iGEM 2025 — Team hizju-china

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Notebook

* [Basic Preparation](#wenben1)
* [And-gate Sensing](#wenben2)
* [Amplification & Feedback Module](#wenben3)
* [Protein Engineering](#wenben4)
* [Biosafety Circuits](#wenben5)
* [Hardware Module](#wenben6)

![](https://static.igem.wiki/teams/5828/toutu/notebook-min.webp)

## **Basic Preparation**

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### March 21th

Bacterial Preservation: Mix 500 μl of 40% glycerol with 500 μl of bacterial solution (or 300 μl of 50% glycerol with 700 μl of bacterial solution). A total of eight tubes of Amp30E (Kanamycin-resistant strain) were preserved and stored at -80 ℃.

Notes: The operation shall be conducted in a biological safety cabinet, and the glycerol must be sterilized in advance. After transferring the mixture into glycerol storage tubes, mix it thoroughly by pipetting up and down or using a vortex mixer.

### April 6th

Medium Preparation: LB medium was prepared for culturing our chassis strain *Escherichia coli* (*E. coli*). Based on the plasmids used (Kanamycin-resistant pSB3K3 and Ampicillin-resistant pSB4A3), we prepared corresponding single-resistant LB solid medium plates, double-resistant LB solid medium plates, and LB liquid medium for shake flask culture of *E. coli*. The formula of LB medium is included in our  [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)  Page.

Sterilization: Sterilize using an autoclave at 121°C for 15 minutes

Notes: When in use, ensure the water level does not exceed the "HIGH" warning line. Regularly drain excess condensed water. When the temperature drops to approximately 70 ℃, the sterilization program can be terminated, and the sterilized medium can be taken out.

## **And-gate Sensing**

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## **Amplification & Feedback Module**

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## **Protein Engineering**

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## **Biosafety Circuits**

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## **Hardware Module**

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Engineering

* [Overview](#wenben1)
* [Part 1: Construction and Optimization of the AND-gate Sensing Circuit](#wenben2)
* [Part 2: Purification and Enrichment of GLP-1(Z)](#wenben3)
* [Part 3: Construction of the OR-gate Dual-input Biosafety Kill-switch](#wenben4)
* [Part 4: Development of a Hydrogel-Based Oral Delivery System for Engineered Bacteria](#wenben5)
* [Conclusions](#wenben6)
* [References](#wenben7)

![](https://static.igem.wiki/teams/5828/toutu/eng-min.webp)

## **Overview**

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![](https://static.igem.wiki/teams/5828/engineering/f0-e.webp)

To develop a long-acting GLP-1-based oral bacterial agent, we systematically conducted **Design-Build-Test-Learn (DBTL) engineering cycles** for each module. Through these cycles, we continuously optimized the functional performance and parameter conditions of our design to meet the practical requirements for engineering applications. These efforts primarily included the optimization of gene expression and biosafety circuits, the purification of the GLP-1 protein, and the improvement of hardware hydrogel carriers, with phased iterative success achieved.

## **Part 1: Construction and Optimization of the AND-gate Sensing Circuit**

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**Goal**: To construct an AND-gate genetic circuit based on intein trans-splicing for spatiotemporal regulation of GLP-1 synthesis.

Cycle 1

Cycle 2

Cycle 3

### Preliminary Characterization of the Protocatechuic Acid Operon and Bile Salt Sensing Device

#### Design

We selected protocatechuic acid (PCA), a natural metabolite of green tea, as the inducer for GLP-1 synthesis, aiming to leverage the daily consumption of green tea as a healthy routine to assist in glucose management and weight loss for people. Since the PCA-responsive operon [1] ( [BBa\_25N5FWAK](https://registry.igem.org/parts/bba-25n5fwak)) is a relatively new iGEM part, systematic characterization was necessary before its integration into the circuit. Additionally, we chose sodium cholate, a bile salt present at characteristic concentrations in the gut, as the second input signal. We identified a bile salt sensing device ( [BBa\_K1962010](https://registry.igem.org/parts/bba-k1962010)) from the iGEM registry for this purpose.

For initial characterization, we used GFP ([BBa\_K2717024](https://registry.igem.org/parts/bba-k2717024)) as a reporter in place of the downstream split intein-transcription factor halves. This allowed us to assess the expression profiles of both operons under preliminary induction conditions derived from literature [2], with results quantified as normalized fluorescence intensity.

![](https://static.igem.wiki/teams/5828/engineering/f1-e.webp)

### Optimizing Expression Levels of Operon Elements

#### Design

To address the issues identified in Cycle 1, we optimized the expression strength of the original systems by modifying the constitutive promoters and RBS sequences, thereby further validating operon functionality. For the PCA-responsive operon, we replaced the upstream promoter of the PcaV repressor with higher-strength promoters J23101 and J23105 (strength: J23101>J23105>J23117) to enhance repression of the downstream P*LV* promoter and reduce leakage. We also optimized the induction time for the PCA operon to assess system responsiveness and provide reference for in vivo applications.

