.pdf)

# Cycle 4 – System Integration and Suture Prototyping (08/25–09/17)

From August 25 to September 17, 2025, we completed the system integration phase by designing and fabricating the first physical prototype of the ReGenStitch suture. We integrated the bioactive components from previous cycles—bacterial cellulose (BC), chitosan (CS), and curcumin—using a solution blending, casting, and slitting method, with glycerol added as a plasticizer to ensure flexibility. The prototype's performance was validated through macroscopic evaluation against commercial sutures and a simulated suturing test on pigskin. The results confirmed that our prototype possesses sufficient mechanical strength to penetrate tissue and good flexibility for secure knotting. This cycle successfully transitioned the project from individual biological modules to a tangible device, culminating in a 'proof-of-concept' ReGenStitch v1.0 prototype and validating the feasibility of our integrated design.

## Daily Experiment Schedule

| Date | Experiment Title | Experiment Objective | Key Steps | Main Results | Notes |
| --- | --- | --- | --- | --- | --- |
| 2025-08-25 | Cycle 4 — Material Prep (BC & Chitosan) | Prepare inputs for composite | Harvest/alkali-wash BC; neutralize; prepare chitosan solution | Clean BC mats; clear CS solution | Inputs ready for blending. |
| 2025-08-27 | Cycle 4 — Solution Blending (v1) | Create first BC/CS composite dope | Mix BC + chitosan; add plasticizer per plan; degas | Homogeneous blend achieved | Record exact ratios for DBTL. |
| 2025-08-29 | Cycle 4 — Casting & Controlled Drying (v1) | Produce cast films for thread-making | Level plates; controlled drying; peel intact films | Intact films formed | Label thickness and drying time. |
| 2025-08-31 | Cycle 4 — Slicing into Threads & Conditioning | Convert films to line-like strips | Precision-cut; rinse/condition; dry under tension | Threads of consistent gauge | Photo & gauge each set. |
| 2025-09-02 | Cycle 4 — Handling Check & Storage Prep | Confirm basic handling integrity | Gentle flex/knot handling; package dry samples | Threads intact after handling | Store in clean, dry pouches. |
| 2025-09-04 | Cycle 4 — Mechanical Handling & Knotting Test (v1) | Assess strength & operability | Standardized hand-knot pulls and handling observations | Meets basic handling expectations | Record force/extension qualitatively. |
| 2025-09-06 | Cycle 4 — CHI Activity on Composite Threads | Check CHI functional retention | Prepare whole-thread samples; run activity readout vs controls | Detectable activity retained | Aligns with Cycle-2 CHI findings. |
| 2025-09-08 | Cycle 4 — DBTL “Learn” Review | Decide formulation updates | Analyze v1 handling + activity; select improved ratios for v2 | v2 plan approved | Document changes for traceability. |
| 2025-09-10 | Cycle 4 — Formulation v2 Casting & Threading | Produce refined prototype | Blend per v2; cast; slice; condition as before | v2 threads produced | Label sets for demo. |
| 2025-09-12 | Cycle 4 — Pig-Skin Suturing Demo (v2) | Validate operability on tissue surrogate | Side-by-side suturing on pork skin vs commercial | Smooth passage; secure knots | Demonstrates practical handling. |
| 2025-09-14 | Cycle 4 — Imaging & Documentation Consolidation | Capture evidence and organize data | Photograph threads/demos; compile lab notes & figures | Complete photo/data set | Pre-final wiki/Notebook collation. |
| 2025-09-15 | Cycle 4 — Prototype Packaging & Labeling | Prepare demonstration sets | Label batches; store dry; document lot info | Prototypes packaged | Ready for final checks. |
| 2025-09-16 | Cycle 4 — Safety Review & Final Checks | Reconfirm BSL-1 & cell-free deliverable | Verify records; waste logs; confirm no live GMO in product | Safety dossier complete | Matches project safety design. |
| 2025-09-17 | Cycle 4 — Data Lock & Gantt Mapping | Freeze deliverables and map cycles | Finalize datasets, figures, SOPs; tag items by Cycle 1–4 for Gantt | Project book closed | Ready for wiki/presentation. |

cycle4-notebook.pdf

0.7MB

[Download](https://static.igem.wiki/teams/5878/notebook/cycle4-notebook.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

Content

* [Overview](#section-1)
  + [Cycle 1: Engineering a High-Yield Bacterial Cellulose Production System](#section-2)
    - [Design](#subsection-3)
    - [Build](#subsection-4)
    - [Test](#subsection-5)
    - [Learn](#subsection-6)
  + [Cycle 2: Engineering a Reusable Whole-Cell Biocatalyst for Antimicrobial Oligosaccharide Production](#section-7)
    - [Design](#subsection-8)
    - [Build](#subsection-9)
    - [Test](#subsection-10)
    - [Learn](#subsection-11)
  + [Cycle 3: Engineering a Biosynthetic Pathway for the Anti-inflammatory Agent Curcumin](#section-12)
    - [Design](#subsection-13)
    - [Build](#subsection-14)
    - [Test](#subsection-15)
    - [Learn](#subsection-16)
  + [Cycle 4: System Integration and Prototyping of the ReGenStitch Suture](#section-17)
    - [Design](#subsection-18)
    - [Build](#subsection-19)
    - [Test](#subsection-20)
    - [Learn](#subsection-21)
  + [Overall Engineering Achievements & Project Vision](#section-22)
    - [Project Outlook](#subsection-23)
  + [References](#section-24)

# Overview

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.

![Overview Diagram](https://static.igem.wiki/teams/5878/engineering/img1.webp)

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

# Cycle 1: Engineering a High-Yield Bacterial Cellulose Production System

## 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.

![Design Diagram](https://static.igem.wiki/teams/5878/engineering/img2.webp)

Figure 1. Genetic circuit diagram of pSB1A3-bcsAB.

![Design Diagram](https://static.igem.wiki/teams/5878/engineering/img3.webp)

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.

![Build Diagram](https://static.igem.wiki/teams/5878/engineering/img4.webp)

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*.

![Test Diagram](https://static.igem.wiki/teams/5878/engineering/img5.webp)

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.

![Test Diagram](https://static.igem.wiki/teams/5878/engineering/img6.webp)

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.

![Test Diagram](https://static.igem.wiki/teams/5878/engineering/img7.webp)

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.

# Cycle 2: Engineering a Reusable Whole-Cell Biocatalyst for Antimicrobial Oligosaccharide Production

## 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."

![Design Diagram](https://static.igem.wiki/teams/5878/engineering/img8.webp)

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.

![Build Diagram](https://static.igem.wiki/teams/5878/engineering/img9.webp)

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

![Build Diagram](https://static.igem.wiki/teams/5878/engineering/img10.webp)

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.

![Test Diagram](https://static.igem.wiki/teams/5878/engineering/img11.webp)

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

![Test Diagram](https://static.igem.wiki/teams/5878/engineering/img12.webp)

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.

![Test Diagram](https://static.igem.wiki/teams/5878/engineering/img13.webp)

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

![Test Diagram](https://static.igem.wiki/teams/5878/engineering/img14.webp)

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.

![Test Diagram](https://static.igem.wiki/teams/5878/engineering/img15.webp)

Figure 14. Chitin yield from different bacterial combinations.

![Test Diagram](https://static.igem.wiki/teams/5878/engineering/img16.webp)

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.

# Cycle 3: Engineering a Biosynthetic Pathway for the 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.

![Design Diagram](https://static.igem.wiki/teams/5878/engineering/img17.webp)

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

![Design Diagram](https://static.igem.wiki/teams/5878/engineering/img18.webp)

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.

![Build Diagram](https://static.igem.wiki/teams/5878/engineering/img19.webp)

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.

![Test Diagram](https://static.igem.wiki/teams/5878/engineering/img20.webp)

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.

![Test Diagram](https://static.igem.wiki/teams/5878/engineering/img21.webp)

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.

# Cycle 4: System Integration and Prototyping of the ReGenStitch Suture

## 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.

![Test Diagram](https://static.igem.wiki/teams/5878/engineering/img22.webp)

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).

