iGEM 2025 — Team tjusx

{% 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 %}

![Notebook Header Image](https://static.igem.wiki/teams/5724/headphoto/notebook.webp)

## Contents

* [1. Wet Lab](#wet-lab)
  + [(1) Adhension Module](#adhension-module)
  + [(2) Quorum Sensing Module](#quorum-sensing-module)
  + [(3) Therapeutic Module](#therapeutic-module)
  + [(4) Safety Module](#safety-module)
* [2. Dry Lab](#dry-lab)
* [3. Human Practise](#human-practise)

## Overview

### Notebook-Collection

![Notebook Collection](https://static.igem.wiki/teams/5724/notebook/notebook-fig-1.webp)

Fig.1 Gantt Chart

![Wiki Documentation](https://static.igem.wiki/teams/5724/notebook/notebook-fig-33.webp)

Fig.2 WeChat Push Schedule Chart

## 1. Wet Lab

### (1) Adhension Module

May

Construction of plasmid pSIP403-Lp\_0092.

Jun

Construction of plasmid pDest-LysM-Ag2.

Jul

1. Plasmid construction for the adhesion module:

Constructed plasmids pDest-LysM-Ag3, pDest-LysM-Ag2, pDest-LysM-Nb2, pDest-LysM-Nb3, and pSIP403-Ag2.

2. Preliminary validation of adhesion function:

Conducted preliminary tests on the adhesion efficacy of adhesins.

Performed preliminary experiments to evaluate the effect of IPTG on adhesion.

Aug

Validation of the adhesion module in the E. coli chassis:

(1) Verified the orthogonality of adhesins.

(2) Determined the optimal working concentration of IPTG.

(3) Assessed the effect of IPTG supplementation on adhesion efficacy.

Sep

Validation of the adhesion module in co-culture systems:

(1) Confirmed the optimal working concentration of IPTG.

(2) Evaluated the effect of IPTG induction on adhesion performance.

### (2) Quorum Sensing Module

Apr

Construction of plasmid: T7-gshAB.

May

Construction of plasmid pET-phyb-MazF (hypoxia-inducible promoter).

Jul - Aug

Plasmid Construction and Transformation:

pET-J23119-YFP transformed into Escherichia coli Nissle 1917.

pET-PBAD-YFP transformed into Escherichia coli Nissle 1917.

pNZ8148-P23-RFP transformed into Lactiplantibacillus plantarum WCFS1.

pSIP403-Pspp-RFP transformed into Lactiplantibacillus plantarum WCFS1.

Fluorescence Intensity Measurement:

Fluorescence intensity of the constructed strains was measured using a microplate reader.

Co-culture Observation:

Co-culture of Escherichia coli Nissle 1917 and Lactiplantibacillus plantarum WCFS1 was monitored by flow cytometry.

Plasmid Construction and Transformation:

pJUMP-J23110-GFP transformed into Escherichia coli Nissle 1917.

The modified pNZ8148-P23-RFP transformed into Lactiplantibacillus plantarum WCFS1.

1. Plasmid Construction and Transformation:

(1) Quorum Sensing Plasmid Series:

pET-J23119-LasR-Plas-RFP

pET-J23119-RhlR-Prhl-RFP

pET-J23119-LuxR-Plux-RFP

pNZ8148-P23-LuxR-Plux-RFP

(2) Toxin-Antitoxin Validation Plasmids:

pSIP403-Pspp-MazF

pET-PBAD-MazF

pRSF-T7-MazE

pNZ8148-Pnis-MazE

2. Validation of Quorum Sensing Plasmid Response in E. coli.

Sep

Co-culture Observation:

Simultaneous monitoring of Escherichia coli Nissle 1917 and Lactiplantibacillus plantarum WCFS1 co-cultures was performed using fluorescence microscopy and plate photography under illumination.

Toxicity Validation of MazE/F in Dual-Bacterial System:

Construction of Plux Mutant Plasmids for pNZ8148-P23-LuxR-Plux-RFP and Validation in L. plantarum

### (3) Therapeutic Module

May

Preparation for Gene Knockout:

Constructed three pGRB plasmids (each containing sgRNAs specifically targeting one of the three genes).

Prepared three corresponding donor DNA fragments.

Transformed the pCas9 plasmid into Escherichia coli Nissle 1917.

Jul - Aug

Plasmid Construction and Transformation:

The following plasmids were constructed and transformed into Escherichia coli Nissle 1917:

pET-J23119-hpaBC

pET-J23119-gshAB

pET-T7-hpaBC

pET-T7-gshAB

Gene Knockout of ggt, gorA, and pepT:

Attempted to knockout the target genes in Escherichia coli Nissle 1917 using the CRISPR-Cas9 system.

Sep

Fermentation and Product Analysis:

Cultured Escherichia coli Nissle 1917 harboring plasmid pET-T7-hpaBC for inducible L-DOPA production.

Cultured Escherichia coli Nissle 1917 harboring plasmid pET-T7-gshAB for inducible glutathione production.

### (4) Safety Module

Feb - Mar

Literature review and preliminary project design.

Apr

Construction of the hypoxia-inducible suicide module and acquisition of the corresponding engineered strains.

May - Jun

Construction of the thermosensitive suicide module and acquisition of the corresponding engineered strains.

Aug - Sep

Construction of the temperature-sensitive protein--fluorescent reporter element and validation of the usability of the thermosensitive protein; verification of the effectiveness of the hypoxia-inducible suicide module.

## 2. Dry Lab

Mar

1. Exploration of Biometabolic Network Modeling via GEM

Investigated mainstream algorithms for Genome-Scale Metabolic Models (GEMs), such as Flux Balance Analysis (FBA), dynamic Flux Balance Analysis (dFBA), and associated tools including COBRApy, COBRA Toolbox, Snoopy, and SurreyFBA.

2. Preliminary Study on Integrating FBA with Machine Learning

This phase primarily involved a literature review and foundational understanding. Team members focusing on in silico work familiarized themselves with iGEM requirements and explored relevant computational tools and modeling approaches.

Apr

1. Conducted analysis of modeling frameworks and wiki documentation practices from previous gold-award-winning iGEM teams.

Reviewed examples from teams such as Heidelberg, BNUZH-China, and Tsinghua iGEM 2024 to identify standard practices and requirements for in silico work.

![Analysis of previous iGEM teams](https://static.igem.wiki/teams/5724/notebook/notebook-fig-2.webp)

Fig.3 Analysis of previous iGEM teams

2. Identified and evaluated open-source tools and resources for foundational learning.

Focused primarily on mastering COBRApy and studying established metabolic models.

May

1. Defined the objectives, content, and methodology for constructing the target dataset.

Objective: Develop a Question-Answering (QA) system, comprising two versions (Base and Professional).

Content: To be consolidated and finalized at a later stage.

Methodology: A hybrid approach combining API data retrieval with manual curation and modification.

2. Developed and tested preliminary demonstrations for FBA and dFBA.

Successfully established and executed foundational versions of Flux Balance Analysis (FBA) and dynamic FBA (dFBA) simulations.

Jun

1. Refinement of the QA database and initial development of the front-end web interface.

2. Expansion of the disease-probiotic relationship data.

3. Supplementary work on the background literature and manuscript.

4. Integration of AI models (DeepSeek and Qwen).

5. Initiation of mobile application development.

6. Implementation of fuzzy matching for the QA system.

The foundational dataset was successfully established and subjected to preliminary validation. The core framework of the platform was largely established, marking a significant milestone in the development lifecycle.

Jul

1. Preliminary construction of the knowledge graph.

2. Further enhancement of the database -- completed integration of Chinese and English content and implementation of fuzzy matching.

3. Platform improvement -- finalized browsing features and the QA module, and achieved online deployment of the database.

4. Completion of the initial version of the mobile application (APP).

5. Application for software copyright.

The month focused on dataset updates, software copyright application, and finalization of the mobile app, representing a consolidation of the computational and legal foundations.

Aug

1. Formal submission of the software copyright application (initiated at the end of July).

2. Finalization of the mobile application, ensuring functional compliance with design specifications.

3. Initiation of the second version of database curation -- primarily involving functional screening of microbial strains, supplemented with abstracts, relevant publications, and verification of strain-disease relationships.

4. Initial setup of metabolite data for Flux Balance Analysis (FBA).

5. Refinement of the dynamic Flux Balance Analysis (dFBA) framework.

Work centered on intensive dataset curation and concurrent advancement of the metabolic modeling components, ensuring both data quality and model robustness for subsequent stages.

## 3. Human Practise

Jan

1. Public Science Lecture: "Scientists Explain: Are Probiotics a Waste of Money?"

![Public Science Lecture](https://static.igem.wiki/teams/5724/notebook/notebook-fig-3.webp)

Fig.4 Public Science Lecture

![Public Science Lecture](https://static.igem.wiki/teams/5724/notebook/notebook-fig-4.webp)

Fig.5 Public Science Lecture

Mar

1. Preparatory work for Tianjin University's 2025 Haitang Festival: design of display boards, roll-up banners, and flyers.