For the bile salt sensing operon, we optimized the RBS sequences upstream of both the transcription factor and the reporter gene, designing three additional RBS combinations to enhance downstream expression levels, improving the dynamic response range and signal-to-noise ratio under sodium cholate gradient induction.

![](https://static.igem.wiki/teams/5828/engineering/f4-e.webp)

Figure 4. Schematic diagram of the operon optimization in Cycle 2.

#### Build

Using the successfully constructed plasmids from previous cycles as templates, we performed site-directed replacement of promoters and RBS sequences via primer design and circular PCR. The modified constructs were assembled, transformed, and screened for positive clones as described in Cycle 1. This yielded three PCA-responsive operon variants with different expression strengths and two bile salt sensing device variants. Despite repeated attempts including Gibson assembly with re-amplified fragments, using high-fidelity PCR enzymes, and multiple rounds of circular PCR with point-mutation templates, we were unable to successfully replace the RBS32 sequence upstream of the transcription factor with RBS30. Consequently, further characterization based on this specific modification was discontinued.

#### Test

Since the sodium cholate operon primarily functions as a “localizer” and the intestine provides a sustained supply of cholate, while PCA serves as the key input signal for limited induction, we first optimized the induction time for the PCA operon. Preliminary tests led us to select TOP10 strains carrying the J23105 promoter for a 9-hour induction experiment with PCA concentrations of 0, 250, and 400 μM, and take samples at regular intervals to measure fluorescence and OD600. Based on the results, we selected 4 hours as the final induction time to ensure sufficient bacterial growth for subsequent processing steps such as centrifugation, pelleting, and washing. We then evaluated the induction performance of the five plasmid variants under this 4-hour induction period, using PCA and sodium cholate concentration gradients similar to those in Cycle 1.

We found that normalized fluorescence intensity gradually increased with PCA concentration before reaching saturation. Higher expression levels of the PcaV repressor resulted in lower leakage, but this was accompanied by reduced sensitivity to changes in PCA concentration, further validating our conclusions from the first iteration. For the cholate-sensing device, modifying the RBS sequences downstream increased leakage expression but did not yield significant cholate-induced activation. Subsequent tests with higher cholate concentrations still showed no notable increase in fluorescence signal and even negatively affected bacterial growth.

![](https://static.igem.wiki/teams/5828/engineering/f5-e.webp)

Figure 5. Characterization of the two optimized operons. (A) Preliminary experiment on PCA induction time of the operon regulated by promoter J23105 over 0-9 hours. (B) Measurement of PCA-induced fluorescence intensity with constitutive promoters of various strengths at 4-hour induction time. (C) Measurement of BS-induced fluorescence intensity with different RBS combinations at 4-hour induction time for the constructed plasmids.

#### Learn

Based on these results, we selected the J23105 promoter to regulate PcaV repressor expression and retained the original RBS combination for the cholate operon in subsequent AND-gate construction. This strategy aims to minimize leakage in the absence of inducers, which could interfere with validation of the AND-gate logic. The PCA-responsive operon performed satisfactorily, whereas the cholate-sensing device continued to underperform. We suspect that the issue may stem from toxicity caused by overexpression of the heterologous transcription activator RamA in *E. coli*, or from insufficient tolerance mechanisms in the host toward cholate, affecting its metabolic regulation and induction [4]. Future work may require deeper investigation into the cholate regulatory mechanism or substitution with alternative gut-specific molecular sensors with improved performance.

### Construction of the Final AND-gate Circuit with the Characterized Operons

#### Design

Following the individual characterization of the operons, we proceeded to construct the final AND-gate circuit ( [BBa\_2581ND93](https://registry.igem.org/parts/bba-2581nd93)) by linking them to orthogonal split intein-transcription factor halves. In accordance with the original reference, we cloned the upstream AND-gate sensing module and the downstream reporter module into two separate plasmids, pSB3K3 and pSB4A3 respectively. These plasmids were subsequently co-transformed into *E. coli* TOP10 for functional characterization of the AND-gate circuit.

![](https://static.igem.wiki/teams/5828/engineering/f6-e.webp)

Figure 6. Schematic diagram of the AND-gate sensing circuit.

#### Build

As illustrated in Figure 6, we placed the two operons upstream of the genes encoding the N-terminal ( [BBa\_259H9ZPU](https://registry.igem.org/parts/bba-259h9zpu)) and C-terminal ( [BBa\_25YZWH1A](https://registry.igem.org/parts/bba-25yzwh1a)) split halves respectively. Concurrently, the P16 promoter and *GFP* reporter were cloned into a separate pSB4A3 plasmid carrying ampicillin resistance ( [BBa\_25MR74ON](https://registry.igem.org/parts/bba-25mr74on)). After successful assembly, transformation, and sequence verification, both plasmids were co-transformed into TOP10 for functional assessment.