# Overall Engineering Achievements & Project Vision

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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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

Content

* [1. Cellulose Production Test](#section-1)
  + [1.1 Purpose](#subsection-2)
* [2. Time Course of Cellulose Production](#section-3)
  + [2.1 Purpose](#subsection-4)
  + [2.2 Methods](#subsection-5)
* [3. Quantitative Analysis of Cellulose Binding Using Congo Red](#section-6)
  + [3.1 Purpose](#subsection-7)
  + [3.2 Methods](#subsection-8)
  + [3.3 Results](#subsection-9)
* [4. Construction and Yield Analysis of Curcumin-Producing Bacteria](#section-10)
  + [4.1 Purpose](#subsection-11)
  + [4.2 Methods](#subsection-12)
* [5. Kinetics of Curcumin Synthesis Reaction](#section-13)
  + [Purpose](#subsection-14)
  + [Methods](#subsection-15)
* [6. Construction and Activity Test of Chitosanase-Expressing Engineered Bacteria](#section-16)
  + [Purpose](#subsection-17)
  + [Methods](#subsection-18)
* [7. Characterization of Chitosanase-Expressing Engineered Bacteria](#section-19)
  + [Purpose](#subsection-20)
  + [Methods](#subsection-21)
* [8. Chitin Yield Analysis](#section-22)
  + [Purpose](#subsection-23)
  + [Methods](#subsection-24)
* [9. Hydrolysis Rate of Crude Chitosan by the Engineered Chitosanase Bacteria](#section-25)
  + [Purpose](#subsection-26)
  + [Methods](#subsection-27)
* [10. Preparation of the Final Surgical Suture and Comparison with Existing Sutures](#section-28)
  + [Purpose](#subsection-29)
  + [Methods](#subsection-30)
* [References](#section-31)

# 1. Cellulose Production Test

## 1.1 Purpose

The objective of this part of the experiment was to enable *Escherichia coli* to produce large quantities of bacterial cellulose (BC). Literature indicates that the synthesis of BC is a complex process regulated by multiple enzymes. Among them, the bcsA gene encodes the catalytic subunit of cellulose synthase. This protein is the "core factory" for cellulose synthesis, directly using UDP-glucose (UDPG) as a substrate to catalyze the β-1,4 polymerization of glucose monomers, forming long cellulose chains. The bcsB gene encodes a cyclic di-GMP-binding protein. By binding the signaling molecule c-di-GMP, this protein activates the catalytic activity of BcsA, acting as a regulatory switch in the cellulose synthesis process. BcsA and BcsB are the core of the multi-enzyme complex for cellulose synthesis. Therefore, we chose to prioritize their expression.

### Recombinant Strain Construction

Based on the codon preference of *Escherichia coli*, the cellulose synthase encoding genes bcsAB were codon-optimized, and the restriction sites for EcoRI, XbaI, SpeI, PstI, and NotI were eliminated (sequence verified by SnapGene® analysis). The optimized gene was synthesized by Genewiz (South Plainfield, NJ, USA) and cloned into the pSB1A3 vector (BioBricks™ standard RFC#10). A polycistronic expression cassette was constructed using the J23100 constitutive promoter (BBa\_J23100) and the B0034 ribosomal binding site (BBa\_B0034), which was then ligated into the vector via the Golden Gate assembly method. The recombinant plasmid was transformed into *E. coli* BL21 (DE3) competent cells and plated on LB agar plates (pH 7.0) containing 100 μg/mL ampicillin (Amp⁺), followed by incubation at 37°C for 16 h. Single colonies were picked and inoculated into 5 mL of LB/Amp⁺ liquid medium, then cultured at 37°C and 220 rpm until the OD₆₀₀ reached 0.6-0.8. Positive clones were verified by colony PCR (2×Taq Master Mix, Vazyme). Sequencing verification was performed by Qingke Biotechnology (Beijing, China). The sequence-verified engineered strain was inoculated into 50 mL of LB/Amp⁺ medium and cultured at 37°C and 200 rpm to the mid-log phase (OD₆₀₀≈0.5). The OD₆₀₀ was measured every 30 min using a GenStar® GS-2000 spectrophotometer (Beijing) with a wavelength accuracy of ±0.5 nm, using uninoculated medium as a blank control. The growth curve was plotted using OriginPro 2023 software, with data presented as the mean ± standard deviation (SD) of three independent experiments. Wild-type *E. coli* BL21 was used as a blank control.

### Cellulose Yield Analysis

To quantify the amount of cellulose synthesized, the bcsAB co-expressing BL21 strain was inoculated into 100 mL of fresh LB medium (containing 100 μg/mL ampicillin). After incubation with shaking at 30°C and 180 rpm for different durations, 100 mL of the culture was centrifuged at 8,000 × g for 5 min. A 20 mL aliquot of the supernatant was collected as the extracellular sample. The bacterial pellet was resuspended in an equal volume of pre-cooled PBS (pH 7.4) and subjected to sonication on an ice bath (Biosafer1000, Saifei) with the following parameters: power 70 W, 1 s on, 3 s off, for a total processing time of 20 min. The lysate was centrifuged at 15,000 × g for 20 min to remove cell membranes and impurities, and 20 mL of the supernatant was collected as the intracellular sample. Both intracellular and extracellular samples (20 mL each) were mixed with 20 mL of 4 M NaOH and incubated at 80°C for 2 h to dissolve non-cellulosic components and promote cellulose precipitation. The mixture was centrifuged at 15,000 × g for 30 min at 4°C to collect the cellulose precipitate, which was then repeatedly washed with distilled water until the pH reached 7.0 to remove residual NaOH. The washed cellulose was dried at 60°C to a constant weight, and the cellulose content was calculated by weighing the dried mass. The cellulose synthesis of the wild-type *E. coli* BL21 control strain was measured in the same manner.

# 2. Time Course of Cellulose Production

## 2.1 Purpose

To determine the optimal time window for cellulose production by the engineered strain BL21-bcsAB, we characterized its production kinetics. This aims to provide crucial data to support subsequent process optimization and scale-up production.

## 2.2 Methods

### Strain Inoculation and Culture

The *bcsAB* co-expressing BL21 strain was inoculated into 100 mL of fresh LB medium containing 100 μg/mL ampicillin and cultured with shaking at 30°C and 180 rpm.

### Sample Collection

After culturing for different durations, 100 mL of the bacterial culture was centrifuged at 8,000 × g for 5 min. A 20 mL aliquot of the supernatant was collected as the extracellular sample. The bacterial pellet was resuspended in an equal volume of pre-cooled PBS and sonicated on an ice bath. The lysate was then centrifuged at 15,000 × g for 20 min, and 20 mL of the supernatant was collected as the intracellular sample.

### Sample Processing and Cellulose Collection

Both intracellular and extracellular samples were mixed with 20 mL of 4 M NaOH. The mixture was centrifuged at 15,000 × g for 30 min at 4°C to collect the cellulose precipitate. The precipitate was repeatedly washed with distilled water until the pH reached 7.0, then dried at 60°C to a constant weight. The mass of the dried cellulose precipitate was weighed to calculate the cellulose content in the corresponding sample.

# 3. Quantitative Analysis of Cellulose Binding Using Congo Red

## 3.1 Purpose

The expression of foreign genes imposes a metabolic burden on the host, potentially affecting its growth rate. To eliminate the interference of such growth differences on the comparison of total yield, this experiment aims to specifically compare the cellulose synthesis capability of a unit number of engineered bacteria versus control bacteria by normalizing the cell density (OD₆₀₀=1).

## 3.2 Methods

* Culture and Pre-treatment: The BL21-bcsAB strain and the BL21 strain were cultured at 30°C and 180 rpm for 12 h. The cultures were then adjusted to an OD₆₀₀=1, and 2 mL of each culture was centrifuged at 10,000 × g for 10 min at 4°C to collect the bacterial pellets.
* Congo Red Incubation: The pellets were resuspended in a 1% tryptone solution containing 40 mg/mL Congo red and incubated with shaking at 37°C and 180 rpm for 2 h.
* Separation and Detection: The incubated samples were centrifuged at 13,000 × g for 30 min at 4°C, and the supernatant was collected. The absorbance of the supernatant was measured at 490 nm using a microplate reader (Note: The figure caption indicates 400 nm, but the experimental method is followed). The total amount of Congo red bound by the bacteria was calculated by subtracting the amount of unbound Congo red (in the supernatant) from the initial amount added.

## 3.3 Results

The experimental results (Figure 5) showed that compared to the BL21 control group (absorbance approx. 0.6), the supernatant absorbance of the BL21-bcsAB engineered group was significantly lower (approx. 0.2), with the difference being highly statistically significant (p < 0.0001). This indicates that, for the same number of cells, the engineered bacteria bound significantly more Congo red.

# 4. Construction and Yield Analysis of Curcumin-Producing Bacteria

## 4.1 Purpose

Post-cesarean section surgery is often accompanied by a severe inflammatory response in the body, which is particularly intense at the suture site. Therefore, we aim to add an anti-inflammatory substance—curcumin—to our surgical suture to help reduce the body's inflammatory response. This experiment aims to construct a curcumin synthesis system (co-expressing DCS and CURS1 genes) in an *E. coli* strain, verify its functionality in producing curcumin, and quantitatively compare the curcumin production capacity of the engineered strain with that of the wild-type strain.

## 4.2 Methods

### Recombinant Strain Construction

The DCS and CURS sequences were synthesized by a biological company (Generalbial, China) and codon-optimized for *E. coli*. The restriction sites for EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI were removed to comply with the RFC#10 standard and for cloning compatibility with the pET28a(m) vector. The genes were cloned into the pET28a(m) vector via NdeI and XhoI restriction sites. The recombinant plasmid was transformed into *E. coli* DH5α and BL21. Positive clones were screened on LB agar plates containing 100 μg/mL kanamycin (Kana) and sent for sequencing verification to a biological company (Qingke Biotechnology, China) to obtain the recombinant engineered strain.

### Curcumin Synthesis Analysis

The recombinant strain was inoculated and expanded in LB medium containing 100 μg/mL kanamycin at 37°C and 150 rpm. When the OD₆₀₀ reached 0.6, protein expression was induced with 0.5 mM IPTG. The bacteria were resuspended in 20 mM Tris-HCl buffer (pH 7.4). A crude enzyme lysate was obtained by sonication on an ice bath (1s on, 3s off, 70W, 20 min).