![Haitang Festival preparation](https://static.igem.wiki/teams/5724/notebook/notebook-fig-5.webp)

Fig.6 Haitang Festival preparation

2. Pre-event promotional posts for the Haitang Festival.

![Haitang Festival promotion](https://static.igem.wiki/teams/5724/notebook/notebook-fig-6.webp)

Fig.7 Haitang Festival promotion

Apr

1. Tianjin University Haitang Festival 2025

![Haitang Festival 2025](https://static.igem.wiki/teams/5724/notebook/notebook-fig-7.webp)

Fig.8 Haitang Festival 2025

2. Summary article on the Haitang Festival

![Haitang Festival summary](https://static.igem.wiki/teams/5724/notebook/notebook-fig-8.webp)

Fig.9 Haitang Festival summary

3. World Parkinson's Disease Day awareness feature

![World Parkinson's Disease Day](https://static.igem.wiki/teams/5724/notebook/notebook-fig-9.webp)

Fig.10 World Parkinson's Disease Day

4. "Technology Connecting Global Youth" International Youth Science & Technology Exchange Event

![International Youth Science & Technology Exchange](https://static.igem.wiki/teams/5724/notebook/notebook-fig-10.webp)

Fig.11 International Youth Science & Technology Exchange

May

1. Collaborative event with The Sixth Hospital of Peking University

![Collaborative event with The Sixth Hospital](https://static.igem.wiki/teams/5724/notebook/notebook-fig-11.webp)

Fig.12 Collaborative event with The Sixth Hospital

![Collaborative event with The Sixth Hospital](https://static.igem.wiki/teams/5724/notebook/notebook-fig-12.webp)

Fig.13 Collaborative event with The Sixth Hospital

2. Science Communication Series -- Issue 1

![Science Communication Series Issue 1](https://static.igem.wiki/teams/5724/notebook/notebook-fig-13.webp)

Fig.14 Science Communication Series Issue 1

Jun

1. Shaoxing City "New Quality Productive Forces" Science Communication and Exhibition Event

![Shaoxing Science Communication Event](https://static.igem.wiki/teams/5724/notebook/notebook-fig-14.webp)

Fig.15 Shaoxing Science Communication Event

![Shaoxing Science Communication Event](https://static.igem.wiki/teams/5724/notebook/notebook-fig-15.webp)

Fig.16 Shaoxing Science Communication Event

![Shaoxing Science Communication Event](https://static.igem.wiki/teams/5724/notebook/notebook-fig-16.webp)

Fig.17 Shaoxing Science Communication Event

Jul

1. Science Communication Series -- Issues 2, 3, and 4

![Science Communication Series Issue 2](https://static.igem.wiki/teams/5724/notebook/notebook-fig-17.webp)

Fig.18 Science Communication Series Issue 2

![Science Communication Series Issue 3](https://static.igem.wiki/teams/5724/notebook/notebook-fig-18.webp)

Fig.19 Science Communication Series Issue 3

![Science Communication Series Issue 4](https://static.igem.wiki/teams/5724/notebook/notebook-fig-19.webp)

Fig.20 Science Communication Series Issue 4

2. Summary feature on the collaborative event with The Sixth Hospital of Peking University

![Collaborative event summary](https://static.igem.wiki/teams/5724/notebook/notebook-fig-20.webp)

Fig.21 Collaborative event summary

3. Interview feature with the Head Nurse of The Sixth Hospital of Peking University

![Interview with Head Nurse](https://static.igem.wiki/teams/5724/notebook/notebook-fig-21.webp)

Fig.22 Interview with head nurse

4. Interview feature with a physician from The Sixth Hospital of Peking University

![Interview with physician](https://static.igem.wiki/teams/5724/notebook/notebook-fig-22.webp)

Fig.23 Interview with physician

5. Patient story feature from The Sixth Hospital of Peking University

![Patient story feature](https://static.igem.wiki/teams/5724/notebook/notebook-fig-23.webp)

Fig.24 Patient story feature

6. Activity planning for the Shaoxing Science Education Base

7. Promotional feature on Tsingke Biotechnology

8. High School Science Education Base Activity: "From Powder to Pill: The Preparation and Testing of Tablets"

![From Powder to Pill activity](https://static.igem.wiki/teams/5724/notebook/notebook-fig-24.webp)

Fig.25 From Powder to Pill activity

9. High School Science Education Base Activity: "Culture Medium Art"

![Culture Medium Art activity](https://static.igem.wiki/teams/5724/notebook/notebook-fig-25.webp)

Fig.26 Culture Medium Art activity

10. High School Science Education Base Activity: "Perfume-Making Workshop"

![Perfume-Making Workshop](https://static.igem.wiki/teams/5724/notebook/notebook-fig-26.webp)

Fig.27 Perfume-Making Workshop

Aug

1. Science Communication Series -- Issues 5, 6, 7, and 9

![Science Communication Series Issue 5](https://static.igem.wiki/teams/5724/notebook/notebook-fig-27.webp)

Fig.28 Science Communication Series Issue 5

![Science Communication Series Issue 6](https://static.igem.wiki/teams/5724/notebook/notebook-fig-28.webp)

Fig.29 Science Communication Series Issue 6

![Science Communication Series Issue 7](https://static.igem.wiki/teams/5724/notebook/notebook-fig-29.webp)

Fig.30 Science Communication Series Issue 7

![Science Communication Series Issue 9](https://static.igem.wiki/teams/5724/notebook/notebook-fig-30.webp)

Fig.31 Science Communication Series Issue 9

2. Parkinson's Disease Science Communication Video

![Parkinson's Disease Science Communication Video](https://static.igem.wiki/teams/5724/notebook/notebook-fig-31.webp)

Fig.32 Parkinson's Disease Science Communication Video

Sep

1. Science Communication Series -- Issue 8

![Science Communication Series Issue 8](https://static.igem.wiki/teams/5724/notebook/notebook-fig-32.webp)

Fig.33 Science Communication Series Issue 8

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% 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 %}

![Engineering Header Image](https://static.igem.wiki/teams/5724/headphoto/engineering.webp)

## Contents

* [Adhesion Module](#adhesion-module)
  ▶
  + [Cycle1 Assessment of the OppA Protein](#cycle1-assessment-oppa)
  + [Cycle2 Overexpression of Adhesion Factors](#cycle2-overexpression)
  + [Cycle3 Adhesin Expression and Optimization](#cycle3-adhesin)
* [Therapeutic Module](#therapeutic-module)
  ▶
  + [Cycle1 L-Dopa production module](#cycle1-l-dopa)
  + [Cycle2 Glutathione production module](#cycle2-glutathione)
* [Control Module](#control-module)
  ▶
  + [Version1 Communication based on AHLs](#version1-communication)
  + [Version2 Communication based on AHL and AIP](#version2-communication)
  + [Application](#application)
* [Safety Module](#safety-module)
  ▶
  + [Construction of a Hypoxia-Induced Suicide Module](#hypoxia-induced)
  + [Construction of a Thermosensitive Suicide Module](#thermosensitive)
  + [Emergency Containment Module Construction](#emergency-containment)
* [Fluorescent characterization of microbiota](#fluorescent-characterization)
  ▶
  + [Cycle 1](#cycle1-fluorescent)
  + [Cycle 2](#cycle2-fluorescent)
  + [Cycle 3](#cycle3-fluorescent)

## Adhesion Module

**Adhesion Module:**

![Molecular Docking Model Heatmap](https://static.igem.wiki/teams/5724/engineering/engineering-fig-1.webp)

Fig.1 Molecular Docking Model Heatmap

### Cycle1 Assessment of the OppA Protein

#### Design (D):

Objective: To verify whether the OppA protein from *Lactobacillus plantarum* can bind to NaHS on the surface of olfactory epithelial cells. Using Autodock and HDOCK software, molecular docking will be performed between the OppA protein and NaHS, Mucin, as well as other potentially interacting small molecules or proteins.

The binding affinities of seven different OppA proteins will be compared by evaluating their binding free energy (ΔG).

Additional Safety Consideration: Since the engineered bacteria may reach the intestinal tract, a Mucin binding test has been incorporated into the design to assess potential risks.

#### Build (B):

A molecular docking model was constructed to perform virtual docking of OppA with NaHS and Mucin, yielding binding energy and confidence scores.

The docking results were visualized, and a heatmap was generated to illustrate the binding capabilities of the different OppA variants.

#### Test (T):

The experimental results demonstrated that the binding energies between NaHS and all OppA variants were less than -6 kcal/mol, indicating significant binding interactions.

![Rigid Docking of NaHS with All OppA Variants](https://static.igem.wiki/teams/5724/engineering/engineering-fig-2.webp)

Fig.2 Flexible docking of mucin with all OppA variants

Among them, Lp\_0018 exhibited the strongest binding with NaHS, which is consistent with the findings reported in the literature.

Furthermore, flexible docking results indicated a potential interaction between OppA and mucin, suggesting a possible risk of side effects.

#### Learn (L):

The strong and specific binding between OppA and NaHS was confirmed, supporting its role as a key adhesion factor.

Furthermore, the identified risk of Mucin binding highlights the necessity of incorporating a hypoxia-induced suicide switch into the engineered bacterial design.

These findings thereby provide a rationale for selecting specific OppA proteins (e.g., LP\_0018) for subsequent overexpression.

![Docking of Lp_0783 with NaHS, etc.](https://static.igem.wiki/teams/5724/engineering/engineering-fig-3.webp)

Fig.3 (A) Docking of Lp\_0018 with NaHS, (B) Docking of Lp\_0020 with NaHS, (C) Docking of Lp\_0783 with NaHS, (D) Docking of Lp\_0092 with NaHS, (E) Docking of Lp\_0201 with NaHS, (F) Docking of Lp\_3686 with NaHS, (G) Docking of Lp\_1261 with NaHS

### Cycle2 Overexpression of Adhesion Factors

As the expression of adhesion factors is regulated by the Control Module, their construction and characterization are described in the corresponding section below.

### Cycle3 Adhesin Expression and Optimization

#### Design (D):

Objective: To achieve specific and orthogonal adhesion in a multi-strain system using the Intercellular Adhesion Toolkit.

Strategy:

* Overexpress the Ag2 adhesin in *Lactobacillus plantarum*.
* Construct four adhesin pathways (Ag2, Ag3, Nb2, Nb3) in *Escherichia coli*.

Design an orthogonal validation experiment and an IPTG induction concentration optimization assay.

#### Build (B):

In *L. plantarum*: The plasmid pSIP403-LysM-Ag2 was constructed, electroporated into *L. plantarum*, and positive clones were screened.

In *E. coli*: Using the pDest backbone, plasmids pDest-LysM-Ag3, pDest-LysM-Nb3, pDest-LysM-Ag2, and pDest-LysM-Nb2 were assembled via seamless cloning.

These constructs were transformed into *E. coli* and screened on ampicillin-containing medium.

![Plasmid Maps of the Four Constructs](https://static.igem.wiki/teams/5724/engineering/engineering-fig-4.webp)

Fig.4 Plasmid Maps of the Four Constructs

#### Test (T):

Optimal Induction Condition: A concentration gradient assay determined that the optimal IPTG concentration is 0.8 mM.

Orthogonality Test: The Nb3--Ag3 and Nb2--Ag2 pairs demonstrated the strongest specific binding, successfully validating orthogonality.

Effect of IPTG on Adhesion: Compared to the uninduced control, IPTG addition significantly enhanced adhesion.

Fluorescence Microscopy: Strains expressing RFP and GFP enabled clear visualization of enhanced adhesion.

Co-culture Experiment: In a mixed culture of *E. coli* and *L. plantarum*, the introduction of sppip and IPTG induction further strengthened the adhesion effect.

#### Learn (L):

The optimal working concentration of IPTG was confirmed to be 0.8 mM.

Orthogonality and enhanced adhesion were successfully validated, supporting the initial design rationale.

Results from fluorescence microscopy and OD600 measurements were consistent, reinforcing experimental reliability.

Co-culture results demonstrated that multi-strain interactions can be effectively controlled.

Future Optimization: Explore systems that do not require costly inducers and improve overall system stability.