![](https://static.igem.wiki/teams/5828/engineering/f7-e.webp)

Figure 7. Construction of the AND-gate plasmids in Cycle 3. A. Plasmid map of pSB3K3-J23105-*PcaV*-P*LV*-*SspGyrB*C-*ECF16*C-J23101-RamA-PacrRA-SspGyrBN-ECF16N. B. Plasmid map of pSB4A3- P16-*GFP*. C, D. Sequencing results of target AND-gate plasmids.

#### Test

We cultured the engineered *E. coli* strain harboring both plasmids overnight, selected using double-antibiotic medium. Then we added inducers at an initial OD600 of 0.02 and cultured for 10 hours. The sodium cholate concentration was fixed at 25 µM, while PCA was applied in a gradient from 0 to 1000 µM, based on literature references [2]. The normalized GFP fluorescence intensity was measured to validate AND-gate functionality and identify the optimal PCA concentration. Results indicated that fluorescence saturation occurred at approximately 400 µM PCA, which was consequently selected for subsequent experiments.

We then fixed the 400 µM PCA and 25 µM sodium cholate concentrations to characterize and optimize the induction time, simulating the duration from PCA induction to GLP-1 production by the gut flora. Negative controls included bacteria carrying the AND-gate plasmids without PCA induction, and bacteria carrying empty plasmids with both inducers. Results demonstrated an approximately 5-fold ON/OFF signal amplitude after 5 hours of induction, confirming proper AND-gate logic function with a relatively short overall induction time.

![](https://static.igem.wiki/teams/5828/results/f6re.webp)

Figure 8. Characterization of the AND-gate genetic circuit in Cycle 3. (A) Measurement of GFP unit fluorescence intensity under 0–1000 µM PCA and fixed 25 µM sodium cholate over 10 hours. (B) Measurement of the unit fluorescence intensity of GFP for verification of the AND-gate logic during 3.5–6.0 hours of induction.

#### Learn

Through these iterative design and validation cycles, we successfully constructed an AND-gate sensing circuit capable of dynamically responding to PCA and cholate concentrations. This system shows potential for application in probiotic bacteria to sense green tea metabolites and trigger target gene expression upon gut colonization. However, several challenges remain, including the in vivo response time to PCA, optimization of the cholate-sensing operon, plasmid stability during bacterial propagation, and residual gene leakage. Addressing these issues will be essential for practical therapeutic development of the circuit.

This iterative process has deepened our understanding of how promoter and RBS sequences influence operon sensitivity, leakage, and dynamic response profiles. By systematically optimizing component expression levels, we enhanced target gene output appropriately. Furthermore, constructing this AND-gate logic has expanded our knowledge and application of split intein splicing systems [5], providing a foundation for future research and insights.

## **Part 2: Purification and Enrichment of GLP-1(Z)**

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**Goal**: To achieve the isolation, enrichment, and purification of the GLP-1(Z) protein in vitro.

## **Part 3: Construction of the OR-gate Dual-input Biosafety Kill-switch**

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**Goal**: To achieve active-passive intelligent suicide regulation of engineered *Escherichia coli* and ensure biosafety.

## **Part 4: Development of a Hydrogel-Based Oral Delivery System for Engineered Bacteria**

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**Goal**: To enable the oral bacterial agents to withstand gastric acid and digestive enzymes, achieve targeted release in the intestine, and enhance its adhesive properties to support long-term colonization of the engineered bacteria.

Cycle 1

Cycle 2

Cycle 3

Cycle 4

### Microspheres Based on Sodium Alginate–Calcium Chloride Hydrogel with Chitosan Coating

#### Design

Following our [Human Practices](https://2025.igem.wiki/hizju-china/human-practices%20) research, we selected the highly biocompatible hydrogel as the carrier for the oral bacterial agents. Through literature and patent surveys, we found a preparation method for sodium alginate–calcium chloride hydrogel microspheres coated with chitosan [18]. This delivery system relies on the fact that the carboxyl groups of alginate remain unprotonated under acidic conditions, reducing intermolecular electrostatic repulsion and forming a relatively dense protective matrix for the bacteria. Under neutral conditions, increased electrostatic repulsion causes the hydrogel network to swell, facilitating bacterial release. The chitosan coating provides a protective layer of protonated amino groups and steric hindrance in acidic environments, shielding the microspheres from hydrogen ions and macromolecules like proteases, thereby creating a favorable microenvironment to maintain bacterial viability [19].