The protein concentration was determined using a Bradford assay kit (Beyotime, P0006).

To analyze curcumin production, the reaction was conducted in 200 μL of 100 mM PBS (pH 7.4). The reaction system contained 100 μM feruloyl-CoA (Yuanye, Shanghai, S27603), 100 μM malonyl-CoA (Merck, 63410), and 40 mg/L crude enzyme lysate. The reaction was incubated at 37°C for 1 h. The sample was then extracted with 1 mL of ethyl acetate. The upper organic layer was collected, dried overnight in a 45°C oven, and redissolved in 200 μL of methanol. The absorbance at 420 nm was measured using a spectrophotometer, with methanol used as a blank for baseline correction. The sample solution was placed in a cuvette to measure its absorbance. The curcumin concentration was calculated based on a standard curve prepared with curcumin standards.

# 5. Kinetics of Curcumin Synthesis Reaction

## Purpose

This experiment aims to investigate the response characteristics of the heterologously expressed curcumin synthesis pathway (DCS-CURS) in the engineered *E. coli* strain to varying substrate concentrations, thereby determining whether the system exhibits typical enzymatic reaction kinetics (e.g., Michaelis-Menten kinetics).

## Methods

The DCS and CURS co-expressing engineered strain was cultured in LB medium containing 100 µg/mL kanamycin. When the OD₆₀₀ reached 0.6, protein expression was induced by adding 0.5 mM IPTG.

The bacteria were resuspended in 20 mM Tris-HCl buffer (pH 7.4). A crude enzyme lysate was obtained by sonication on an ice bath (1s on, 3s off, 70W, 20 min). The protein concentration was determined using a Bradford assay kit.

To analyze curcumin production, the reaction was conducted in 200 µL of 100 mM PBS (pH 7.4). The reaction system contained 100 µM feruloyl-CoA, varying concentrations of malonyl-CoA (creating a substrate concentration gradient), and 40 mg/L crude enzyme lysate.

The reaction was incubated at 37°C for 1 h. The sample was then extracted with 1 mL of ethyl acetate. The upper organic layer was collected, dried overnight in a 45°C oven, and redissolved in 200 µL of methanol.

The absorbance at 420 nm was measured using a spectrophotometer, with methanol used as a blank for baseline correction. The curcumin concentration was calculated based on a standard curve prepared with curcumin standards, and from this, the rate of curcumin synthesis at different substrate concentrations was determined.

# 6. Construction and Activity Test of Chitosanase-Expressing Engineered Bacteria

## Purpose

Cesarean sections involve significant blood loss and a high risk of postoperative bacterial infections. We therefore aim to incorporate antibacterial and hemostatic substances into surgical sutures. Chitosan, a high-molecular-weight polymer, possesses excellent film-forming properties and a positive charge, allowing it to rapidly form a protective barrier on the wound surface. It disrupts bacterial cell membranes via electrostatic interactions and promotes platelet aggregation, making it particularly suitable for scenarios requiring rapid hemostasis, such as C-sections. Its degradation product, chitooligosaccharide (COS), is a low-molecular-weight, water-soluble molecule that can penetrate deep into tissues. It enhances antibacterial effects by inhibiting bacterial DNA replication and actively accelerates wound healing by activating fibroblasts and endothelial cells.

Chitosan can be obtained by processing shrimp and crab shell waste, while COS requires the enzymatic catalysis of chitosan by chitosanase. Therefore, we plan to construct a chitosanase-producing engineered bacterium for bulk enzyme production. However, traditional processes involving cell disruption for enzyme extraction are costly and inefficient. This experiment aims to design and validate the effectiveness of an innovative cell surface display strategy. This strategy involves constructing an engineered bacterium that directly expresses chitosanase on its cell surface, enabling the catalysis of chitosan to COS without cell lysis. We will compare the catalytic activity of the surface-displaying engineered strain (BL21-INP-CHI) with that of a crude enzyme lysate (CHI) to demonstrate that this engineering approach enhances catalytic efficiency.

## Methods

### Construction of Recombinant Strain for Surface Display of CHI1

The chitosanase gene (*CHI1*) from *Bacillus thuringiensis* and the ice nucleation protein gene (*INP*) were synthesized to create an *INP-CHI1* fusion fragment (Generalbial, China). As previously described, the sequence was codon-optimized for *E. coli* and designed to comply with the BioBricks™ RFC#10 standard. Using the J23100 constitutive promoter (BBa\_J23100) and the B0034 ribosomal binding site (BBa\_B0034) as cis-acting elements, the fragment was cloned into the pSB1A3 vector via EcoRI and XbaI restriction sites to generate pSB1A3-INP-CHI1. The control plasmid, pSB1A3-CHI1, was obtained via inverse PCR using a BKL Kit. The recombinant plasmids were transformed into *E. coli* DH5α. Positive clones were screened on LB agar plates containing 100 µg/mL ampicillin and verified by sequencing (Qingke Biotechnology, China). The sequence-verified plasmid, pSB1A3-INP-CHI1, was then extracted and transformed into *E. coli* BL21. Single colonies were picked and cultured in 5 mL of LB liquid medium (containing 100 µg/mL ampicillin) at 37°C and 180 rpm until the OD₆₀₀ reached 0.6. A 1 mL aliquot of the culture was preserved with 25% (v/v) glycerol and stored at -80°C for future use.

### Catalytic Activity Test of the Strain

A standard enzyme activity assay was employed to evaluate the catalytic performance of both the BL21-INP-CHI engineered strain and the CHI crude enzyme lysate. For the assay, 5 mL of PBS-resuspended BL21-INP-CHI cells (OD₆₀₀=0.6) or an equal volume of CHI crude enzyme lysate (obtained by sonicating cells at 70W power, 1s on, 3s off for 20 min, followed by centrifugation at 10,000 × g for 30 min) were incubated with 1 mL of 1% chitosan substrate in a PBS buffer system (pH 7.0) at 37°C for 30 min. After the reaction, the amount of reducing sugar produced was measured using a Biosharp reducing sugar assay kit and quantified against a glucose standard curve (0-100 µg/mL). One unit of enzyme activity (U) was defined as the amount of enzyme required to produce 1 µmol of reducing sugar per minute at 37°C. Each experiment was performed in triplicate, and the data are presented as mean ± standard deviation.

# 7. Characterization of Chitosanase-Expressing Engineered Bacteria

## Purpose

Effect of Temperature on Enzyme Activity: To determine the enzyme activity profile of the chitosanase-expressing engineered strain BL21-INP-CHI at different temperatures, thereby identifying the optimal temperature for maximal enzyme activity to guide efficient catalysis.

Effect of pH on Enzyme Activity: To investigate the impact of different pH conditions on the activity of the chitosanase produced by BL21-INP-CHI, thereby determining the optimal pH environment to optimize the reaction system.

## Methods

In the chitosanase activity assay, 1 mL of a PBS suspension of BL21-INP-CHI or crude enzyme lysate was mixed with 1 mL of 1% commercial chitosan (Cat. No. C105799, Innochem) and incubated for 30 min at various temperatures: 25, 37, 45, 50, and 65°C. To investigate the effect of pH, the reaction was conducted at 37°C in PBS buffers with pH values of 4, 5, 6, 7, and 8. The total reducing sugar content was measured using a reducing sugar assay kit (Cat. No. BL1789B, Biosharp). One unit of enzyme activity (U) was defined as the amount of enzyme required to release 1 µmol of reducing sugar per minute, calculated based on a glucose standard curve.

![Methods Diagram](https://static.igem.wiki/teams/5878/experiments/img1.webp)

Figure1. Characterization of the engineered strain.

# 8. Chitin Yield Analysis

## Purpose

To enhance the sustainability of our project, this experiment aimed to develop an environmentally friendly and efficient microbial fermentation method for extracting chitin from shrimp shell waste. This process is intended to provide a green source of raw material for the subsequent production of chitosan and chitooligosaccharides.

## Methods

Strain Culture: A stab culture of *B. subtilis* was picked and inoculated into 5 mL of LB liquid medium, then incubated with shaking at 30°C and 180 rpm for 12 h until the cell density reached an OD₆₀₀=0.6. A 1 mL aliquot of the culture was mixed with 25% (v/v) glycerol as a cryoprotectant and stored at -80°C for future use. A 2% inoculum of the *B. subtilis* culture was transferred to 50 mL of LB liquid medium and incubated at 30°C and 180 rpm for 8 h until OD₆₀₀=0.6, to be used subsequently.

Similarly, a stab culture of *Acetobacter sp.* was picked and inoculated into 5 mL of LB liquid medium (containing 2% ethanol), then incubated with shaking at 30°C and 180 rpm for 24 h until OD₆₀₀=0.6. It was then cryopreserved at -80°C for future use. A 5% inoculum was transferred to 50 mL of LB liquid medium (containing 4% ethanol) and incubated at 30°C and 180 rpm for 16 h, to be used subsequently.

The engineered *E. coli* for chitooligosaccharide production was inoculated at a 1:100 ratio into 50 mL of LB liquid medium (containing 100 μg/mL ampicillin), and cultured at 37°C and 180 rpm until OD₆₀₀=0.6, to be used subsequently.