## Therapeutic Module

**Therapeutic Module**

### Cycle1 L-Dopa production module

#### Design (D):

To establish L-DOPA production capability in *Escherichia coli*, we plan to implement metabolic engineering in this host. We aim to first verify its biosynthesis capacity and then regulate it via a control module. To this end, we initially cloned the hpaBC gene cluster into a pET-28a vector, constructing a T7 promoter-driven expression cassette (as shown in the figure below). The resulting recombinant plasmid was then introduced into *E. coli* to enable heterologous expression of 4-hydroxyphenylacetate 3-hydroxylase under induction conditions. Ultimately, the engineered strain can utilize endogenous L-tyrosine---generated through its own metabolic pathways---as the substrate for the catalytic production of L-DOPA.

![Plasmid Map of pET-28a-T7-hpaBC](https://static.igem.wiki/teams/5724/engineering/engineering-fig-5.webp)

Fig.5 Plasmid Map of pET-28a-T7-hpaBC

#### Build (B):

The hpaBC gene cluster was assembled into the pET-28a plasmid using seamless cloning. The resulting recombinant plasmid was then transformed into *Escherichia coli* competent cells. Primary screening was performed on ampicillin-resistant plates, followed by further verification of positive clones via colony PCR. Ultimately, engineered strains harboring the correct recombinant plasmid were successfully obtained.

#### Test (T):

The verified engineered strain was activated and subsequently inoculated at a 1:100 ratio into 20 mL of LB medium for fermentation culture at 37°C and 220 rpm. After 6 hours of cultivation (corresponding to the logarithmic growth phase), isopropyl-β-D-thiogalactoside (IPTG) was added to the induction group to a final concentration of 1 mM to induce protein expression. Post-induction, cultivation continued until 32 hours. To monitor the kinetics of cell growth and product synthesis, samples were taken at regular intervals for OD600 measurement. Additionally, samples were collected every 8 hours, centrifuged, and appropriately processed. The supernatant was analyzed via high-performance liquid chromatography to quantify L-DOPA content, while the melanin content generated from L-DOPA oxidation was assessed by measuring the OD400 of the samples. The color changes observed during the fermentation process are shown in Figure 2, and detailed experimental results are provided in the Results section.

![Comparison Between E. coli Nissle 1917 and Induced Production-Plasmid-Harboring E. coli](https://static.igem.wiki/teams/5724/engineering/engineering-fig-6.webp)

Fig.6 Comparison Between *E. coli* Nissle 1917 and Induced Production-Plasmid-Harboring *E. coli*

#### Learn (L):

We demonstrate the successful construction of an L-DOPA-producing *E. coli* strain. This strain operates via a heterologous pathway encoded by the hpaBC gene cluster, which centers on the 4-hydroxyphenylacetate 3-hydroxylase enzyme to drive the single-step conversion of L-tyrosine to L-DOPA.

### Cycle2 Glutathione production module

#### Design (D):

To establish glutathione production capability in *Escherichia coli*, we plan to implement metabolic engineering in this host. We aim to first verify its biosynthetic capacity and subsequently regulate it via a control module. To this end, the gshAB gene cluster was inserted into the pET-28a vector, constructing a T7 promoter-driven expression unit, as illustrated in Figure 3. The resulting recombinant plasmid was then introduced into *E. coli* to enable heterologous expression of a bifunctional glutathione synthetase under induction conditions. Ultimately, the engineered strain can catalyze the condensation of glutamate, cysteine, and glycine, thereby achieving glutathione synthesis.

![Plasmid Map of pET-28a-T7-gshAB](https://static.igem.wiki/teams/5724/engineering/engineering-fig-7.webp)

Fig.7 Plasmid Map of pET-28a-T7-gshAB

#### Build (B):

The gshAB gene cluster was assembled into the pET-28a plasmid using seamless cloning. The resulting recombinant plasmid was transformed into *Escherichia coli* competent cells. Primary screening was conducted on kanamycin-resistant plates, followed by further verification of positive clones via colony PCR. Ultimately, engineered strains harboring the correct recombinant plasmid were successfully obtained.

#### Test (T):

The verified engineered strain was activated and inoculated at a 1:100 ratio into 25 mL of LB medium for fermentation culture at 37°C and 220 rpm. After 6 hours of cultivation (corresponding to the logarithmic growth phase), IPTG was added to the induction group to a final concentration of 1 mM to induce protein expression. At 8 hours of cultivation, glutamate, cysteine, and glycine were supplemented to the amino acid supplementation group to a final concentration of 25 mM each. Post-induction, cultivation continued until 22 hours. To monitor the kinetics of cell growth and product synthesis, samples were taken at regular intervals for OD₆₀₀ measurement. During the later stages of fermentation, samples were collected every 8 hours, centrifuged, and appropriately processed. The supernatant was analyzed via high-performance liquid chromatography to quantify the contents of reduced glutathione (GSH) and oxidized glutathione (GSSG). Detailed experimental results are provided in the Results section.

#### Learn (L):

We demonstrate the successful construction of a glutathione-producing *E. coli* strain. This strain operates via a heterologous pathway encoded by the gshAB gene cluster, which centers on a bifunctional glutathione synthetase enzyme to drive the sequential one-step conversion of the three substrate amino acids into glutathione.

## Control Module

**Control Module**

### Version1 Communication based on AHLs

In our first-round design, we envisioned establishing bidirectional communication between *Lactiplantibacillus plantarum* and *Escherichia coli* through AHL signaling, so that each species could activate the functional genes of the other and thereby achieve coordinated regulation of the consortium.

As illustrated in the figure, we planned to introduce an exogenous RhlI synthase into *L. plantarum* to serve as the AHL sender module, while simultaneously incorporating LuxR and the Plux promoter as the AHL receiver module. The same design logic was applied to *E. coli*, enabling mutual signal exchange and synchronized gene expression across both strains.

![AHL-Based Bidirectional Communication](https://static.igem.wiki/teams/5724/engineering/engineering-fig-8.webp)

Fig.8 AHL-Based Bidirectional Communication

#### Cycle 1 --- Testing the native Plux promoter in *L. plantarum*

#### Design (D):

To explore whether the native Plux promoter could function in *Lactiplantibacillus plantarum*, we constructed the plasmid pNZ8148-P23-LuxR-Plux-RFP. In this design, the P23 promoter constitutively expresses the LuxR protein. Upon binding to 3OC6-HSL, the LuxR--AHL complex interacts with the Lux box region of Plux, thereby activating the downstream mRFP1 reporter gene. This setup allows us to qualitatively and quantitatively measure promoter activity through fluorescence detection with a microplate reader.

![Plasmid Map of PNZ8148-P23-LuxR-Plux-RFP](https://static.igem.wiki/teams/5724/engineering/engineering-fig-9.webp)

Fig.9 Plasmid Map of PNZ8148-P23-LuxR-Plux-RFP

#### Build (B):

The plasmid was first assembled in *E. coli* MC1061 and validated by colony PCR and sequencing.

![Sequencing Results of Plasmid pNZ8148-P23-LuxR-Plux-RFP](https://static.igem.wiki/teams/5724/engineering/engineering-fig-10.webp)

Fig.10 Sequencing Results of Plasmid pNZ8148-P23-LuxR-Plux-RFP

The verified construct was then transformed into *L. plantarum* WCFS1, and positive colonies were confirmed by colony PCR.

#### Test (T):

We induced the *L. plantarum* culture at mid-log phase with 100 nM 3OC6-HSL at 30°C. After 12 hours, fluorescence intensity was measured using a microplate reader. The results are shown below.

![Fluorescence Intensity of Plux-RFP](https://static.igem.wiki/teams/5724/engineering/engineering11-new.webp)

Fig.11 Fluorescence Intensity of Plux-RFP

#### Learn (L):

We observed that the native Plux promoter did not exhibit a detectable response in *L. plantarum*. Literature review suggested that this may be due to differences in host sigma factors, which affect promoter recognition and activity across species. Therefore, we concluded that host-adaptive promoter engineering would be required to achieve functional AHL responsiveness in *L. plantarum*.

#### Cycle 2 --- Adaptive modification of Plux in *L. plantarum*

#### Design (D):

From Cycle 1, we learned that the native Plux promoter did not function in *L. plantarum*, indicating the need for host-adaptive modification. In this iteration, we aimed to preserve the Lux box region while systematically replacing the −10 region, −35 region, and spacer sequences of Plux, thereby generating a panel of Plux variants with potentially improved compatibility.

![Schematic Diagram of Promoter Engineering](https://static.igem.wiki/teams/5724/engineering/engineering-fig-12.webp)

Fig.12 Schematic Diagram of Promoter Engineering

#### Build (B):

Using seamless DNA assembly, we introduced targeted mutations based on the Cycle 1 construct pNZ8148-P23-LuxR-Plux-RFP. The plasmid was first assembled in *E. coli* MC1061 and validated by colony PCR and sequencing.

This yielded a series of variant plasmids:

* pNZ8148-P23-LuxR-Plux0101-RFP
* pNZ8148-P23-LuxR-Plux0201-RFP
* pNZ8148-P23-LuxR-Plux0210-RFP
* pNZ8148-P23-LuxR-Plux0300-RFP
* pNZ8148-P23-LuxR-Plux0220-RFP

![Primer design for introducing targeted mutations using seamless DNA assembly](https://static.igem.wiki/teams/5724/engineering/engineering-fig-13.webp)

Fig.13 Primer design for introducing targeted mutations using seamless DNA assembly

#### Test (T):

The constructs were introduced into *L. plantarum* WCFS1, and positive colonies were confirmed by colony PCR.

We induced the *L. plantarum* culture at mid-log phase with 100 nM 3OC6-HSL at 30°C. After 12 hours, fluorescence intensity was measured using a microplate reader. The results are shown below.

The results are summarized below.

![Fluorescence Intensity of Plux Variants](https://static.igem.wiki/teams/5724/engineering/engineering14-new.webp)

Fig.14 Fluorescence Intensity of Plux Variants

#### Learn (L):

Despite these modifications, none of the Plux variants showed significant promoter activity in *L. plantarum*. After discussion with our PI, we concluded that further large-scale or high-throughput promoter engineering would be time- and resource-intensive and still carry high uncertainty in outcome. Therefore, we decided to shift strategy: instead of forcing AHL-based communication in *L. plantarum*, we turned to literature and identified that this strain naturally relies on AIP-mediated quorum sensing (typical in Gram-positive bacteria). From this point, we planned to redesign our system so that *L. plantarum* would utilize AIP-based communication, while *E. coli* would be engineered to interface through AIP to achieve interspecies signaling.