We followed the protocol using 1% (w/v) sodium alginate and 0.1 M calcium chloride solution to prepare hydrogel microspheres, which were subsequently coated with 0.5% (w/v) chitosan. The protective and release performance was preliminarily assessed by OD600 measurement.

![](https://static.igem.wiki/teams/5828/engineering/f39-e.webp)

Figure 39. "Egg-box" model of sodium alginate–calcium chloride cross-linking. [20]

#### Build

We first prepared sterile solutions of 1% (w/v) sodium alginate, 0.1 mol/L CaCl2, and 0.5% (w/v) chitosan with 1% (v/v) acetic acid in deionized water, followed by filter sterilization using syringes and sterile membrane filters. Overnight cultures of engineered EcN were then added in gradients of 10, 20, 50, 100, and 200 µL to the sterile sodium alginate solution. The mixtures were manually dispensed dropwise from syringes into the calcium chloride solution. After standing for 10 minutes, calcium alginate microcapsules gradually formed. The microspheres were then collected with a sterilized spoon and transferred into the chitosan solution, incubated for approximately 1.5 hours to obtain chitosan-coated hydrogel microspheres.

![](https://static.igem.wiki/teams/5828/engineering/f40-e.webp)

Figure 40. Preparation of sodium alginate–calcium chloride hydrogel microspheres. (A) Hydrogel microspheres prepared with different bacterial culture volumes. (B) Microstructure of hydrogels observed under optical microscopy at 400× magnification.

#### Test

We successfully prepared chitosan-coated sodium alginate–calcium chloride hydrogel microspheres. Optical microscopy revealed relatively uniform morphology, with color intensity increasing with higher bacterial loadings. The microspheres were incubated separately in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) (turn to [Experiments](https://2025.igem.wiki/hizju-china/experiments%20) page for preparation details), washed thoroughly with PBS buffer, and then transferred into liquid LB medium for 6 hours of culture. The protective effect was evaluated by measuring the OD600 of the cultures. Tubes containing microspheres treated with SIF showed significant turbidity. In contrast, tubes with microspheres treated with SGF exhibited almost no increase in OD600 (close to blank control), indicating minimal resistance of the hydrogel system to simulated gastric conditions and an inability to fulfill the oral delivery function.

#### Learn

The sodium alginate–calcium chloride hydrogel prepared according to the patented method failed to protect the bacteria effectively from gastric acid and digestive enzymes as intended. We hypothesized that the cross-linked alginate-calcium network and the chitosan coating were too loose, insufficiently dense, or too thin, allowing gastric acid to penetrate the microspheres and kill the encapsulated *E. coli* [20]. The inherent cationic nature of chitosan, attributable to its positive surface charge, may confer toxicity toward the indigenous gut microbiota, necessitating strict control over its dosage [21]. Consequently, this system proved inadequate for oral delivery. Future efforts will focus on designing hydrogel systems with better encapsulation efficiency or smaller pore sizes to improve protection.

### Interfacial Engineering of Living Bacteria Using Carboxymethyl Starch Sodium

#### Design

In the first cycle, we suspected that the spatial grid structure of the alginate‑calcium chloride coating was too loose, allowing gastric acid to penetrate the hydrogel and compromise bacterial viability. To address this, we investigated strategies to reduce diffusion and permeation within the gel. One approach to improve protection is to increase the gel’s coverage ratio per unit surface area, or to directly form a protective layer over the bacterial surface. Accordingly, we adopted a surface engineering strategy based on carboxymethyl starch sodium (CMS-Na).

CMS-Na is commonly used as a natural excipient and, similar to alginate, can resist gastric acid and enzyme penetration via protonation of its carboxyl groups. Upon reaching the intestine, it swells and dissociates, releasing the bacteria. By cross‑linking CMS-Na with calcium ions on the bacterial surface, a hydrogel coating can be formed to enable intestinal delivery and release.

![](https://static.igem.wiki/teams/5828/engineering/f41-e.webp)

Figure 41. Schematic diagram of the interfacial engineering for living drugs[22].

#### Build

We pre-cultured a 4 mL tube of EcN overnight at 37 °C and 220 rpm as the encapsulation stock. The culture was then centrifuged at 6000 rpm for 5 min and washed twice with 1× PBS. The pellets were resuspended in 1 mL of sterile PBS containing 12.5 mM CaCl2 and incubated for 5 min. After that, 50 µL of sterile 10 mg/mL CMS‑Na solution was added, and the mixture was vortexed for another 5 min. Finally, the sample was centrifuged and washed twice with PBS to obtain the gel‑coated living drug formulation, denoted LD(P). A negative control (LD) was prepared via only centrifugation and PBS washing.