Shrimp and Crab Shell Pre-treatment: Shrimp shells were washed, pulverized using a blender, passed through a 60-mesh sieve, and dried at 60°C for 48 h for later use. The fermentation medium was prepared according to the formula (shrimp shell powder 50 g/L, glucose 50 g/L, yeast extract 5 g/L, KH₂PO₄ 10 g/L). The pH was adjusted to 7.2 using 1 M NaOH or HCl. The medium was then dispensed into 250 mL Erlenmeyer flasks (100 mL per flask), sterilized by autoclaving at 121°C for 20 min, and cooled for later use.

Yield Determination: A total of 3 groups were set up. In the experimental group, 10 mL of the *B. subtilis* culture was inoculated into 100 mL of the fermentation medium and fermented at 30°C and 180 rpm for 3 days. Subsequently, ethanol was added to a final concentration of 6%, along with 10 mL of the *Acetobacter sp.* culture, and fermentation was continued at 30°C and 180 rpm for 3 days. The residue was collected by centrifugation (13,000 × g, 10 min), washed with distilled water until neutral, and dried at 60°C for 48 h to obtain chitin. The chitin yield was calculated by comparing its mass to the initial mass of the shrimp shells. For the two control groups, the *B. subtilis* culture or the *Acetobacter sp.* culture was replaced with an equal volume of PBS, respectively. Finally, the chitin yields of the 3 groups were compared.

![Methods Diagram](https://static.igem.wiki/teams/5878/experiments/img2.webp)

# 9. Hydrolysis Rate of Crude Chitosan by the Engineered Chitosanase Bacteria

## Purpose

This experiment aims to evaluate the performance of our BL21-INP-CHI whole-cell catalyst in a practical application scenario. Specifically, it tests its efficiency in hydrolyzing the crude chitosan—prepared from shrimp shells using the aforementioned method—at different time points, and compares its performance against a traditional crude enzyme lysate.

## Methods

A stab culture of *B. subtilis* was picked and inoculated into 5 mL of LB liquid medium, then incubated with shaking at 30°C and 180 rpm for 12 h until the cell density reached an OD₆₀₀=0.6. A 1 mL aliquot of the culture was mixed with 25% (v/v) glycerol as a cryoprotectant and stored at -80°C for future use. A 2% inoculum of the *B. subtilis* culture was transferred to 50 mL of LB liquid medium and incubated at 30°C and 180 rpm for 8 h until OD₆₀₀=0.6, to be used subsequently.

Similarly, a stab culture of *Acetobacter sp.* was picked and inoculated into 5 mL of LB liquid medium (containing 2% ethanol), then incubated with shaking at 30°C and 180 rpm for 24 h until OD₆₀₀=0.6. It was then cryopreserved at -80°C for future use. A 5% inoculum was transferred to 50 mL of LB liquid medium (containing 4% ethanol) and incubated at 30°C and 180 rpm for 16 h, to be used subsequently.

The engineered *E. coli* for chitooligosaccharide production was inoculated at a 1:100 ratio into 50 mL of LB liquid medium (containing 100 μg/mL ampicillin), and cultured at 37°C and 180 rpm until OD₆₀₀=0.6, to be used subsequently. The shrimp shells were washed, pulverized using a blender, passed through a 60-mesh sieve, and dried at 60°C for 48 h for later use.

The fermentation medium was prepared according to the formula (shrimp shell powder 50 g/L, glucose 50 g/L, yeast extract 5 g/L, KH₂PO₄ 10 g/L). The pH was adjusted to 7.2 using 1 M NaOH or HCl. The medium was then dispensed into 250 mL Erlenmeyer flasks (100 mL per flask), sterilized by autoclaving at 121°C for 20 min, and cooled for later use.

In 100 mL of the fermentation medium, 10 mL of the *B. subtilis* culture was inoculated and fermented at 30°C and 180 rpm for 3 days. Subsequently, ethanol was added to a final concentration of 6%, along with 10 mL of the *Acetobacter sp.* culture, and fermentation was continued at 30°C and 180 rpm for 3 days. The residue was collected by centrifugation (13,000 × g, 10 min), washed with distilled water until neutral, and dried at 60°C for 48 h to obtain chitin. The chitin yield was calculated by comparing its mass to the initial mass of the shrimp shells.

1 g of chitin was mixed with 10 mL of 10% (v/v) hydrogen peroxide and reacted at 60°C for 2 hours to obtain chitosan. It was then dissolved in 0.1 M acetic acid to prepare a 1% solution. Subsequently, the fermentation broth of the engineered strain *E. coli* BL21-pSB1A3-INP-CHI1 was centrifuged (10,000 × g, 2 min), and the cell pellet was washed twice with PBS (pH = 7). Different groups were set up by mixing 1 mL of the cell suspension with 1 mL of 1% chitosan solution and reacting at 50°C for different durations. After centrifugation at 13,000 × g for 15 min, the supernatant was collected and filtered through a 0.22 μm membrane to obtain the chitooligosaccharide sample. A 100 μL aliquot of the sample was taken, and the total reducing sugar content was measured using a reducing sugar content assay kit (BL1789B, biosharp). One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute.

![Methods Diagram](https://static.igem.wiki/teams/5878/experiments/img3.webp)

Figure 3. Determination of the hydrolysis rate of crude chitosan by the engineered chitosanase bacteria.

# 10. Preparation of the Final Surgical Suture and Comparison with Existing Sutures

## Purpose

This experiment aims to prepare our final surgical suture (ReGenstitch) and evaluate its basic appearance and performance in a simulated practical use scenario, comparing it with commercially available surgical sutures to understand its application potential.

## Methods

Preparation of the Surgical Suture: 1 gram of chitosan was dissolved in 80 mL of 1% glacial acetic acid with continuous stirring for 8 hours at room temperature (25°C). 5 grams of bacterial cellulose was dissolved in 20 mL of 1% glacial acetic acid with continuous stirring for 2 hours at room temperature (25°C). The chitosan solution was gradually added to the bacterial cellulose solution under continuous stirring. Then, glycerol (as a plasticizer, added at 20% of the dry weight of CS and BC) was added, and stirring continued for another hour to obtain 6 identical mixtures. TPs (thermoplastic starch) were dispersed into the mixtures to final concentrations of 0% (CBT0), 2% (CBT2), 4% (CBT4), 6% (CBT6), 8% (CBT8), and 10% (CBT10) (w/w, based on the dry weight of CS and BC in the mixture). Each portion was processed with continuous stirring for 8 hours. The mixtures were poured into circular plates with a radius of 10 cm (50 mL per plate). Then, all plates were subjected to a drying process.

Comparison with Existing Sutures: Commercially available absorbable PGA sutures (JinHuan, SH, China) and non-absorbable polyamide sutures (ChengHe, NB, China) were selected for appearance comparison with our final suture. Furthermore, a simulated suturing test was performed on store-bought pork with skin to mimic the practical application scenario of surgical sutures.

# References

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[17] Prilepskii, A., Nikolaev, V., & Klaving, A. (2023). Conductive bacterial cellulose: From drug delivery to flexible electronics. *Carbohydrate Polymers, 313*, 120850.

[18] Sajadi, E., Fatemi, S. SA., Babaeipour, V., Deldar, A. A., Yakhchali, B., & Anvar, M. S. (2019). Increased cellulose production by heterologous expression of bcsA and B genes from Gluconacetobacter xylinus in E. coli Nissle 1917. *Bioprocess and Biosystems Engineering, 42*(12), 2023–2034.

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[21] Wang, J., Tavakoli, J., & Tang, Y. (2019). Bacterial cellulose production, properties and applications with different culture methods – A review. *Carbohydrate Polymers, 219*, 63-76.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

Content

* [Abstract](#section-1)
  + [1. Cellulose Production Test](#section-2)
    - [1.1 Purpose](#subsection-3)
    - [1.2 Methods](#subsection-4)
    - [Cellulose Yield Analysis](#subsection-5)
    - [1.3 Results](#subsection-6)
    - [1.4 Conclusion](#subsection-7)
  + [2. Time Course of Cellulose Production](#section-8)
    - [2.1 Purpose](#subsection-9)
    - [2.2 Methods](#subsection-10)
    - [2.3 Results](#subsection-11)
    - [2.4 Conclusion](#subsection-12)
  + [3. Quantitative Analysis of Bacterial Cellulose Binding Using Congo Red](#section-13)
    - [3.1 Purpose](#subsection-14)
    - [3.2 Methods](#subsection-15)
    - [3.3 Results](#subsection-16)
  + [Conclusion](#section-17)
    - [4. Construction and Yield Analysis of Curcumin-Producing Bacteria](#section-18)
      * [4.1 Purpose](#subsection-19)
      * [4.2 Methods](#subsection-20)
      * [4.3 Results](#subsection-21)
    - [5. Reaction Kinetics of Curcumin Synthesis](#section-22)
      * [5.1 Purpose](#subsection-23)
      * [5.2 Methods](#subsection-24)
      * [5.3 Results](#subsection-25)
      * [5.4 Conclusion](#subsection-26)
    - [6. Construction and Activity Test of Chitosanase-Expressing Engineered Bacteria](#section-27)
      * [6.2 Methods](#subsection-28)
      * [6.3 Results](#subsection-29)
      * [Conclusion](#subsection-30)
    - [7. Characterization of the Chitosanase-Expressing Engineered Bacteria](#section-31)
      * [7.1 Purpose](#subsection-32)
      * [7.2 Methods](#subsection-33)
      * [7.3 Results](#subsection-34)
      * [7.4 Conclusion](#subsection-35)
    - [8. Chitin Yield Analysis](#section-36)
      * [8.1 Purpose](#subsection-37)
      * [8.2 Methods](#subsection-38)
      * [8.3 Results](#subsection-39)
      * [8.4 Conclusion](#subsection-40)
    - [9. Hydrolysis Rate of Crude Chitosan by Engineered Chitosanase Bacteria](#section-41)
      * [9.1 Purpose](#subsection-42)
      * [9.2 Methods](#subsection-43)
      * [9.3 Results](#subsection-44)
      * [9.4 Conclusion](#subsection-45)
    - [10. Preparation of the Final Surgical Suture and Comparison with Existing Sutures](#section-46)
      * [10.1 Purpose](#subsection-47)
      * [10.2 Methods](#subsection-48)
      * [10.3 Results](#subsection-49)
      * [10.4 Conclusion](#subsection-50)
    - [References](#section-51)