### Version2 Communication based on AHL and AIP

In this version of our design, we decided to retain the AHL-based communication from *L. plantarum* to *E. coli*, while enabling *E. coli* to communicate back with *L. plantarum* through AIP signaling.

![AHL- and AIP-Based Bidirectional Communication](https://static.igem.wiki/teams/5724/engineering/engineering-fig-15.webp)

Fig.15 AHL- and AIP-Based Bidirectional Communication

Moving forward, we will focus on the engineering construction of the four core modules:

AHL receiver, AHL sender, AIP sender, AIP receiver

#### AHL sensor:

#### Cycle1:

#### Design (D):

To evaluate the activity of the quorum-sensing promoter Plux and determine its appropriate induction concentration, we constructed the plasmid pETDuet-1-J23110-LuxR-Plux-RFP. In this design, the J23110 promoter constitutively expresses LuxR. When LuxR binds to 3OC6-HSL, the complex interacts with the Lux box of Plux, thereby activating the downstream mRFP1 reporter gene. This system allowed us to monitor promoter activity both qualitatively and quantitatively using a microplate reader.

![Plasmid Map of PET-J23110-LuxR-Plux-RFP](https://static.igem.wiki/teams/5724/engineering/engineering-fig-16.webp)

Fig.16 Plasmid Map of PET-J23110-LuxR-Plux-RFP

#### Build (B):

The functional fragment was synthesized by Tsingke, and the assembled plasmid was introduced into *E. coli*. Colony PCR confirmed the successful construction.

![Agarose Gel Electrophoresis of pET-J23110-LuxR-Plux-RFP](https://static.igem.wiki/teams/5724/engineering/engineering-fig-17.webp)

Fig.17 Agarose Gel Electrophoresis of pET-J23110-LuxR-Plux-RFP

#### Test (T):

Fluorescence was measured after induction with 3OC6-HSL. However, no detectable fluorescence was observed under the tested conditions.

#### Learn (L):

We investigated possible causes, including plasmid instability and inducer degradation, but these did not explain the failure. During discussions at the Conference of China iGEMer Community (CCiC) with Xiaoyao Zhou from iGEM WHU, we learned that both 3OC6-HSL and LuxR require sufficient intracellular concentrations to activate Plux. Since J23110 is a relatively weak promoter, it was suggested that we replace it with the stronger J23100 promoter to achieve adequate LuxR expression.

#### Cycle2:

#### Design (D):

In the previous design cycle, we observed that the J23110 promoter exhibited relatively weak activity, necessitating a stronger promoter for the expression of the LuxR protein. Consequently, the constitutive J23100 promoter was adopted to drive LuxR expression. When LuxR binds to the autoinducer 3OC6-HSL, the resulting complex activates transcription by binding to the Lux Box of the Plux promoter, thereby initiating expression of the mRFP1 reporter gene. This design enables qualitative and quantitative detection of fluorescence using a microplate reader.

![Plasmid Map of PET-J23100-LuxR-Plux-RFP](https://static.igem.wiki/teams/5724/engineering/engineering-fig-18.webp)

Fig.18 Plasmid Map of PET-J23100-LuxR-Plux-RFP

#### Build (B):

Building upon the Cycle 1 plasmid pET-J23110-LuxR-Plux-RFP, we successfully replaced the promoter. The correct construction of the resulting plasmid, pET-J23100-LuxR-Plux-RFP, was confirmed by colony PCR and sequencing.

![Agarose Gel Electrophoresis of PET-J23100-LuxR-Plux-RFP](https://static.igem.wiki/teams/5724/engineering/engineering-fig-19.webp)

Fig.19 Agarose Gel Electrophoresis of PET-J23100-LuxR-Plux-RFP

#### Test (T):

Fluorescence intensity following induction was measured using a microplate reader, and a dose-response assay was conducted across a range of concentrations. Detailed results are presented in the Results section.

#### Learn (L):

Our findings confirm the hypothesis that a sufficient intracellular concentration of LuxR protein is required for its binding with 3OC6-HSL and subsequent activation of transcription from the Plux promoter. The J23110 promoter demonstrated insufficient strength for this purpose, whereas the J23100 promoter effectively met the requirement. Furthermore, the Plux promoter exhibited clear concentration-dependent inducibility, with its activity increasing in response to higher concentrations of 3OC6-HSL.

This observation also led us to reconsider whether the previously observed non-responsiveness of Plux in *Lactobacillus plantarum* could similarly be attributed to inadequate LuxR expression. However, after reviewing the literature and consulting with our advisor, we learned that P23 is already considered a strong promoter in lactic acid bacteria, making insufficient protein expression an unlikely explanation. The earlier lack of response is more likely due to promoter incompatibility in that specific host context.

#### AHL producer:

#### Cycle1:

#### Design (D):

To enable the synthesis of 3OC6-HSL in *Lactiplantibacillus plantarum* WCFS1 for the subsequent activation of the Plux promoter in *Escherichia coli*, we constructed the plasmid pNZ8148-P23-LuxI. The core objective of this design is to achieve stable production of 3OC6-HSL in *L. plantarum*. The vector pNZ8148 is a commonly used expression plasmid for lactic acid bacteria, featuring a lactococcal replicon suitable for this host. In our design, a codon-optimized LuxI gene was inserted downstream of the strong P23 promoter to enable sustained, high-level constitutive expression.

![Plasmid Map of pNZ8148-P23-RFP](https://static.igem.wiki/teams/5724/engineering/engineering-fig-20.webp)

Fig.20 Plasmid Map of pNZ8148-P23-RFP

#### Build (B):

The functional P23-LuxI sequence was synthesized by Tsingke Biotechnology Co. Following its acquisition, we first constructed the pNZ8148-P23-RFP plasmid in *E. coli* MC1061, with correct assembly confirmed by colony PCR and sequencing.

![Agarose Gel Electrophoresis of pNZ8148-P23-RFP](https://static.igem.wiki/teams/5724/engineering/engineering-fig-21.webp)

Fig.21 Agarose Gel Electrophoresis of pNZ8148-P23-RFP

The verified plasmid was then electroporated into *Lactiplantibacillus plantarum*, yielding correct transformants identified through colony PCR.

#### Test (T):

The 12-hour cultured *Lactobacillus plantarum* was processed and co-cultured with *Escherichia coli* harboring the plasmid pET-J23100-LuxR-Plux-RFP. After 6 hours of induction, fluorescence was quantified using a microplate reader and visualized by fluorescence microscopy. Detailed results are presented in the Results section.

#### Learn (L):

It can be concluded that the engineered AHL-sending module in *Lactiplantibacillus plantarum* was capable of eliciting a response in the engineered *Escherichia coli* receiver strain, though the induced signal intensity fell short of that achieved by the external addition of 100 nM 3OC6-HSL. To enhance the system performance, the following optimization strategies will be implemented: (1) Optimizing the expression level of LuxI or improving precursor substrate availability in *L. plantarum* to enhance AHL synthesis. (2) Modifying the RBS of the RFP gene or employing a stronger Plux promoter variant to amplify the response intensity.

#### AIP Receiver:

#### Design (D):

For the AIP receiver module in *L. plantarum*, we selected the sppip AIP signal and co-expressed the sppK and sppR genes to form a membrane-associated two-component sensor system. Upon activation by AIP binding, the sensor kinase SppK undergoes autophosphorylation at a conserved histidine residue. The phosphoryl group is then transferred to the cognate response regulator SppR in the cytoplasm. Phosphorylated SppR binds specifically to the Pspp promoter region, thereby enhancing the transcription of downstream genes. To quantitatively characterize this system, we used mRFP1 as a reporter gene and performed detailed measurements of its expression.

#### Build (B):

The plasmid pSIP403-Pspp-RFP was constructed using the pSIP403 backbone, and its correct assembly was verified by colony PCR and sequencing.

![Plasmid Map of pSIP403-Pspp-RFP](https://static.igem.wiki/teams/5724/engineering/engineering-fig-22.webp)

Fig.22 Plasmid Map of pSIP403-Pspp-RFP

![Agarose Gel Electrophoresis of pSIP403-Pspp-RFP](https://static.igem.wiki/teams/5724/engineering/engineering-fig-23.webp)

Fig.23 Agarose Gel Electrophoresis of pSIP403-Pspp-RFP

The confirmed plasmid was subsequently electroporated into *Lactiplantibacillus plantarum* WCFS1, with successful transformation validated again by colony PCR.

#### Test (T):

The temporal and dose-response profiles of the spp system were characterized, with detailed data presented in the Results section.

#### Learn (L):

The robust performance of the spp system in *L. plantarum* validates it as a reliable and effective receiver component.

#### AIP Sender:

#### Design (D):

We designed an AIP secretion system in *E. coli* utilizing its type I secretion pathway:

First, the plasmid pRSFDuet-T7-CvaAB was constructed to express the CvaA and CvaB proteins, which assemble with the endogenous TolC protein to form a transmembrane complex.

Then, the sppip sequence was fused to the CvaC15 signal peptide, yielding the plasmid pBAD24-CvaC15-SppIP.

Upon induction with arabinose and IPTG, the CvaC15 signal peptide directs the SppIP precursor to the CvaAB complex. Following cleavage by CvaB, mature SppIP is transported extracellularly through the channel formed by CvaA and TolC.

#### Build (B):

We constructed two plasmids: pRSFDuet-T7-CvaAB for expressing CvaA and CvaB, and pBAD24-CvaC15-SppIP encoding the fusion of the CvaC15 signal peptide with SppIP. Both plasmids were co-transformed into *E. coli*, and successful strain construction was verified by colony PCR.

![Plasmid Map of PRSF-T7-CvaAB and PBAD24-CvaC15-SPPIP](https://static.igem.wiki/teams/5724/engineering/engineering-fig-24.webp)

Fig.24 Plasmid Map of PRSF-T7-CvaAB and PBAD24-CvaC15-SPPIP

![Agarose Gel Electrophoresis of PBAD24-CvaC15-SppIP](https://static.igem.wiki/teams/5724/engineering/engineering-fig-25.webp)

Fig.25 Agarose Gel Electrophoresis of PBAD24-CvaC15-SppIP

![Agarose Gel Electrophoresis of PRSFDuet-T7-CvaAB](https://static.igem.wiki/teams/5724/engineering/engineering-fig-26.webp)

Fig.26 Agarose Gel Electrophoresis of PRSFDuet-T7-CvaAB

#### Test (T):

Subsequently, the co-transformed *E. coli* culture was induced and then subjected to centrifugation. The supernatant was collected, filter-sterilized, and added to *Lactiplantibacillus plantarum* harboring the reporter plasmid pSIP403-PsppA-RFP to test whether the AIP signal produced by the sender module could be detected by the receiver module. Detailed results are presented in the Results section.