#### Test

We adjusted the OD600 of both suspensions to 1 and incubated them in SGF and SIF for 0–4 hours. After incubation, the samples were centrifuged, washed with PBS, and approximately 100 µL of each was spread on LB plates for overnight culture. Colony counts were compared to evaluate the acid‑ and enzyme‑resistance of the hydrogel. Visually, after centrifugation, the LD control treated with SGF showed almost no pellet, whereas LD(P) remained in hydrogel form. Plate results qualitatively indicated that the CMS‑Na coating improved the acid tolerance of EcN.

![](https://static.igem.wiki/teams/5828/engineering/f42-e.webp)

Figure 42. Protective effect of LD(P) against SGF. (A) Centrifuged pellets after PBS washing. (B) Colony growth on plates from samples incubated in SIF and SGF for 2 hours.

#### Learn

Our experiments preliminarily verified that CMS‑Na‑based surface engineering can enhance bacterial resistance to gastric acid. However, several practical issues arose: 12.5 mM CaCl2 in PBS readily formed gelatinous precipitates due to reaction between calcium and phosphate ions; the coating was uneven, leading to poor batch-to-batch reproducibility and difficulty in tracking protection dynamics over 0–4 hours; LD(P) storage stability was uncertain; and the quality of the CMS‑Na used was variable, necessitating stricter control of post‑preparation pH and other parameters. Therefore, a more reproducible and quantitatively reliable protocol is needed to accurately evaluate the effects of the coating on bacterial viability and release, while minimizing systematic bias.

### Combined Strategy of Enteric Capsules and Sodium Alginate–Calcium Chloride Hydrogel Microspheres

#### Design

To improve batch-to-batch reproducibility and achieve precise control over the release profile, enabling targeted intestinal delivery and prolonged colonization, we selected a combination of enteric capsules and sodium alginate (SA)-calcium chloride hydrogel microspheres as the carrier [23]. This combination strategy enhances gastric acid resistance while leveraging the sustained-release properties of hydrogels to deliver probiotics to specific intestinal regions. Moreover, it also facilitates standardized preparation of live bacterial formulations based on gel quality and encapsulation efficiency.

![](https://static.igem.wiki/teams/5828/engineering/f43-e.webp)

Figure 43. Schematic diagram of our hydrogel microsphere delivery system with gelatin capsules.

#### Build

EcN was first cultured overnight at 37 °C. Bacterial cells were then harvested and diluted to approximately 1×10⁹ CFU/mL, washed twice with 1× PBS buffer, and resuspended in 1% (w/v) sodium alginate (SA) solution. The mixture was extruded dropwise using syringes into a 0.1 M calcium chloride solution to form hydrogel microspheres. After 10 minutes of crosslinking, the microspheres were collected and encapsulated into gelatin enteric capsules to form the final oral delivery formulation.

To further optimize controlled release, we prepared a series of microspheres by varying the concentrations of SA and calcium chloride to adjust crosslinking density.

![](https://static.igem.wiki/teams/5828/engineering/f44-e.webp)

Figure 44. Preparation of enteric capsule-encapsulated hydrogel microspheres for oral delivery. (A) Sodium alginate–calcium chloride hydrogel microspheres. (B) Microspheres encapsulated within a gelatin enteric capsule.

#### Test

We evaluated the protective and release performance of the formulation by incubating samples in SGF for 1 hour, then transferring the microspheres to SIF for extended release. Samples were taken hourly, centrifuged, washed with PBS, serially diluted, and plated on LB agar. After 24-hour incubation at 37 °C, colonies were carefully counted. Microspheres without capsule protection served as the negative control. Results indicated that enteric capsules maintained their structural integrity in SGF but dissolved almost completely within 10 minutes upon transfer to SIF at 37 °C. Release profiles showed that capsule-protected microspheres released probiotics more slowly.

For microspheres with different crosslinking densities, minimal release was observed within the first 4 hours, followed by rapid release of encapsulated bacteria, demonstrating a clear delayed-release effect. Varying sodium alginate and calcium chloride concentrations did not significantly alter this release pattern.

![](https://static.igem.wiki/teams/5828/engineering/f45-e.webp)

Figure 45. Characterization of the protective and release properties of capsule-encapsulated hydrogel microspheres. (A) Differences of the capsule structural integrity in SGF and SIF. (B) Effect of enteric capsule encapsulation on the release profile of hydrogel microspheres. (C) Viability and accumulation of *E. coli* released from microspheres with different crosslinker concentrations. (D) Release profiles of probiotic carriers with different cross-linking degrees. (E) CFU measurement during the first 4 hours with 10-3 dilution.

#### Learn

After 7 hours in SIF, capsule-encapsulated microspheres had released only 54.2% of their load relative to controls, suggesting the release had not yet reached a plateau. We hypothesized that gelatin degradation products may engage in weak electrostatic interactions or hydrogen bonding with carboxyl groups on the alginate microsphere surface, thereby altering release kinetics.