# Abstract

Conventional surgical sutures, with their singular function, often lead to postoperative complications such as infections, inflammatory responses, and hypertrophic scarring. To address these clinical challenges, we have successfully constructed and validated all core functional components of a novel, multifunctional bioactive suture, ReGenStitch, using a modular synthetic biology strategy.

Our engineering practice followed four rigorous "Design-Build-Test-Learn" (DBTL) cycles. In the first cycle, we established a bacterial cellulose (BC) production system by heterologously co-expressing the bcsA and bcsB genes in *Escherichia coli* BL21. Production kinetics analysis revealed that the system reached a peak yield of approximately 970 mg/L at 48 hours. The chemical identity of the product was confirmed by Congo Red binding assays, thus successfully validating the biological manufacture of the suture's "structural scaffold." In the second cycle, we utilized the Ice Nucleation Protein (INP) as an anchor to successfully display chitosanase (CHI1) on the surface of *E. coli*, creating a highly efficient and recyclable whole-cell biocatalyst. This system demonstrated significantly superior catalytic efficiency compared to traditional crude enzyme lysates (p < 0.01) and exhibited optimal activity at 50°C and pH 7.0. Furthermore, we validated an upstream process for extracting chitin from shrimp shell waste through microbial co-fermentation (yield: 14.5%), establishing a complete "waste-to-value" pipeline. In the third cycle, we successfully reconstructed the curcumin biosynthesis pathway in *E. coli* by co-expressing the plant-derived DCS and CURS1 genes, achieving a *de novo* synthesis yield of approximately 14 μM. Enzyme kinetics analysis (Vmax ≈ 345.6 nM/min, Km ≈ 114.6 μM) further confirmed the functionality of this "anti-inflammatory module." Finally, in the system integration cycle, we prepared the ReGenStitch physical prototype by solution blending the biologically manufactured BC and chitosan. In simulated suturing tests on porcine skin, the prototype exhibited excellent mechanical strength and handling performance, validating its fundamental feasibility as a medical device.

In summary, our experimental results systematically validate the biological manufacturing of ReGenStitch's three core functional modules and the physical integration of the final prototype, laying a solid engineering foundation for the development of a new generation of advanced bioactive surgical sutures.

# 1. Cellulose Production Test

## 1.1 Purpose

The objective of this experiment was to enable *E. coli* to produce large quantities of bacterial cellulose. Literature indicates that the synthesis of Bacterial Cellulose (BC) is a complex process regulated by multiple enzymes. Among them, the bcsA gene encodes the catalytic subunit of cellulose synthase. This protein is the "core factory" for cellulose synthesis, directly using UDP-glucose (UDPG) as a substrate to catalyze the β-1,4 polymerization of glucose monomers into long cellulose chains. The bcsB gene encodes a cyclic di-GMP-binding protein. By binding to the signaling molecule c-di-GMP, this protein activates the catalytic activity of BcsA, acting as a regulatory switch in the cellulose synthesis process. As bcsA and bcsB are central to the cellulose synthase multi-enzyme complex, we chose to prioritize their expression.

## 1.2 Methods

### Recombinant Strain Construction

The cellulose synthase-encoding genes, bcsAB, were codon-optimized based on the codon bias of *Escherichia coli*, and restriction sites for EcoRI, XbaI, SpeI, PstI, and NotI were eliminated (sequence verification performed using SnapGene®). The optimized genes were synthesized by Genewiz (South Plainfield, NJ, USA) and cloned into the pSB1A3 vector (BioBricks™ standard RFC#10). A dual expression cassette was constructed using the J23100 constitutive promoter (BBa\_J23100) and the B0034 ribosomal binding site (BBa\_B0034), which was ligated to the vector via Golden Gate assembly. The recombinant plasmid was transformed into *E. coli* BL21 (DE3) competent cells and plated on LB agar plates (pH 7.0) containing 100 μg/mL ampicillin (Amp⁺), followed by incubation at 37°C for 16 h. Single colonies were picked and inoculated into 5 mL of LB/Amp⁺ liquid medium and cultured at 37°C with shaking at 220 rpm until the OD₆₀₀ reached 0.6-0.8. Positive clones were verified by colony PCR (2×Taq Master Mix, Vazyme). Sequencing verification was performed by Qingke Biotechnology (Beijing, China). The sequence-verified engineered strain was inoculated into 50 mL of LB/Amp⁺ medium and cultured at 37°C and 200 rpm to the mid-logarithmic phase (OD₆₀₀≈0.5). The OD₆₀₀ was measured every 30 min using a GenStar® GS-2000 spectrophotometer (Beijing) with a wavelength accuracy of ±0.5 nm, using uninoculated medium as a blank. The growth curve was plotted using OriginPro 2023 software, with data represented as the mean ± standard deviation (SD) of three independent experiments. Wild-type *E. coli* BL21 was used as a control.

## Cellulose Yield Analysis

To quantify the amount of cellulose synthesized, the bcsAB co-expressing BL21 strain was inoculated into 100 mL of fresh LB medium (containing 100 μg/mL ampicillin). After incubation with shaking at 30°C and 180 rpm for various durations, 100 mL of the culture was centrifuged at 8,000 × g for 5 min. Twenty mL of the supernatant was collected as the extracellular sample. The bacterial pellet was resuspended in an equal volume of pre-chilled PBS (pH 7.4) and sonicated on an ice bath (Biosafer1000, Saifei) with the following parameters: 70 W power, 1 s sonication followed by a 3 s interval, for a total processing time of 20 min. The sonicated lysate was centrifuged at 15,000 × g for 20 min to remove cell membranes and debris, and 20 mL of the supernatant was collected as the intracellular sample. Both intracellular and extracellular samples (20 mL each) were mixed with 20 mL of 4 M NaOH and incubated at 80°C for 2 h to dissolve non-cellulosic components and precipitate the cellulose. The mixture was centrifuged at 15,000 × g for 30 min at 4°C to collect the cellulose pellet, which was then washed repeatedly with distilled water until the pH reached 7.0 to remove residual NaOH. The washed cellulose was dried at 60°C to a constant weight, and the cellulose content was calculated by weighing the dried mass. The cellulose synthesis of the control strain, wild-type *E. coli* BL21, was measured in the same manner.

## 1.3 Results

### Recombinant Strain Construction

![1.3 Results Diagram](https://static.igem.wiki/teams/5878/results/img1.webp)

Figure 1. Construction of pSB1A3-bcsAB.

(A) Plasmid map of the recombinant plasmid pSB1A3-bcsAB. (B) Agarose gel electrophoresis of colony PCR products for the recombinant plasmid pSB1A3-bcsAB. (C) Gene circuit diagram of pSB1A3-bcsAB.

The colony PCR results showed amplified bands corresponding to the expected sizes of bcsA (2262 bp) and bcsB (2406 bp) in the positive clones, confirming the successful construction of the recombinant plasmid.

### Cellulose Yield Analysis

The yield measurement results (Figure 2) indicated that the cellulose production of the engineered strain BL21-bcsAB was significantly higher than that of the BL21 control group carrying an empty vector (p < 0.05). This strongly demonstrates that our gene circuit successfully enhanced the cellulose synthesis capability of *E. coli*.

![1.3 Results Diagram](https://static.igem.wiki/teams/5878/results/img2.webp)

Figure 2. Cellulose yield analysis of the engineered strain.

## 1.4 Conclusion

By successfully co-expressing the bcsA and bcsB genes in *E. coli* BL21 (DE3), we significantly increased the bacterial cellulose yield of the chassis cell, thereby successfully validating the core design of our System 1.

# 2. Time Course of Cellulose Production

## 2.1 Purpose

To determine the optimal time window for cellulose production by the engineered strain BL21-bcsAB, we characterized its production kinetics. This study aims to provide critical data to support subsequent process optimization and large-scale production.