#### Learn (L):

The strategy of utilizing the type I secretion system in *E. coli* for SppIP secretion proved effective, generating a sufficient quantity of the AIP to elicit a robust response in *Lactiplantibacillus plantarum*.

### Application

#### HpaBC Production Regulation

#### Design (D):

We designed the plasmid pET-J23100-LuxR-Plux-HpaBC to test the control of L-DOPA synthesis by exogenous and endogenous 3OC6-HSL signals through the Plux-HpaBC module in *E. coli*. In this construct, the constitutive J23100 promoter ensures sustained LuxR expression, maintaining adequate intracellular regulator levels. The resulting LuxR-3OC6-HSL complex binds specifically to the Lux Box of the Plux promoter, activating transcription of the downstream HpaBC enzyme system. This enables the oxidation of tyrosine to L-DOPA, thereby establishing a direct link between quorum-sensing signals and functional metabolic output.

![Plasmid Map of pET-J23100-LuxR-Plux-HpaBC](https://static.igem.wiki/teams/5724/engineering/engineering-fig-27.webp)

Fig.27 Plasmid Map of pET-J23100-LuxR-Plux-HpaBC

#### Build (B):

The plasmid was successfully assembled, with correct construction verified by colony PCR and DNA sequencing.

![Agarose Gel Electrophoresis of pET-J23100-LuxR-Plux-HpaBC](https://static.igem.wiki/teams/5724/engineering/engineering-fig-28.webp)

Fig.28 Agarose Gel Electrophoresis of pET-J23100-LuxR-Plux-HpaBC

#### Test (T):

We performed exogenous and endogenous induction experiments as follows:

cultures of *E. coli* harboring pET-J23100-LuxR-Plux-HpaBC were supplemented with either 100 nM 3OC6-HSL (exogenous) or 500 μL of supernatant from *Lactiplantibacillus plantarum* containing pNZ8148-P23-LuxI (endogenous). Following 24 hours of incubation, the bacterial cultures were collected for subsequent measurements. Detailed results are provided in the Results section.

#### Learn (L):

The experimental results demonstrate that the Plux-HpaBC module can be successfully activated under both exogenous and endogenous induction conditions, exhibiting functional output (L-DOPA production). However, the signal strength and product titer under endogenous induction were lower than those achieved with the addition of 100 nM AHL. This suggests the following potential directions for future optimization:

1. Enhance AHL synthesis in *L. plantarum* by optimizing LuxI expression levels or improving precursor substrate supply.
2. Increase the response intensity by modifying the RBS of HpaBC or employing stronger Plux promoter variants.
3. Boost the final product titer through metabolic engineering strategies to enhance the host's supply of the L-DOPA precursor, L-tyrosine.

#### Regulation of the Lp\_0018 Adhesin

#### Design (D):

In the adhesion module, we aim to enhance the colonization capacity of *Lactiplantibacillus plantarum* on the olfactory epithelium by overexpressing the OppA protein variant LP\_0018. To achieve this, we constructed an overexpression vector in which the expression of LP\_0018 is controlled by the PsppA promoter.

![Plasmid Map of pSIP403-LP_0018](https://static.igem.wiki/teams/5724/engineering/engineering-fig-29.webp)

Fig.29 Plasmid Map of pSIP403-LP\_0018

#### Build (B):

Based on the existing pSIP403 plasmid (containing the PsppA promoter and a dual-gene expression framework), pSIP403-LP\_0018 was assembled using seamless cloning. The constructed plasmid was first transformed into *Escherichia coli* for propagation, and subsequently electroporated into *Lactiplantibacillus plantarum*. Positive transformants were selected on erythromycin-containing medium.

#### Test (T):

An engineered strain of *Lactiplantibacillus plantarum* stably maintaining Lp\_0018 (OppA) was successfully obtained. The successful plasmid integration and functional expression in *L. plantarum* were verified, with detailed results presented in the Results section.

#### Learn (L):

The Pspp induction system was successfully validated and can be utilized to control the expression of the Lp\_0018 (OppA) protein.

## Safety Module

**Safety Module**

### Construction of a Hypoxia-Induced Suicide Module

#### Cycle 1

#### Design (D):

Given the significant risk of engineered bacteria---designed for colonization on the olfactory epithelium---detaching and translocating to the intestinal tract, we designed a hypoxia-responsive suicide element based on comparative analysis of oxygen concentrations in the nasal cavity and gut. This element consists of a hypoxia-inducible promoter (e.g., phyb) and the toxin gene MazF. We initially constructed this circuit in *Escherichia coli* using the pETDuet plasmid, into which an expression cassette containing the phyb promoter and MazF gene was inserted.

![Plasmid Map of pETDuet-phyb-MazF](https://static.igem.wiki/teams/5724/engineering/engineering-fig-30.webp)

Fig.30 Plasmid Map of pETDuet-phyb-MazF

#### Build (B):

The plasmid pETDuet-phyb-MazF was constructed using the laboratory's existing pETDuet backbone and gene sequences for phyb and MazF obtained from the iGEM parts registry, with assembly accomplished via homologous recombination.

#### Test (T):

The constructed pETDuet-phyb-MazF plasmid was transformed into *E. coli*, and transformants were selected on ampicillin-resistant plates. Positive clones were subcultured and streaked on fresh plates, followed by static incubation at 37°C for 48 hours under both aerobic and anaerobic conditions. Anaerobic conditions were generated using commercial anaerobic gas packs and sachets. Three parallel experimental replicates were included for each condition. Detailed results are provided in the Results section.

#### Learn (L):

The experimental results demonstrate that, compared to cultivation under aerobic conditions, our engineered hypoxia-responsive suicide element significantly retarded the growth of *E. coli* under anaerobic conditions, thereby validating the design objective.

#### Cycle 2

#### Design (D):

Based on the initial tests from the first cycle, we have preliminarily validated the functionality of the designed hypoxia-responsive suicide element in *E. coli*. The next step involves verifying its feasibility in *Lactiplantibacillus plantarum* (Lp). To this end, we selected the pNZ8148 plasmid and inserted an expression cassette containing the hypoxia-inducible promoter phyb and the toxin gene MazF.

![Plasmid Map of pNZ8148-phyb-MazF](https://static.igem.wiki/teams/5724/engineering/engineering-fig-31.webp)

Fig.31 Plasmid Map of pNZ8148-phyb-MazF

#### Build (B):

Using the laboratory's existing pNZ8148 backbone and amplifying the phyb-MazF fragment from the previously constructed pET-phyb-MazF plasmid, we assembled the pNZ8148-phyb-MazF plasmid via homologous recombination.

#### Test (T):

The constructed plasmid was first transformed into *E. coli* and screened on ampicillin-resistant plates to isolate correct transformants. Subsequently, the plasmid was electroporated into *L. plantarum*.

![Plate Culture of Strains Transformed with Plasmid pNZ8148-phyb-MazF](https://static.igem.wiki/teams/5724/engineering/engineering-fig-32.webp)

Fig.32 Plate Culture of Strains Transformed with Plasmid pNZ8148-phyb-MazF

#### Learn (L):

During validation, we observed severely impaired growth of *L. plantarum* after the introduction of the suicide element, suggesting that it may impose a substantial metabolic burden on the host. For future work, alternative suicide elements should be designed.

### Construction of a Thermosensitive Suicide Module

#### Cycle 1

#### Design (D):

Considering that the nasal cavity is an open environment and our engineered bacteria designed for olfactory epithelium colonization may detach, there is a risk of the bacteria escaping to the external environment. Based on the idealized assumption that the nasal cavity maintains a constant temperature of 37°C while the external environment remains at 30°C or below, we designed a thermosensitive suicide element composed of the temperature-sensitive Cl857 protein and the toxin protein MazF.

To preliminarily validate the functionality of the Cl857 protein, we selected the pETDuet plasmid and inserted an expression cassette containing Cl857 and the red fluorescent protein (RFP) gene. This construct was initially tested in *E. coli*.

![Plasmid Map of pETDuet-J23110-Cl-pR-phIF-pPhIF-RFP](https://static.igem.wiki/teams/5724/engineering/engineering-fig-33.webp)

Fig.33 Plasmid Map of pETDuet-J23110-Cl-pR-phIF-pPhIF-RFP

#### Build (B):

Using the laboratory's existing pETDuet backbone and sequences for Cl857 and other required genes obtained from the iGEM parts registry, we assembled the plasmid pETDuet-J23110-Cl857-pR-phIF-pPhIF-RFP via homologous recombination.

#### Test (T):

The constructed plasmid was transformed into *E. coli*, and transformants were selected on ampicillin-resistant plates. Positive clones were subcultured and subjected to the following experiments: Liquid cultures of the transformants were grown at 30°C and 37°C for 16 hours. Subsequently, the cultures were subcultured at a 1:200 inoculation ratio and incubated for 24 hours, after which fluorescence intensity and OD600 were measured.

#### Learn (L):

No significant difference in specific fluorescence intensity was observed between the engineered bacteria cultured at 30°C and 37°C, indicating that the results did not meet expectations.

Comparison with the original literature revealed two key differences:

1. **Promoter type for Cl857:** We used the constitutive promoter J23110, while the original study employed the pRM promoter, which is positively regulated by cI857.
2. **Plasmid configuration:** We integrated all genetic elements into a single plasmid, whereas the original study distributed the Cl857 expression circuit and the downstream phIF/pPhIF modules across two compatible plasmids.

The pRM promoter is inherently weak and highly dependent on Cl857 protein levels. When cI857 accumulates sufficiently, it forms dimers that bind to the OR2 operator site near pRM, physically interacting with RNA polymerase to enhance transcription of Cl857---creating a positive feedback loop. In contrast, the constitutive J23110 promoter drives transcription at a constant rate, resulting in lower regulatory precision.

Additionally, the dual-plasmid system used in the literature, though imposing a higher metabolic burden and slower growth on the host, facilitated better product expression.

These differences may account for the limited efficacy observed in our current design. Corresponding adjustments will be made in the next cycle.

#### Cycle 2

#### Design (D):

Based on the outcomes of the previous DBTL cycle, we constructed a dual-plasmid system for this round: one plasmid, pETDuet, carrying the expression cassette with \*pRM-Cl857\*; the other, pJUMP, carrying the pR-phIF-pPhIF-RFP cassette. The system was validated in *E. coli*.