All crosslinking concentrations tested exhibited similar release profiles, with a consistent delay of approximately 4 hours before the onset of rapid release. This might be due to insufficient variation in concentration, which did not significantly alter hydrogel porosity or crosslinking topology. Future work could explore alternative crosslinkers to further optimize release kinetics.

In summary, combining hydrogel microspheres with enteric capsules provides effective protection against gastric conditions and enables sustained intestinal release, offering a promising platform for oral probiotic delivery. Further optimization of release kinetics and adhesive properties may be required to enhance the long-term colonization and drug production of engineered bacteria.

### Thiol-Modified Hydrogels for Enhanced Bacterial Adhesion and Colonization

#### Design

To extend colonization and reduce dosing frequency, we sought to improve the adhesive properties of the hydrogel carrier. Literature indicates that probiotic adhesion relies on surface protein interactions and specific receptors. The intestinal mucus layer is rich in cysteine residues [24], and thiolated hydrogels can form strong disulfide bonds with both bacterial pili as well as mucosal cysteines, prolonging retention of engineered probiotics [25]. So we also preliminarily performed thiolation modification on carboxymethyl cellulose (CMC), which is a hydrogel material similar to sodium alginate and carboxymethyl starch (CMS), and characterized the adhesion properties of the resulting CMC-SH, laying a foundation for the development of long-acting hydrogel-based oral bacterial formulations.

![](https://static.igem.wiki/teams/5828/engineering/f46-e.webp)

Figure 46. Schematic diagram of thiol-modified hydrogels for enhanced bacterial adhesion and intestinal colonization.

#### Build

We fortunately obtained approximately 0.1 g of CMC-SH from Professor Zhuxian Zhou’s lab (synthesis methods as detailed in the literature [26]) and combined it with sodium alginate to form composite films for evaluating bacterial adhesion. Due to the complexity of CMC-SH synthesis, we prepared a limited batch by dissolving 0.07 g CMC-SH and 0.04 g sodium alginate in 1.4 mL distilled water, incubating at 25 °C for 4 hours, adding 21 mg glycerol, and sonicating to mix thoroughly [27]. The gel was then spread on one half of a split LB plate, leaving the other half uncoated. After solidification, both sides were streaked with pre-cultured EcN using an inoculating loop. Plates were inverted and incubated overnight at 37 °C and unmodified CMC served as a control.

#### Test

We assessed the adhesion property using a simple rinsing assay (see our [experimental protocol](%E2%80%99https%3A//static.igem.wiki/teams/5828/experiment/protocol/hydrogel.pdf%E2%80%99). In both types of plates, bacteria on uncoated agar were easily rinsed off. In contrast, CMC-SH-coated areas and the bacteria adhering to its surface were much harder to be rinsed off compared with the CMC coating, showing little difference from the unwashed ones. We preliminarily conclude that thiolated CMC is capable of enhancing its adhesive capacity, even for surface-associated cells, which is conducive to the long-term colonization of engineered bacteria in the intestine.

![](https://static.igem.wiki/teams/5828/engineering/f47-e.webp)

Figure 47. Adhesion performance of CMC and CMC-SH hydrogel coatings for EcN on LB agar based on the running water rinsing experiment.

#### Learn

After establishing a delivery system that protects probiotics and enables intestinal release, we further explored thiolation as a strategy to prolong colonization. Owing to time constraints and experimental conditions, while we have not conducted in vivo experimental verification, and simple water flow impact experiments cannot achieve quantitative evaluation of hydrogel adhesion properties, this strategy has been fully characterized in the literature [27] and exhibits practical feasibility. Subsequently, we could conduct further verification by performing bacterial encapsulation experiments based on modified hydrogel carriers, or experiments designed for the thiolation modification of components such as sodium alginate.

Through these studies, we have systematically optimized and combined commonly used hydrogel materials to develop an oral bacterial formulation that offers gastric protection, targeted release, sustained colonization, storage stability, and reproducible production. Our project aims to provide preliminary validation of an oral live drug delivery platform, bringing us closer to a user-acceptable, compliance-friendly solution for weight management and glucose control.

## **Conclusion**

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Through persistent iteration across all modules, we have integrated and refined our initial designs, making concrete progress toward engineering a practical therapeutic system. While many challenges remain and further improvements are needed, we have successfully validated the core functionalities of each component. These efforts, combined with our work on long-acting GLP-1 live biotherapeutics, provide an integrated framework and valuable lessons including constructive failures for future iGEM teams and similar projects.

## **References**

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[12] Rottinghaus, A. G., Ferreiro, A., Fishbein, S. R. S., Dantas, G., & Moon, T. S. (2022). Genetically stable CRISPR-based kill switches for engineered microbes. *Nature Communications*, 13(1), 672.