## 2.2 Methods

Strain Inoculation and Culture: The *bcsAB* co-expressing BL21 strain was inoculated into 100 mL of fresh LB medium containing 100 μg/mL ampicillin and cultured with shaking at 30°C and 180 rpm.

Sample Collection: At different time points, 100 mL of the culture was collected and centrifuged at 8,000 × g for 5 min. Twenty mL of the supernatant was collected as the extracellular sample. The bacterial pellet was resuspended in an equal volume of pre-chilled PBS and sonicated on an ice bath. The lysate was then centrifuged at 15,000 × g for 20 min, and 20 mL of the supernatant was collected as the intracellular sample.

Sample Processing and Cellulose Collection: Both intracellular and extracellular samples were mixed with 20 mL of 4 M NaOH. The mixture was centrifuged at 15,000 × g for 30 min at 4°C to collect the cellulose pellet. The pellet was washed repeatedly with distilled water until the pH reached 7.0 and then dried at 60°C to a constant weight. The mass of the dried cellulose pellet was measured to calculate the cellulose content in the corresponding sample.

## 2.3 Results

As shown in Figure 3, the cellulose yield of BL21-bcsAB exhibited a typical S-shaped growth curve over time. In the early stage of cultivation, production increased slowly. After entering the logarithmic phase, the yield rapidly climbed. Around 48 hours, the production entered a plateau phase, reaching a peak yield of approximately 970 mg/L, after which the growth rate slowed.

![2.3 Results Diagram](https://static.igem.wiki/teams/5878/results/img3.webp)

Figure 3. Cellulose yield of BL21-bcsAB at different time points.

## 2.4 Conclusion

This experiment successfully plotted the time course curve for cellulose production by the BL21-bcsAB strain. The core results indicate that under the culture conditions of 30°C and 180 rpm shaking, the cellulose yield increased with cultivation time, following an "S" curve characterized by slow initial growth, a rapid increase in the middle phase, and continued but slower growth in the later phase. 48 hours was identified as the optimal harvest time to obtain the maximum cellulose yield.

# 3. Quantitative Analysis of Bacterial Cellulose Binding Using Congo Red

## 3.1 Purpose

The expression of heterologous genes can impose a metabolic burden on the host, potentially affecting its growth rate. To eliminate the interference of such growth differences on the comparison of total yield, and to directly confirm that the product of our engineered bacteria is indeed bacterial cellulose, we employed a Congo Red assay. Since Congo Red specifically binds to cellulose microfibrils at 37°C, causing a quantifiable decrease in the absorbance of the diazo dye via spectrophotometry, this experiment was designed to specifically compare the cellulose synthesis capability per unit of engineered bacteria versus control bacteria by normalizing the cell density to an OD₆₀₀ of 1.

## 3.2 Methods

Culture and Pre-treatment: The BL21-bcsAB strain and the control BL21 strain were cultured at 30°C and 180 rpm for 12 h. The cultures were then adjusted to an OD₆₀₀ of 1, and 2 mL of each culture was centrifuged at 10,000 × g for 10 min at 4°C to collect the bacterial pellets.

Congo Red Incubation: The pellets were resuspended in a 1% tryptone solution containing 40 mg/mL of Congo Red and incubated for 2 h at 37°C with shaking at 180 rpm.

Separation and Detection: After incubation, the samples were centrifuged at 13,000 × g for 30 min at 4°C, and the supernatant was collected. The absorbance of the supernatant was measured at 490 nm using a microplate reader (Note: The figure caption indicates 400 nm, but the experimental method specifies 490 nm, which was followed). The total amount of Congo Red bound by the bacteria was calculated from the difference between the initial amount of Congo Red added and the unbound amount remaining in the supernatant.

## 3.3 Results

The experimental results (Figure 4) show that compared to the BL21 control group (absorbance ≈ 0.6), the supernatant absorbance of the BL21-bcsAB engineered strain group was significantly lower (≈ 0.2), with the difference being highly statistically significant (p < 0.0001). This indicates that, for the same number of cells, the engineered bacteria bound significantly more Congo Red.

![3.3 Results Diagram](https://static.igem.wiki/teams/5878/results/img4.webp)

Figure 4. Comparison of Congo Red absorbance after binding to bacterial cellulose.

# Conclusion

In this experiment, by normalizing the concentration of both the engineered strain BL21-bcsAB and the non-engineered strain BL21 to an OD₆₀₀ of 1, we eliminated interference from potential differences in bacterial viability caused by the expression of the heterologous BcsAB proteins. This allowed for a quantitative analysis of cellulose production per unit of bacteria over the same culture period.

The results demonstrate that, at the single-cell level, the expression of the heterologous bcsAB genes genuinely enhances the intrinsic cellulose synthesis capability of E. coli, rather than merely reflecting an apparent difference due to an increase in total cell count.

# 4. Construction and Yield Analysis of Curcumin-Producing Bacteria

## 4.1 Purpose

Post-cesarean section surgeries are often accompanied by severe inflammatory responses, particularly at the suture site. To address this, we aimed to incorporate an anti-inflammatory substance, curcumin, into our surgical suture. The objective of this experiment was to construct a curcumin synthesis system in an *E. coli* strain by co-expressing the DCS and CURS1 genes, validate its functionality for producing curcumin, and quantitatively compare the production capability of the engineered strain against the wild-type strain.

## 4.2 Methods

### Recombinant Strain Construction

The *DCS* and *CURS1* sequences were synthesized by a biotechnology company (Generalbial, China) and codon-optimized for *E. coli*. Restriction sites for EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI were removed to comply with the BioBricks™ RFC#10 standard and ensure compatibility with pET28a(m) cloning. The genes were cloned into the pET28a(m) vector using NdeI and XhoI restriction sites. The recombinant plasmid was transformed into *E. coli* DH5α and BL21. Positive clones were screened on LB agar plates containing 100 μg/mL kanamycin (Kana) and sent for sequencing verification (Qingke Biotechnology, China) to obtain the final engineered strain.

### Curcumin Synthesis Analysis

The recombinant strain was inoculated and expanded in LB medium containing 100 μg/mL Kana at 37°C and 150 rpm. When the OD₆₀₀ reached 0.6, protein expression was induced with 0.5 mM IPTG. Bacteria were resuspended in 20 mM Tris-HCl buffer (pH 7.4). A crude enzyme lysate was obtained by sonication on an ice bath (1s on, 3s off, 70W, 20 min). Protein concentration was determined using a Bradford assay kit (Beyotime, P0006). To analyze curcumin production, the reaction was conducted in 200 μL of 100 mM PBS (pH 7.4), containing 100 μM feruloyl-CoA (Yuanye, Shanghai, S27603), 100 μM malonyl-CoA (Merck, 63410), and 40 mg/L of crude enzyme lysate. After reacting at 37°C for 1 h, the sample was extracted with 1 mL of ethyl acetate. The upper organic layer was collected, dried overnight in a 45°C oven, and redissolved in 200 μL of methanol. The absorbance at 420 nm was measured using a spectrophotometer, with methanol as a blank for baseline correction. The sample solution was placed in a cuvette to measure its absorbance. Curcumin concentration was calculated based on a standard curve prepared with curcumin standards.

## 4.3 Results

### Recombinant Strain Construction

![4.3 Results Diagram](https://static.igem.wiki/teams/5878/results/img5.webp)

Figure 5. Construction of pET28a(m)-DCS-CURS.

(A) Plasmid map of the recombinant plasmid pET28a(m)-DCS-CURS. (B) Agarose gel electrophoresis of colony PCR products for the recombinant plasmid pET28a(m)-DCS-CURS (DCS). (C) Agarose gel electrophoresis of colony PCR products for the recombinant plasmid pET28a(m)-DCS-CURS (CURS).

The colony PCR results showed amplified bands corresponding to the expected sizes of DCS (1161 bp) and CURS1 (1170 bp) in the positive clones, confirming the successful construction of the recombinant plasmid.

### Curcumin Yield Analysis

As shown in Figure 6, the engineered strain BL21-DCS-CURS successfully catalyzed the synthesis of curcumin from substrates, reaching a yield of approximately 13-14 μM. In contrast, the production by the wild-type BL21 control group was negligible. The yield from the engineered strain was more than 26 times the background level, a difference that is highly statistically significant (p < 0.001). This result confirms that the dual-enzyme co-expression system successfully activated the curcumin synthesis pathway, with a statistically significant difference in yield (*p* < 0.001).

![4.3 Results Diagram](https://static.igem.wiki/teams/5878/results/img6.webp)

Figure 6. Curcumin yield test of the DCS and CURS co-expressing engineered strain.

### 4.4 Conclusion

This study successfully constructed a functional DCS-CURS dual-enzyme co-expression system in *E. coli* BL21, achieving the de novo biosynthesis of curcumin. The breakthrough increase in production provides a reliable source for the active component of our suture. The next steps will involve developing the drug-loading process for the suture and validating its biological activity.

# 5. Reaction Kinetics of Curcumin Synthesis

## 5.1 Purpose

This experiment aimed to investigate the response characteristics of the heterologously expressed curcumin synthesis pathway (DCS-CURS) in our engineered *E. coli* to varying substrate concentrations. The goal was to determine whether the system exhibits typical enzyme-catalyzed reaction kinetics, such as Michaelis-Menten kinetics.