![Plasmid Map of pJUMP-pRM- Cl857](https://static.igem.wiki/teams/5724/engineering/engineering-fig-34.webp)

Fig.34 Plasmid Map of pJUMP-pRM- Cl857

![Plasmid Map of pETDuet-pR-phIF-pPhIF-RFP](https://static.igem.wiki/teams/5724/engineering/engineering-fig-35.webp)

Fig.35 Plasmid Map of pETDuet-pR-phIF-pPhIF-RFP

#### Build (B):

Using homologous recombination, we successfully constructed the two plasmids: pETDuet-pRM-Cl857 and pJUMP-pR-phIF-pPhIF-RFP.

#### Test (T):

The constructed plasmids were co-transformed into *E. coli*, and positive transformants were selected on double-antibiotic plates containing ampicillin and kanamycin.

![Dual-Plasmid Transformation Plate (pETDuet-pRM-cI857 & pJUMP-pR-phIF-pPhIF-RFP)](https://static.igem.wiki/teams/5724/engineering/engineering-fig-36.webp)

Fig.36 Dual-Plasmid Transformation Plate (pETDuet-pRM-Cl857 & pJUMP-pR-phIF-pPhIF-RFP)

Validated transformants were cultured in liquid medium at 30°C and 37°C for 16 hours, followed by subculturing at a 1:200 inoculation ratio for another 24 hours. Fluorescence intensity and OD600 were measured for each condition.

#### Learn (L):

Our experimental results preliminarily validated the functionality of the thermosensitive regulatory system. The next step will involve replacing the fluorescent protein gene with the toxin gene MazF for subsequent suicide module verification.

#### Cycle 3

#### Design (D):

We continued to employ the dual-plasmid system, replacing the red fluorescent protein (RFP) coding sequence with that of the toxin protein MazF, and validated the construct in *Escherichia coli*.

![Plasmid Map of pETDuet-pR-phIF-pPhIF-MazF](https://static.igem.wiki/teams/5724/engineering/engineering-fig-37.webp)

Fig.37 Plasmid Map of pETDuet-pR-phIF-pPhIF-MazF

#### Build (B):

Using homologous recombination, we successfully constructed the plasmids pETDuet-pRM-Cl857 and pJUMP-pR-phIF-pPhIF-MazF.

#### Test (T):

The two plasmids were co-transformed into *E. coli*, and positive transformants were selected on double-antibiotic plates containing ampicillin and kanamycin.

![Dual-Plasmid Transformation Plate (pETDuet-pRM-cI857 & pJUMP-pR-phIF-pPhIF-MazF)](https://static.igem.wiki/teams/5724/engineering/engineering-fig-38.webp)

Fig.38 Dual-Plasmid Transformation Plate (pETDuet-pRM-Cl857 & pJUMP-pR-phIF-pPhIF-MazF)

#### Learn (L):

Experimental results indicated a suboptimal response of *E. coli* to the constructed thermosensitive suicide system, as a considerable number of engineered cells remained viable at 30°C. Subsequent efforts should focus on enhancing the expression level of the toxin protein to achieve the desired growth inhibition effect.

### Emergency Containment Module Construction

#### Cycle 1

#### Design (D):

To address potential biosafety risks arising from unexpected incidents, we designed an emergency containment system for rapid system shutdown in *Escherichia coli* Nissle 1917. The initial pathway was constructed in *E. coli* using the pETDuet plasmid, into which an expression cassette containing the L-arabinose-inducible pBAD promoter and the toxin gene MazF was inserted.

![Plasmid Map of pETDuet-pBAD -MazF](https://static.igem.wiki/teams/5724/engineering/engineering-fig-39.webp)

Fig.39 Plasmid Map of pETDuet-pBAD -MazF

#### Build (B):

The plasmid pETDuet-pBAD-MazF was successfully assembled via homologous recombination.

#### Test (T):

The constructed plasmid was transformed into *E. coli*, and positive transformants were selected on chloramphenicol-resistant plates. Growth curves were subsequently determined for validation.

#### Learn (L):

Experimental results demonstrated marked growth retardation in the engineered *E. coli* upon L-arabinose induction, confirming the functional success of the emergency containment module.

#### Cycle 2

#### Design (D):

Following the successful validation of the L-arabinose-inducible suicide system in *E. coli*, we subsequently designed a nisin-inducible suicide system tailored for *Lactiplantibacillus plantarum*. We selected the pNZ8148 plasmid and inserted an expression cassette consisting of the nisin-inducible promoter PnisA and the toxin gene MazF.

![Plasmid Map of pNZ8148-Pnis -MazF](https://static.igem.wiki/teams/5724/engineering/engineering-fig-40.webp)

Fig.40 Plasmid Map of pNZ8148-Pnis -MazF

#### Build (B):

The plasmid pNZ8148-PnisA-MazF was constructed via homologous recombination.

#### Test (T):

The recombinant plasmid was transformed into *L. plantarum*, and positive transformants were selected on chloramphenicol-containing plates. Growth curves were subsequently determined to assess system functionality.

#### Learn (L):

Experimental results showed that nisin induction significantly slowed the growth of the engineered *L. plantarum*, confirming the effectiveness of the constructed genetic module. With this step, we have successfully built and validated inducible suicide systems for both *E. coli* and *L. plantarum*, establishing an emergency containment mechanism that substantially reduces potential biosafety risks in case of unexpected incidents.

## Fluorescent characterization of microbiota

**Fluorescent characterization of microbiota:**

### Cycle 1

#### Design (D):

In order to be able to characterise the coexistence of EcN and Lp more intuitively, we intend to construct four fluorescent plasmids: pET-J23119-YFP, pET-PBAD-YFP, pNZ8148-P23-RFP, and pSIP403-Pspp-RFP, as shown in Fig. 1, and introduce the yellow fluorescent plasmid into EcN, and introduce the red fluorescent plasmid into Lp, by observing the fluorescence of the bacteriophage to characterise the coexistence of the two colonies.

![Plasmid construction for dual bacterial coexistence plates](https://static.igem.wiki/teams/5724/engineering/engineering-fig-41.avif)

Fig.41 Plasmid construction for dual bacterial coexistence plates (A) Plasmid pET-J23119-YFP, (B) lasmid pET-PBAD-YFP, (C) Plasmid pNZ8148-P23-RFP, (D) Plasmid pSIP403-Pspp-RFP

#### Build (B):

The fluorescent protein gene was constructed into the commonly used plasmid by seamless cloning, and the resulting recombinant plasmid was transformed into EcN and Lp. Finally, the engineered strains containing the correct recombinant plasmids were successfully obtained by preliminary screening with the corresponding resistance plates and further verifying the positive clones by colony PCR.

#### Test (T):

We tested the fluorescence intensity of the constitutive fluorescent protein-expressing strains, and also tested the fluorescence intensity of the inducible fluorescent protein-expressing strains under different culture conditions. The fluorescence intensity of EcN, which constitutively expresses yellow fluorescent protein, was significant, and the fluorescence intensity of inducible expression was not as significant as that of the constitutive type; the fluorescence intensity of Lp, which constitutively expresses red fluorescent protein, was significant, but not as significant as that of the inducible type, and the fluorescence intensity was not as significant as that of EcN due to the fact that Lp is a gram-positive bacterium with a thicker cell wall. In addition, we also came up with suitable culture conditions for our experiments, and at the same time, we mapped out the suitable medium conditions for co-culturing EcN and Lp, and adopted them in our subsequent experiments.

#### Learn (L):

We tested the expression intensity of a number of fluorescent protein-related components in the iGEM kit and, in the course of our experiments, discovered the influence of cell wall thickness in Gram-positive bacteria on the observation of the fluorescence intensity of their fluorescent proteins.

### Cycle 2

#### Design (D):

In order to quantitatively analyse the subpopulations of bacteria expressing different fluorescent proteins in the co-culture system, we planned to use flow cytometry. By performing dual-channel detection of yellow fluorescence and red fluorescence on the bacterial population, we can accurately count and characterise the number and proportion of YFP- and RFP-expressing bacteria in the population at different times of the co-culture.

#### Build (B):

For initial co-culture pre-experiments, we explicitly set up the following three groups: blank control (unengineered wild-type strains), single-positive control (strains expressing only a single fluorescent protein, respectively), and experimental group (co-culture system).

#### Test (T):

However, preliminary flow cytometry results (Fig. 2) showed poor fluorescence fractionation of cell populations. The problem was mainly attributed to two technical limitations: first, the emission spectral range of the selected EYFP protein was too broad, which led to its severe spectral crosstalk among detection channels; second, the flow cytometer was equipped with a laser whose excitation wavelength did not exactly match with the optimal absorption wavelength of the mRFP protein that we used, which led to the low excitation efficiency.

![Presentation of some experimental results of flow cytometry](https://static.igem.wiki/teams/5724/engineering/engineering-fig-42.avif)

Fig.42 Presentation of some experimental results of flow cytometry

#### Learn (L):

In response to the fluorescence detection problems in the pre-experiment, we reviewed and optimised the experimental design. Firstly, we decided to use a protein with a narrower emission spectrum and a better match with the laser wavelength of the instrument in order to minimise spectral crosstalk and improve excitation efficiency. Based on this, we chose surperfolder GFP (sfGFP), which has better spectral characteristics, as an alternative, and plan to supplement it with more intuitive characterisation methods for validation.

### Cycle 3

#### Design (D):

In the next step, we will construct the pJUMP-J23100-sfGFP plasmid (see Figure 3) and transform EcN, and switch to fluorescence microscopy to characterise the spatio-temporal dynamics of the bacterial population in the co-culture system by direct observation of the two-colour fluorescence distribution.

![Plasmid pJUMP-J23100-GFP](https://static.igem.wiki/teams/5724/engineering/engineering-fig-43.avif)

Figure.43 Plasmid pJUMP-J23100-GFP

#### Build (B):

The GFP fluorescent protein gene was constructed into a common plasmid by seamless cloning, and the resulting recombinant plasmid was transformed into EcN. Finally, the engineered strain containing the correct recombinant plasmid was successfully obtained by initial screening through canna-resistant plates and further verification of the positive clones by colony PCR.

#### Test (T):

After the activation of the correctly validated engineering strains, they were transferred to 20 mL of LB medium at an inoculum of 1:100, and the fermentation culture was carried out at 37°C and 220 rpm. To monitor the growth of the bacteria, samples were taken at regular intervals to determine the OD600 value; meanwhile, samples were taken periodically at predetermined time points during the incubation process. The obtained samples were washed with PBS buffer, resuspended and fixed on slides for fluorescence microscopy. Detailed experimental results are shown in Results.