[13] Schleif R. (2000). Regulation of the L-arabinose operon of Escherichia coli. *Trends in Genetics*, 16(12), 559–565. [https://doi.org/10.1016/s0168-9525(00)02153-3](https://doi.org/10.1016/s0168-9525%2800%2902153-3)

[14] Sotiropoulos, V., & Kaznessis, Y. N. (2007). Synthetic tetracycline-inducible regulatory networks: computer-aided design of dynamic phenotypes. *BMC Systems Biology*, 1, 7. <https://doi.org/10.1186/1752-0509-1-7>

[15] Clifton, K. P., Jones, E. M., Paudel, S., Marken, J. P., Monette, C. E., Halleran, A. D., Epp, L., & Saha, M. S. (2018). The genetic insulator RiboJ increases expression of insulated genes. *Journal of Biological Engineering*, 12, 23. <https://doi.org/10.1186/s13036-018-0115-6>

[16] Jiangnan University. (2024). An operon for fucose-induced gene expression, its construction method, and application [China Patent No. CN202311579074.0].

[17] Bernard, P., Kézdy, K. E., Van Melderen, L., Steyaert, J., Wyns, L., Pato, M. L., Higgins, P. N., & Couturier, M. (1993). The F plasmid CcdB protein induces efficient ATP-dependent DNA cleavage by gyrase. *Journal of Molecular Biology*, 234(3), 534–541. <https://doi.org/10.1006/jmbi.1993.1609>

[18] Xuzhou Medical University. (2024). A probiotic-hydrogel oral delivery system and its preparation method [Patent]. CN Patent No. 2023/1306/10.

[19] Wang, M., Zang, Y., Hong, K., Zhao, X., Yu, C., Liu, D., An, Z., Wang, L., Yue, W., & Nie, G. (2021). Preparation of pH-sensitive carboxymethyl cellulose/chitosan/alginate hydrogel beads with reticulated shell structure to deliver Bacillus subtilis natto. *International journal of biological macromolecules*, 192, 684–691. <https://doi.org/10.1016/j.ijbiomac.2021.10.019>

[20] Lai, J., Azad, A. K., Sulaiman, W. M. A. W., Kumarasamy, V., Subramaniyan, V., & Alshehade, S. A. (2024). Alginate-Based Encapsulation Fabrication Technique for Drug Delivery: An Updated Review of Particle Type, Formulation Technique, Pharmaceutical Ingredient, and Targeted Delivery System. *Pharmaceutics*, 16(3), 370. <https://doi.org/10.3390/pharmaceutics16030370>

[21] Tang, H., Zhang, P., Kieft, T. L., Ryan, S. J., Baker, S. M., Wiesmann, W. P., & Rogelj, S. (2010). Antibacterial action of a novel functionalized chitosan-arginine against Gram-negative bacteria. *Acta Biomaterialia*, 6(7), 2562–2571. <https://doi.org/10.1016/j.actbio.2010.01.002>

[22] Feng, P., Bai, X., Ma, X., Kong, H., & Yang, R. (2024). Interfacial-engineered living drugs with "ON/OFF" switching for oral delivery. *Nanoscale*, 16(28), 13399–13406. <https://doi.org/10.1039/d4nr01927j>

[23] Patarroyo, J. L., Fonseca, E., Cifuentes, J., Salcedo, F., Cruz, J. C., & Reyes, L. H. (2021). Gelatin-Graphene Oxide Nanocomposite Hydrogels for Kluyveromyces lactis Encapsulation: Potential Applications in Probiotics and Bioreactor Packings. *Biomolecules*, 11(7), 922. <https://doi.org/10.3390/biom11070922>

[24] Coleman, O. I., & Haller, D. (2021). Microbe-Mucus Interface in the Pathogenesis of Colorectal Cancer. *Cancers*, 13(4), 616. <https://doi.org/10.3390/cancers13040616>

[25] Jiang, Z., Li, M., McClements, D. J., Liu, X., & Liu, F. (2022). Recent Advances in the Design and Fabrication of Probiotic Delivery Systems to Target Intestinal Inflammation. *Food Hydrocolloids*, 125, 107438. <https://doi.org/10.1016/j.foodhyd.2021.107438>

[26] Deng, S., Li, X., Yang, W., He, K., & Ye, X. (2018). Injectable in situ cross-linking hyaluronic acid/carboxymethyl cellulose based hydrogels for drug release. *Journal of Biomaterials Science, Polymer Edition*, 29(13), 1643–1655. <https://doi.org/10.1080/09205063.2018.1481005>

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Experiments

* [Protocols](#wenben1)
* [Sequences](#wenben2)
* [Competent Cells](#wenben3)

![](https://static.igem.wiki/teams/5828/toutu/exep-min.webp)

## **Protocols**

---

We have compiled an extensive set of standard protocols to optimize our experiments, covering standard reagent preparation, molecular biology experiments, protein engineering, hardware testing, and equipment operation.