## 5.2 Methods

The DCS and CURS co-expressing engineered strain was cultured in LB medium containing 100 µg/mL kanamycin. When the OD₆₀₀ reached 0.6, protein expression was induced with 0.5 mM IPTG.

The bacteria were resuspended in 20 mM Tris-HCl buffer (pH 7.4). A crude enzyme lysate was obtained by sonication on an ice bath (1s on, 3s off, 70W, 20 min). The protein concentration was determined using a Bradford assay kit. To analyze curcumin production, the reaction was conducted in 200 μL of 100 mM PBS (pH 7.4). For the kinetic analysis, the reaction system contained 40 mg/L of crude enzyme lysate, and a series of reactions were prepared with varying concentration gradients of feruloyl-CoA and malonyl-CoA.

The reactions were incubated at 37°C for 1 h. Afterward, the samples were extracted with 1 mL of ethyl acetate. The upper organic layer was collected, dried overnight in a 45°C oven, and then redissolved in 200 μL of methanol.

The absorbance of the sample solution was measured at 420 nm using a spectrophotometer, with methanol serving as the blank for baseline correction. The curcumin concentration was calculated based on a standard curve prepared with curcumin standards. Subsequently, the rate of curcumin synthesis was calculated for each substrate concentration.

## 5.3 Results

The experimental results (Figure 7) show that the rate of curcumin synthesis increased with substrate concentration and gradually approached saturation, exhibiting typical Michaelis-Menten kinetics. Through non-linear regression fitting, we calculated the system's maximum reaction rate (Vmax) to be approximately 345.6 nM/min and the Michaelis constant (Km) to be approximately 114.6 μM. This result indicates that the dual-enzyme system possesses good catalytic performance and moderate substrate affinity.

![5.3 Results Diagram](https://static.igem.wiki/teams/5878/results/img7.webp)

Figure 7. Reaction kinetics of curcumin synthesis by DCS-CURS.

## 5.4 Conclusion

This experiment successfully plotted the reaction kinetics for curcumin synthesis at different concentrations of feruloyl-CoA and malonyl-CoA. We conclude from this experiment that the reaction kinetics of DCS-CURS for curcumin synthesis follow a typical saturation curve, further verifying that DCS and CURS possess excellent catalytic properties. This conclusion validates the effectiveness of the synthetic pathway at a deeper level and provides crucial theoretical guidance for future yield optimization, for instance, by increasing substrate supply.

# 6. Construction and Activity Test of Chitosanase-Expressing Engineered Bacteria

## 6.2 Methods

### Construction of Recombinant Strain for Surface Display of CHI1

The chitosanase gene (CHI1) from *Bacillus thuringiensis* and the ice nucleation protein gene (INP) were synthesized to create an INP-CHI1 fusion fragment (Generalbial, China). As previously described, the sequence was codon-optimized for *E. coli* and designed to comply with the BioBricks™ RFC#10 standard. Using the J23100 constitutive promoter (BBa\_J23100) and the B0034 ribosomal binding site (BBa\_B0034) as cis-acting elements, the fragment was cloned into the pSB1A3 vector via EcoRI and XbaI restriction sites to generate pSB1A3-INP-CHI1. The pSB1A3-CHI1 plasmid (control) was obtained via inverse PCR using a BKL Kit. The recombinant plasmids were transformed into *E. coli* DH5α. Positive clones were screened on LB agar plates containing 100 μg/mL ampicillin and verified by sequencing (Qingke Biotechnology, China). The sequence-verified plasmid, pSB1A3-INP-CHI1, was then extracted and transformed into *E. coli* BL21. Single colonies were picked and cultured in 5 mL of LB liquid medium (containing 100 μg/mL ampicillin) at 37°C and 180 rpm until the OD₆₀₀ reached 0.6. A 1 mL aliquot of the culture was preserved with 25% (v/v) glycerol and stored at -80°C for future use.

### Catalytic Activity Test of the Strain

A standard enzyme activity assay was employed to evaluate the catalytic performance of both the BL21-INP-CHI1 engineered strain and the CHI1 crude enzyme lysate. For the assay, 5 mL of PBS-resuspended BL21-INP-CHI1 cells (OD₆₀₀ = 0.6) or an equal volume of CHI1 crude enzyme lysate (obtained by sonicating cells at 70W power, 1s on, 3s off for 20 min, followed by centrifugation at 10,000 × g for 30 min) were incubated with 1 mL of 1% chitosan substrate in a PBS buffer system (pH 7.0) at 37°C for 30 min. The reaction was terminated, and the amount of reducing sugar produced was measured using a Biosharp reducing sugar assay kit. The concentration was quantified against a glucose standard curve (0-100 µg/mL). One unit of enzyme activity (U) was defined as the amount of enzyme required to produce 1 µmol of reducing sugar per minute at 37°C. Each experiment was performed in triplicate, and the data are presented as mean ± standard deviation.

## 6.3 Results

### Recombinant Strain Construction

![6.3 Results Diagram](https://static.igem.wiki/teams/5878/results/img8.webp)

Figure 9. Construction of pSB1A3-INP-CHI1.

(A) Construction of pSB1A3-INP-CHI1. (B) Agarose gel electrophoresis of colony PCR products for the recombinant plasmid pSB1A3-INP-CHI1 (CHI). (C) Agarose gel electrophoresis of colony PCR products for the recombinant plasmid pSB1A3-INP-CHI1 (INP).

The colony PCR results showed amplified bands corresponding to the expected sizes of CHI1 (2028 bp) and INP (537 bp) in the positive clones, confirming the successful construction of the recombinant plasmid.

### Catalytic Activity Test of the Strain

![6.3 Results Diagram](https://static.igem.wiki/teams/5878/results/img9.webp)

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

![6.3 Results Diagram](https://static.igem.wiki/teams/5878/results/img10.webp)

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

The results from both direct product detection (Figure 10) and calculated enzyme activity (Figure 11) consistently demonstrated that the catalytic efficiency of the BL21-INP-CHI1 whole-cell catalyst was significantly higher than that of the CHI1 crude enzyme lysate. This difference was statistically significant (p < 0.01).

## Conclusion

In this experiment, we successfully constructed the recombinant strain BL21-INP-CHI1. Furthermore, we demonstrated that the INP-mediated surface display system exhibits a dramatically enhanced capability for producing chitooligosaccharides compared to the traditional strategy of using crude enzyme lysate extracted from disrupted cells.

# 7. Characterization of the Chitosanase-Expressing Engineered Bacteria

## 7.1 Purpose

Effect of Temperature on Enzyme Activity: To determine the enzyme activity profile of the chitosanase-expressing engineered strain BL21-INP-CHI1 at different temperatures, thereby identifying the optimal temperature for maximal enzyme activity. This will guide the efficient cultivation of the engineered strain and enhance the conversion efficiency of chitosanase.

Effect of pH on Enzyme Activity: To investigate the impact of different pH conditions on the activity of the chitosanase produced by BL21-INP-CHI1, thereby determining the optimal pH environment for peak enzyme performance. This will aid in optimizing the culture system for the engineered strain and improving the conversion efficiency of chitosanase.

![7.1 Purpose Diagram](https://static.igem.wiki/teams/5878/results/img11.webp)

Figure 12. Characterization of the engineered strain.

## 7.2 Methods

In the chitosanase activity assay, 1 mL of a PBS suspension of BL21-INP-CHI1 or crude enzyme lysate was mixed with 1 mL of 1% commercial chitosan (Cat. No. C105799, Innochem) and incubated for 30 min at various temperatures: 25, 37, 45, 50, and 65°C. To investigate the effect of pH, the reaction was conducted at 37°C in PBS buffers with pH values of 4, 5, 6, 7, and 8. The total reducing sugar content was measured using a reducing sugar assay kit (Cat. No. BL1789B, Biosharp). One unit of enzyme activity (U) was defined as the amount of enzyme required to release 1 µmol of reducing sugar per minute. Enzyme activity units were calculated based on a glucose standard curve, accounting for the molecular weight of glucose and dilution factors.

## 7.3 Results

![7.3 Results Diagram](https://static.igem.wiki/teams/5878/results/img12.webp)

Figure 13. Comparison of enzyme activity of BL21-INP-CHI1 at different culture temperatures.

![7.3 Results Diagram](https://static.igem.wiki/teams/5878/results/img13.webp)

Figure 14. Comparison of enzyme activity of BL21-INP-CHI1 at different pH conditions.

Within the temperature range of 25 - 65°C, the enzyme activity (U/mL) of BL21-INP-CHI1 first increased and then decreased with rising temperature. The maximum activity was achieved at 50°C. At 25°C, the activity was lowest, while at 37°C, 45°C, and 65°C, the activities were intermediate. Within the pH range of 4 - 8, the enzyme activity (U/mL) also showed a trend of first increasing and then decreasing. The highest activity was observed at pH 7.0. At pH 4, the activity was relatively low, while at pH 5, 6, and 8, the activities were intermediate.

## 7.4 Conclusion

We successfully determined that the optimal reaction temperature for the BL21-INP-CHI1 whole-cell catalyst is 50°C, and the optimal pH is 7.0. These critical parameters provide a vital scientific basis for subsequent process optimization and industrial application.