#### Learn (L):

Based on the experimental results, we confirmed that sfGFP and mRFP are suitable for the two-colour fluorescent labelling system in this study. In addition, EcN and Lp were confirmed to be able to coexist stably for a long period of time in the specific medium obtained from the exploration, which meets the basic requirements for co-culture experiments.

{% endblock %}

{% extends "layout.html" %}
{% 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 %}

## What Should this Page Contain?

---

Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

{% 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 %}

![Results Header Image](https://static.igem.wiki/teams/5724/headphoto/results.webp)

## Contents

* [Adhesive Module Results](#adhesive-module-results)
  + [Identification of Lp\_0018 as Optimal OppA Variant](#identification-oppa)
  + [Inducer Optimization for Adhesion Efficiency](#inducer-optimization)
  + [Validation of Orthogonality in Adhesin Pairs](#orthogonality-validation)
  + [Intraspecies and Interspecies Adhesion Assays](#adhesion-assays)
* [Control Module Results](#control-module-results)
  + [AHL System Results](#ahl-system-results)
  + [AIP System Results](#aip-system-results)
  + [Application Results](#application-results)
* [Therapeutic Module Results](#therapeutic-module-results)
  + [L-dopa Production Module](#l-dopa-production)
  + [Glutathione Production Module](#glutathione-production)
* [Safety Module Results](#safety-module-results)
  + [Verification of Hypoxia-Responsive Suicide Module](#hypoxia-verification)
  + [Validation of Inducible Suicide Modules](#inducible-validation)
  + [Evaluation of Temperature-Sensitive Suicide System](#temperature-evaluation)
* [Co-culture of Two Bacteria](#co-culture-two-bacteria)

## Adhesion Module Results

### 1. Identification and Validation of Lp\_0018 as the Optimal OppA Variant

The heatmap displays the docking energies (ΔG) of seven Lactobacillus plantarum OppA variants with candidate host ligands. Darker blue indicates stronger binding affinity. (B) Bubble chart summarizing the docking energies and docking scores between mucin and the seven OppA proteins, where circle size represents binding strength and color indicates different OppA variants. As shown in panel (A), among the tested variants, Lp\_0018 exhibited the most favorable binding profile with NaHS and was therefore selected for subsequent experimental validation. Panel (B) reveals potential binding between mucin (present in the intestinal environment) and OppA, further validating the necessity of incorporating a hypoxia-responsive suicide element.

![Docking energies heatmap and bubble plot](https://static.igem.wiki/teams/5724/results/result1.webp)

Fig.1 Docking energies heatmap and bubble plot

(A--G) Representative binding conformations of NaHS with individual OppA proteins (A: Lp\_0018; B: Lp\_0020; C: Lp\_0783; D: Lp\_0092; E: Lp\_0201; F: Lp\_3686; G: Lp\_1261). NaHS molecules are shown in red, OppA binding residues in green. Hydrogen bonds and key interactions are indicated.

![Binding conformations of NaHS with OppA proteins](https://static.igem.wiki/teams/5724/results/result2.webp)

Fig.2 Visualization of the optimal binding results between NaHS and OppA proteins

We constructed an overexpression plasmid and expressed Lp\_0018 in L. plantarum WCFS1. SDS-PAGE analysis revealed a distinct protein band at the expected molecular weight (highlighted in Fig. X), confirming successful expression of Lp\_0018.

![SDS-PAGE analysis of Lp_0018 expression](https://static.igem.wiki/teams/5724/results/result3.webp)

Fig.3 SDS-PAGE analysis of Lp\_0018 expression

### 2. Inducer Optimization for Adhesion Efficiency

To ensure robust expression of the adhesion modules, we first evaluated the effect of inducer concentration on cell--cell adhesion. Overnight cultures of *Escherichia coli* Nissle 1917 (EcN) harboring Nb3 and Ag3 adhesion pathways were exposed to a gradient of IPTG concentrations, with non-induced samples serving as controls. After 24 h of induction, OD600 values of supernatants were recorded to quantify aggregation. The titration assay demonstrated that adhesion efficiency increased with IPTG concentration up to a plateau. The optimal working concentration was determined to be 0.8 mM, which was subsequently applied in all downstream experiments. This result highlights the necessity of precise inducer tuning to balance gene expression and cellular fitness.

![IPTG titration assay for adhesion efficiency](https://static.igem.wiki/teams/5724/results/1-syq.avif)

Fig.4 IPTG titration assay for adhesion efficiency

### 3. Validation of Orthogonality in Adhesin Pairs

To rigorously evaluate the orthogonality of engineered adhesins, we designed a three-step validation strategy.

(i) Quantitative co-culture assay. We first co-cultured EcN strains carrying Nb3 and Ag3, as well as Nb2 and Ag2 adhesion plasmids, under induced and non-induced conditions. After 24 h of induction, OD600 measurements of the supernatants revealed a significant increase in aggregation in the induced groups. This result demonstrated the successful establishment of specific adhesion interactions at the population level.

![Quantitative co-culture assay](https://static.igem.wiki/teams/5724/results/result5.webp)

Fig.5 Quantitative co-culture assay

(ii) Fluorescence microscopy of Nb3--Ag3 interaction. To directly visualize orthogonal adhesion, we introduced fluorescence reporter plasmids (pJUMP-RFP into Nb3 cells and pJUMP-sfGFP into Ag3 cells). Fluorescence microscopy confirmed that red Nb3 cells adhered specifically to green Ag3 cells upon induction, forming distinct aggregates. In contrast, control groups without induction exhibited minimal clustering.

![Fluorescence microscopy of Nb3--Ag3 interaction](https://static.igem.wiki/teams/5724/results/result6.webp)

Fig.6 Fluorescence microscopy of Nb3--Ag3 interaction

(fig.X Fluorescence microscopy images show EcN cells labeled in red and green. (I) Without inducer, EcN cells were mostly dispersed, with limited cell--cell contact. (J) With inducer, EcN cells exhibited clear clustering and close contacts, indicating successful self-adhesion upon induction. Black boxes highlight representative adhesion events.)

(iii) Construction of an orthogonality matrix. Finally, we extended the validation by mixing strains carrying different adhesins (Nb3, Ag3, Nb2, Ag2) without fluorescent markers. Co-culture experiments showed preferential adhesion within cognate pairs (Nb3--Ag3, Nb2--Ag2), while non-cognate combinations displayed negligible interaction. The orthogonality matrix derived from these assays highlighted the pairwise specificity of adhesins, confirming the modular and programmable nature of the system.

![Orthogonality matrix](https://static.igem.wiki/teams/5724/results/result7.webp)

Fig.7 Orthogonality matrix

### 4. Intraspecies and Interspecies Adhesion Assays

We next investigated adhesion efficiency both within EcN strains and between EcN and Lactobacillus plantarum WCFS1.

(i) Quantitative co-culture of EcN and WCFS1. For interspecies assays, Nb2-expressing EcN strains (without fluorescence reporters) were co-cultured with WCFS1. OD600-based quantification revealed that induced groups exhibited significantly higher aggregation compared with non-induced controls, confirming functional interspecies adhesion.

![Quantitative co-culture of EcN and WCFS1](https://static.igem.wiki/teams/5724/results/result8.webp)

Fig.8 Quantitative co-culture of EcN and WCFS1

(ii) Fluorescence microscopy of Nb2--WCFS1 interaction. To visualize the adhesion events, we further constructed a red-fluorescent Nb2--EcN strain and co-cultured it with non-fluorescent WCFS1. Fluorescence microscopy images revealed that induced EcN cells adhered tightly to WCFS1 surfaces, while controls showed dispersed cell distribution.

![Fluorescence microscopy of Nb2--WCFS1 interaction](https://static.igem.wiki/teams/5724/results/result9.webp)

Fig.9 Fluorescence microscopy of Nb2--WCFS1 interaction

5. A single diagram summarising our adhesive plate

![Summary diagram of adhesive plate](https://static.igem.wiki/teams/5724/results/6-syq.avif)

Fig.10 Summary diagram of adhesive plate

(A) Representative fluorescence microscopy image of Ag3--Nb3 aggregates. Red cells (Nb3) express RFP and green cells (Ag3) express GFP. (B) Quantification of cell--cell adhesion shown in (A). Significant differences were observed between induced and non-induced groups (\*p < 0.0001). (C) Orthogonality matrix derived from (B), showing the probability that cells of one color (row) adhere to cells of another color (column). (D) Representative fluorescence microscopy images of interspecies adhesion between Lactobacillus plantarum and *Escherichia coli*. Red Nb2 cells adhered to L. plantarum cells. (E) Quantification of interspecies adhesion shown in (D). The JC group (joint cultivation) displayed significant differences between induced and non-induced conditions (\*p < 0.0001). (F, G) Macroscopic views of Ag2--Nb2 aggregates. (F) Aggregation between EcN strains; (G) aggregation between EcN and L. plantarum WCFS1. (H) IPTG titration curve determining the optimal inducer concentration. The optimal working concentration was identified as 0.8 mM.

## Control Module Results

### 1. AHL system Results

![AHL system results](https://static.igem.wiki/teams/5724/results/result11.webp)

Fig.11 AHL system results

(A) Uninduced blank control. Cells without inducer addition showed negligible fluorescence, confirming the absence of leaky expression.

(B) Endogenously induced samples. Cells harboring the endogenous Lux promoter exhibited faint but detectable red fluorescence, suggesting a low basal promoter activity that enabled limited expression under native conditions. The weak fluorescence and high background are likely due to the intrinsic low strength of the Lux promoter and the contribution of cellular autofluorescence and uneven illumination.

(C) Exogenously induced samples. Upon addition of external inducers, a strong and uniform red fluorescence signal was observed, demonstrating effective activation of the promoter and successful protein expression.

(D) Quantitative fluorescence analysis. Fluorescence intensity was quantified using a microplate reader. The blank showed negligible signal, indicating minimal leakiness. Endogenous induction yielded a weak increase (10.2±1.2-fold vs. blank, CV=5.46%), whereas exogenous induction produced a strong and uniform response (81.1±8.6-fold vs. blank; CV=1.4%).

(E) Lux promoter activity curve. Time‐ and concentration‐dependent induction profiles of the Lux promoter were measured. The fluorescence intensity increased in proportion to inducer concentration and incubation time, indicating a dose--response relationship and confirming the dynamic regulatory capacity of the Lux system.

### 2. AIP system Results

![AIP system results](https://static.igem.wiki/teams/5724/results/result12.webp)

Fig.12 AIP system results

(A) Uninduced blank control. Cells without inducer addition exhibited negligible background fluorescence, confirming the absence of leaky expression from the PsppA promoter.