### reagent preparation

[LB medium](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-preparations-for-lb-medium.pdf)
[GMML medium](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-preparations-for-gmml-medium.pdf)
[Agarose Gel](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-preparations-for-agarose-gel.pdf)
[Antibiotics](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-preparations-for-antibiotic-stock-solutions.pdf)

[SIF](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-preparations-for-simulated-intestinal-fluid.pdf)
[SGF](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-preparations-for-simulated-gastric-fluid.pdf)

### molecular biology experiments

[PCR](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-pcr.pdf)
[Plasmid Miniprep](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-plasmid-miniprep.pdf)
[DNA Purification](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-dna-purification-from-agarose-gels.pdf)
[DpnI](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-removing-template-dna-in-pcr-reactions-using-dpni.pdf)

[Gibson Assembly](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-gibson-assembly.pdf)
[Preservation and Revival](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-bacterial-glycerol-stock-preservation-and-revival.pdf)

### protein engineering

[Protein Purification](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-protein-purification.pdf)
[Protein Nanofiltration](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-protein-nanofiltration.pdf)
[SDS-PAGE](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-sds-page.pdf)

### hardware testing

[Adhesion assay](https://static.igem.wiki/teams/5828/experiment/protocol/adhesion-assay.pdf)
[Gelatine capsule](https://static.igem.wiki/teams/5828/experiment/protocol/gelatine-capsule.pdf)
[Hydrogel](https://static.igem.wiki/teams/5828/experiment/protocol/hydrogel.pdf)
[LD(P)](https://static.igem.wiki/teams/5828/experiment/protocol/ld-p.pdf)

### equipment operation

[UV-Vis Spectrophotometer](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-measuring-bacterial-culture-od600-value-using-a-uv-vis-spectrophotometer.pdf)
[Microplate Reader](https://static.igem.wiki/teams/5828/experiment/protocol/experiment-absorbance-and-fluorescence-measurement-using-a-microplate-reader-1.pdf)

### Calibration

[OD600](https://static.igem.wiki/teams/5828/experiment/protocol/od600.pdf)
[Fluorescence](https://static.igem.wiki/teams/5828/experiment/protocol/fluorescence.pdf)

## **Sequences**

---

To facilitate molecular biology experiments, constructing a comprehensive sequence library enables the combination of various elements and exploration of their functions.

[Oligonucleotides](https://static.igem.wiki/teams/5828/experiment/sequences/experiment-oligonucleotides-for-pcr-and-sequencing.pdf)
[Company](https://static.igem.wiki/teams/5828/experiment/sequences/experiment-sequences-synthesized-by-the-company.pdf)
[Other labs](https://static.igem.wiki/teams/5828/experiment/sequences/experiment-sequences-acquired-from-other-labs.pdf)
[Plasmid Backbones](https://static.igem.wiki/teams/5828/experiment/sequences/experiment-plasmid-backbones.pdf)

[HiZJU-China](https://static.igem.wiki/teams/5828/experiment/sequences/experiment-gene-fragments-constructed-for-our-experiment.pdf)

## **Competent Cells**

---

Most of our experiments involve gene transformation and different types of competent cells, and thus the systematic selection of an appropriate chassis for experimental use is crucial.

[TOP10](https://static.igem.wiki/teams/5828/experiment/competentcells/experiment-transformation-of-top10-chemically-competent-cells.pdf)
[Trelief® 5α](https://static.igem.wiki/teams/5828/experiment/competentcells/experiment-transformation-of-trelief5.pdf)
[BL21(DE3)](https://static.igem.wiki/teams/5828/experiment/competentcells/experiment-transformation-of-bl21-de3-chemically-competent-cells.pdf)
[EcN](https://static.igem.wiki/teams/5828/experiment/competentcells/experiment-preparation-of-ecn-competent-cells-and-electroporation.pdf)

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![](https://static.igem.wiki/teams/5828/picture/bjcs.webp)
[![](https://static.igem.wiki/teams/5828/home/dhk1.webp)

Module 1:
AAF Sensing Circuits](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27result1%27%29%20%7D%7D)
[![](https://static.igem.wiki/teams/5828/home/dhk2.webp)

Module 2:
Protein Engineering of GLP-1](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27result2%27%29%20%7D%7D)
[![](https://static.igem.wiki/teams/5828/home/dhk3.webp)

Module 3:
OR-gate Biosafety](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27result3%27%29%20%7D%7D)
[![](https://static.igem.wiki/teams/5828/home/dhk4.webp)

Module 4:
Hardware For Delivery &
Characterization](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27result4%27%29%20%7D%7D)

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