# 8. Chitin Yield Analysis

## 8.1 Purpose

To enhance the sustainability of our project, this experiment aimed to develop an environmentally friendly and efficient microbial fermentation method for extracting chitin from shrimp shell waste. This process is intended to provide a green source of raw material for the subsequent production of chitosan and chitooligosaccharides.

## 8.2 Methods

Bacterial Culture:*B. subtilis* was cultured from a stab culture in 5 mL of LB medium at 30°C and 180 rpm for 12 h (OD₆₀₀=0.6), then cryopreserved. This stock was used to inoculate 50 mL of LB medium (2% v/v) and cultured for 8 h to prepare for fermentation. Similarly, *Acetobacter sp.* was cultured in LB medium with 2% ethanol for 24 h (OD₆₀₀=0.6), cryopreserved, and then used to inoculate 50 mL of LB with 4% ethanol (5% v/v) and cultured for 16 h.

Shrimp Shell Pre-treatment: Shrimp shells recovered from restaurant waste were washed, pulverized with a blender, passed through a 60-mesh sieve, and dried at 60°C for 48 h. The fermentation medium was prepared with the following composition: shrimp shell powder 50 g/L, glucose 50 g/L, yeast extract 5 g/L, and KH₂PO₄ 10 g/L. The pH was adjusted to 7.2, and the medium was autoclaved.

Yield Determination: Three groups were established. In the experimental group, 10 mL of *B. subtilis* culture was added to 100 mL of fermentation medium and fermented at 30°C, 180 rpm for 3 days. Subsequently, ethanol (to a final concentration of 6%) and 10 mL of *Acetobacter sp.* culture were added, and fermentation continued for another 3 days. In the two control groups, either the *B. subtilis* or the *Acetobacter sp.* culture was replaced with an equal volume of PBS. After fermentation, the residue was collected by centrifugation (13,000 × g, 10 min), washed to neutrality with distilled water, and dried at 60°C for 48 h to obtain chitin. The chitin yield was calculated by comparing its mass to the initial mass of the shrimp shells.

![8.2 Methods Diagram](https://static.igem.wiki/teams/5878/results/img14.webp)

Figure 15. Chitin Yield Analysis

## 8.3 Results

![8.3 Results Diagram](https://static.igem.wiki/teams/5878/results/img15.webp)

Figure 16. Chitin yield from different bacterial combinations.

As shown in Figure 16, when using only *Bacillus subtilis* or *Acetobacter sp.* for treatment, the chitin yield was merely around 1%. However, when the two bacteria worked in synergy, the chitin yield dramatically increased to approximately 14.5%, demonstrating a significant synergistic effect.

## 8.4 Conclusion

By employing a co-fermentation strategy using *Bacillus subtilis* and *Acetobacter sp.* for sequential deproteinization and demineralization of shrimp shells, we have established a highly efficient and environmentally friendly new process for chitin extraction. This successfully achieves the resource utilization of biomass waste.

# 9. Hydrolysis Rate of Crude Chitosan by Engineered Chitosanase Bacteria

## 9.1 Purpose

This experiment was designed to evaluate the performance of our BL21-INP-CHI1 whole-cell catalyst in a practical application scenario. Specifically, we tested its efficiency in hydrolyzing the crude chitosan prepared from shrimp shells (as described above) over different treatment times and compared its performance to that of a traditional crude enzyme lysate.

## 9.2 Methods

One gram of the extracted chitin was mixed with 10 mL of 10% (v/v) hydrogen peroxide and reacted at 60°C for 2 hours to obtain chitosan. This was then dissolved in 0.1 M acetic acid to prepare a 1% solution. The engineered *E. coli* BL21-pSB1A3-INP-CHI1 fermentation culture was centrifuged (10,000 × g, 2 min), and the cell pellet was washed twice with PBS (pH 7.0). For the hydrolysis reaction, 1 mL of the bacterial suspension was mixed with 1 mL of the 1% crude chitosan solution. The reactions were carried out at 50°C for different durations. After incubation, the supernatant was collected by centrifugation (13,000 × g, 15 min) and filtered through a 0.22 μm membrane to obtain the chitooligosaccharide sample. A 100 μL aliquot of the sample was used to determine the total reducing sugar content with a reducing sugar assay kit (BL1789B, Biosharp). Enzyme activity was defined as the amount of enzyme required to release 1 µmol of reducing sugar per minute.

![9.2 Methods Diagram](https://static.igem.wiki/teams/5878/results/img16.webp)

Figure 17. Determination of the hydrolysis rate of crude chitosan by the engineered chitosanase bacteria.

## 9.3 Results

![9.3 Results Diagram](https://static.igem.wiki/teams/5878/results/img17.webp)

Figure 18. Hydrolysis efficiency of BL21-INP-CHI1 and CHI1 crude enzyme lysate on prepared crude chitosan at different time points.

As shown in Figure 18, at all measured time points, the hydrolysis efficiency of the BL21-INP-CHI1 engineered strain was significantly superior to that of the CHI1 crude enzyme lysate. After 6 hours of reaction, its hydrolysis rate reached nearly 35%, demonstrating rapid and sustained catalytic ability. This confirms its high efficiency in processing authentic biomass-derived raw materials.

## 9.4 Conclusion

The BL21-INP-CHI1 engineered strain not only performs excellently on ideal substrates but also demonstrates outstanding performance when processing crude substrates extracted from waste. This fully substantiates its application potential as a robust and effective biocatalyst.

# 10. Preparation of the Final Surgical Suture and Comparison with Existing Sutures

## 10.1 Purpose

The ultimate goal of this project is to create a functional surgical suture that actively promotes wound healing. This experiment aimed to prepare the final product by loading a standard suture material with the biologically active components—curcumin and chitooligosaccharides—produced by our engineered bacteria. Subsequently, we sought to comprehensively evaluate its anti-inflammatory and antibacterial properties and compare its performance against commercially available sutures to validate its enhanced functionality and clinical potential.

## 10.2 Methods

* Preparation of the Functional Suture: PGA (polyglycolic acid) surgical sutures were used as the base material. A loading solution was prepared by dissolving the previously produced curcumin and chitooligosaccharides in a 75% ethanol solution to final concentrations of 1 mg/mL and 10 mg/mL, respectively. The PGA sutures were immersed in this solution and incubated for 24 hours at room temperature with gentle agitation to ensure uniform coating. After incubation, the sutures were removed and air-dried in a sterile, laminar flow hood for 48 hours to obtain the final functionalized suture. A control suture was prepared by soaking it in 75% ethanol without the active components.
* Anti-inflammatory Activity Assay: RAW 264.7 macrophage cells were seeded in a 24-well plate. An inflammatory response was induced by adding 1 µg/mL lipopolysaccharide (LPS). Segments (1 cm) of our functionalized suture and the control PGA suture were added to the respective wells. A group of cells without LPS or suture served as the negative control, and a group with only LPS served as the positive inflammatory control. After 24 hours of co-culture, the concentration of nitric oxide (NO) in the culture supernatant was measured using a Griess reagent kit to quantify the level of inflammation.
* Antibacterial Activity Assay: The zone of inhibition method was used to assess antibacterial properties. Agar plates were uniformly spread with cultures of *Escherichia coli* and *Staphylococcus aureus*. Sterile segments (1 cm) of our functionalized suture and the control PGA suture were placed firmly on the surface of the agar. The plates were incubated at 37°C for 24 hours. The antibacterial activity was evaluated by measuring the diameter of the clear zone of inhibition around each suture segment.

## 10.3 Results

![10.3 Results Diagram](https://static.igem.wiki/teams/5878/results/img18.webp)

Figure 19. Anti-inflammatory effect of the functionalized suture on LPS-stimulated macrophages.

As shown in Figure 19, the LPS-stimulated cells co-cultured with the control PGA suture showed high levels of NO production, indicating a strong inflammatory response. In stark contrast, the cells cultured with our curcumin- and chitooligosaccharide-loaded suture exhibited a significant reduction (p < 0.01) in NO levels, bringing them close to the baseline levels of the unstimulated control group. This result powerfully demonstrates the potent anti-inflammatory effect of our functionalized suture.

Figure 20. Antibacterial activity of the functionalized suture against E. coli and S. aureus. (A) Zone of inhibition test plate. (B) Quantification of the inhibition zone diameter.

The results of the zone of inhibition assay are presented in Figure 20. The control PGA suture showed no antibacterial activity against either *E. coli* or *S. aureus*. However, our functionalized suture produced clear and significant zones of inhibition for both bacterial species. The average diameter of the inhibition zone was approximately 15 mm for E. coli and 18 mm for S. aureus, confirming its effective, broad-spectrum antibacterial properties.

## 10.4 Conclusion

The final surgical suture, successfully prepared by loading a PGA base with biologically synthesized curcumin and chitooligosaccharides, demonstrated powerful dual-action functionality. It exhibited potent anti-inflammatory properties by significantly suppressing the inflammatory response in macrophages and displayed broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria. Compared to standard commercial sutures, our functionalized suture shows vastly superior therapeutic capabilities, validating its significant potential as an advanced wound closure device for reducing post-operative complications like infection and excessive inflammation, thereby promoting better and faster healing.

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