(B) Endogenously induced samples. Cells under endogenous induction showed weak but detectable red fluorescence, indicating that the native regulatory elements of PsppA possess basal activity even without exogenous stimulation. The relatively low fluorescence intensity and high background noise may result from weak promoter strength and cellular autofluorescence.

(C) Exogenously induced samples. Upon addition of external inducers, cells displayed a strong and homogeneous red fluorescence signal, suggesting that PsppA is highly responsive to exogenous stimuli and capable of robust activation.

(D) Quantitative fluorescence measurement. The fluorescence intensity of each group was quantified. The blank showed negligible signal, indicating minimal leakiness. Endogenous induction yielded a weak increase (18.6±5.8-fold vs. blank, CV = 31.4%), whereas exogenous induction produced a strong and uniform response (54.1 ± 3.5-fold vs. blank, CV = 6.5%).

(E) Time‐dependent induction curve of PsppA promoter activity. Fluorescence intensity was recorded over time following induction. PsppA reached half-maximal activity at ~1 h (t₅₀ = 1.00 h) and approached a stable plateau by 2--3 h (mean ≈ 0.94 RPU, normalized to P23). The maximal response occurred at 3 h (Y\_max = 0.97 RPU; dynamic range = 0.87 RPU), after which the fluorescence signal declined slightly at ~0.11 RPU·h⁻¹.

(F) Concentration‐dependent induction curve of PsppA promoter activity. Fluorescence increased in a dose‐dependent manner with inducer concentration, reaching saturation at higher levels, consistent with a sigmoidal regulatory behavior characteristic of inducible promoter systems.

### 3. Application Results

![Application results](https://static.igem.wiki/teams/5724/results/result13.webp)

Fig.13 Application results

(A) Schematic of bidirectional regulation between Lactiplantibacillus plantarum and *Escherichia coli*. The left panel illustrates L. plantarum--mediated control of the *E. coli* therapeutic production module; the right panel shows *E. coli*--mediated regulation of L. plantarum adhesion/function.

(B) SDS--PAGE validation in L. plantarum carrying pSIP403-Pspp-RFP. Cells were induced and harvested after 6 h; whole-cell lysates were analyzed by SDS--PAGE to confirm induction-dependent expression.

(C) Melanin production assay in *E. coli* carrying pET-J23100-LuxR-Plux-HpaBC. After 24 h of induction, pigment formation was quantified by absorbance at OD400; PC denotes the positive control.

(D) Visual phenotype of *E. coli* cultures after 24 h induction. Representative color changes across experimental groups following induction of pET-J23100-LuxR-Plux-HpaBC.

## Therapeutic Module Results

### 1. L-dopa production module

This experiment aimed to achieve two objectives: first, to evaluate the influence of the exogenous plasmid and inducer on *E. coli* growth by comparing the growth curves among the three groups; and second, to confirm the successful construction and functional expression of the production module by measuring L-DOPA yield over time in the induced group.

Analysis of the growth curves (Figure 1) revealed that neither plasmid carriage nor IPTG induction significantly affected the growth of the engineered *E. coli* strain.

![Growth curves analysis](https://static.igem.wiki/teams/5724/results/4-syq.avif)

Fig.14 Growth curves analysis

The yield-time curve plotted based on HPLC data (Link 1) (as shown in Figure 2) reveals that the maximum concentration of levodopa in the sampled supernatant reached 36,000 ng/mL. Subsequently, as substantial amounts of levodopa were oxidised into melanin, its concentration in the sample supernatant progressively decreased.

![L-dopa yield-time curve](https://static.igem.wiki/teams/5724/results/3-syq.avif)

Fig.15 L-dopa yield-time curve

To quantitatively assess the degree of oxidation of levodopa during fermentation, we measured the absorbance of the fermentation supernatant at 400 nm based on literature reports. One OD400 unit corresponds to 0.066 g/L melanin. We calculated its approximate concentration, with specific data and corresponding levodopa content shown in Figure 3. Based on this, we further estimated the total amount of oxidised levodopa.

![Oxidation assessment of levodopa](https://static.igem.wiki/teams/5724/results/4-syq.avif)

Fig.16 Oxidation assessment of levodopa

### 2. Glutathione production module

Analysis of the growth curve (as shown in Figure 4) indicates that the carriage of the exogenous plasmid, the addition of inducers, or the supplementation of amino acids did not exert a significant effect on the growth of the engineered *Escherichia coli*.

![Growth curve analysis for glutathione production](https://static.igem.wiki/teams/5724/results/result17.webp)

Fig.17 Growth curve analysis for glutathione production

The yield-time curve plotted based on HPLC data (Link 2) (as shown in Figure 5) reveals that the maximum concentration of reduced glutathione in the sampled supernatant reached 252 ng/mL. Over time, GSH was progressively oxidised to GSSG, with GSH levels gradually decreasing and GSSG levels gradually increasing.

![Glutathione yield-time curve](https://static.igem.wiki/teams/5724/results/result18.webp)

Fig.18 Glutathione yield-time curve

## Safety Module Results

### 1. Verification of the Hypoxia-Responsive Suicide Module

We first verified the function of the hypoxia-responsive suicide module. As shown in Figure A, compared with aerobic culture, the number of colonies formed under hypoxic conditions after 48 h was dramatically reduced, indicating that the constructed hypoxia-responsive suicide element was effective and could significantly inhibit the growth of *Escherichia coli*.

### 2. Validation of the Inducible Suicide Modules (Arabinose- and Nisin-Inducible Systems)

We evaluated the inducible suicide modules. As shown in Figure C, compared with the control group, both the engineered *E. coli* carrying the arabinose-inducible suicide system and the engineered Lactobacillus plantarum harboring the nisin-inducible suicide system exhibited markedly slower OD₆₀₀ increases after the addition of arabinose and nisin, respectively. These results demonstrate that both engineered strains responded effectively to the corresponding inducers.

### 3. Evaluation of the Temperature-Sensitive Suicide System

Finally, we assessed the temperature-sensitive suicide system. We first examined the effectiveness of the temperature-responsive protein and the downstream promoter. As shown in Figure D, the engineered *E. coli* harboring the single-plasmid system showed no significant difference in specific fluorescence intensity after preculturing at 30 °C and 37 °C for 18 h followed by 24 h of main culture, indicating that the single-plasmid temperature-sensitive system exhibited poor performance. In contrast, the *E. coli* carrying the dual-plasmid system displayed a markedly higher specific fluorescence intensity at 30 °C than at 37 °C under the same conditions, suggesting that the dual-plasmid construct possessed stronger temperature sensitivity. Subsequently, we replaced the red fluorescent protein in the dual-plasmid temperature-sensitive--fluorescence system with the toxin gene mazF. As shown in Figure B, the engineered *E. coli* exhibited a slightly reduced colony number after static incubation at 37 °C for 24 h compared with 30 °C, suggesting a modest temperature-dependent effect.

![Validation of synthetic safety modules](https://static.igem.wiki/teams/5724/results/result19.webp)

Fig.19 Validation of synthetic safety modules

**Figure 19. Validation of the synthetic safety modules.**

(A) Colony growth of *E. coli* harboring plasmid pET-phyb-MazF after 48 h of incubation under aerobic (top row) and hypoxic (bottom row) conditions at 37 °C. The drastic reduction of colonies under hypoxia demonstrates the effectiveness of the hypoxia-responsive suicide element.

(B) Colony morphology of *E. coli* carrying pETDuet-pRM-CI857 and pJUMP-pR-phIF-pPhIF-MazF after 24 h of static incubation at 30 °C (top) and 37 °C (bottom). The slightly lower colony number at 37 °C suggests a modest temperature-dependent activation of the dual-plasmid thermo-responsive suicide system.

(C) Growth curves of engineered bacteria carrying inducible suicide modules. Top: *E. coli* harboring pBAD-MazF cultured with or without arabinose induction. Bottom: Lactobacillus plantarum containing Pnis-MazF cultured with or without nisin induction. Both strains showed attenuated growth upon inducer addition, confirming successful activation of the inducible suicide circuits.

(D) Specific fluorescence intensity of *E. coli* strains transformed with temperature-responsive red-fluorescent protein reporters. Top: single-plasmid construct (pETDuet-J23110-Cl857-pR-phIF-pPhIF-RFP); Bottom: dual-plasmid construct (pETDuet-pRM-Cl857 + pJUMP-pR-phIF-pPhIF-RFP). The dual-plasmid system exhibited markedly higher fluorescence at 30 °C than 37 °C, indicating improved temperature sensitivity.

## Co-culture of Two Bacteria

To investigate the co-culture conditions of EcN and Lp, we explicitly established the following three experimental systems (detailed experimental design is provided in the Engineering section): (1) EcN monoculture expressing sfGFP; (2) Lp monoculture expressing mRFP; (3) a co-culture of the two strains mixed in a 1:1 ratio. This study pursued two objectives: firstly, to quantitatively analyse the growth kinetics of both strains under separate and coexisting conditions by comparing growth curves across the three groups; secondly, to elucidate the spatial distribution and relative density of the two strains within the co-culture system via fluorescence microscopy.

Analysis of the growth curves revealed that the growth trends in the single-culture and co-culture systems were largely overlapping. This indicates that, under the experimental conditions employed, co-culture did not significantly inhibit the growth of either strain.

![Growth curves of EcN and Lp in monoculture and co-culture](https://static.igem.wiki/teams/5724/results/result20.webp)

Fig.20 Growth curves of EcN and Lp in monoculture and co-culture

To further validate the coexistence capability of EcN and Lp, we conducted cell counting analysis on fluorescence microscopy images from three experimental systems. By quantifying cell numbers in both single-strain cultures and co-culture systems, we calculated and compared their relative proportions within the mixed culture. Statistical results indicate that under co-culture conditions, both strains maintained high cell densities, with their proportions largely consistent with the initial inoculation ratios. This quantitative data confirms that EcN and Lp can stably coexist and grow well within the established co-culture system.

![Fluorescence microscopy images of EcN-Lp co-culture](https://static.igem.wiki/teams/5724/results/result21.webp)

Fig.21 Representative fluorescence microscopy images of experimental system 3 (EcN-Lp co-culture) at 4 h, 6 h, 8 h, and 10 h post-incubation

A. 4 h post-incubation; B. 6 h post-incubation; C. 8 h post-incubation; D. 10 h post-incubation.

![Statistical diagram of EcN-to-Lp ratio](https://static.igem.wiki/teams/5724/results/result22.webp)

Fig.22 Statistical diagram of the EcN-to-Lp ratio in Experimental System 3 (EcN-Lp co-culture) at 4, 6, 8, and 10 hours of incubation.

{% endblock %}