iGEM 2025 — Team gec-guangzhou

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

Content

* [System 1: Construction of the AHL Biosensor Engineered Bacteria and Performance Testing of AHL Biosensors - 34 pages](#section-1)
  + [Detailed Daily Experiment Plan (June 11, 2025 - July 1, 2025)](#subsection-2)
  + [Lab notebook - system1（Total 34 pages）](#subsection-3)
* [System 2:Construction and Functional Validation of an AHL-Responsive Indigo Chromogenic Bacterial Strain - 48 pages](#section-4)
  + [Detailed Daily Experiment Plan (July 2, 2025 - July 20, 2025)](#subsection-5)
  + [Lab notebook - system2（Total 48 pages）](#subsection-6)
* [System 3:Construction of the Salicylic Acid Overexpression Engineered Strain and Production Experiment - 32 pages](#section-7)
  + [Detailed Daily Experiment Plan (July 24, 2025 - August 18, 2025)](#subsection-8)
  + [Lab notebook - system3（Total 32 pages）](#subsection-9)

# System 1: Construction of the AHL Biosensor Engineered Bacteria and Performance Testing of AHL Biosensors - 34 pages

This experiment aims to construct and validate a genetically engineered E. coli strain to function as a biosensor for detecting acyl-homoserine lactone (AHL), a key signaling molecule of the Ralstonia solanacearum pathogen. The construction first involves using molecular cloning techniques to assemble components—specifically, the AHL receptor protein QscR and its responsive promoter PQscR from Pseudomonas aeruginosa PAO1, coupled with elements controlling the reporter gene mRFP—into the pSB1A3 vector. The entire project, spanning 21 days, comprises four main stages: construction, verification, functional testing, and data analysis of the engineered strain.

## Detailed Daily Experiment Plan (June 11, 2025 - July 1, 2025)

The table below provides a summary of the daily plan, followed by subsequent daily protocols containing detailed steps, results, and chart locations.

| Date | Experiment Title | Key Steps | Experimental Record (Summary) |
| --- | --- | --- | --- |
| 2025-06-11 | Engineering Strain Construction Initiation: Cloning Preparation | Sequence verification, restriction digest of vector & insert (XbaI/SpeI), gel purification, ligation reaction setup. | Restriction digest gel electrophoresis showed correct bands; ligation reaction proceeded overnight. |
| 2025-06-12 | Transformation and Plating | Transformation of ligation product into *E. coli* BL21(DE3) via heat shock, plating on Amp+ LB plates, incubation at 37°C overnight. | Single, isolated colonies appeared on plates. |
| 2025-06-13 | Colony Picking and Pre-culture | Picking 5-10 single colonies, inoculating into LB/Amp+ liquid medium, culturing to mid-log phase. | Culture turned turbid; OD600 reached 0.6-0.8. |
| 2025-06-14 | Colony PCR Verification and Sequencing Submission | Performing colony PCR using culture as template, identifying positive clones via gel electrophoresis, sending positive clone cultures for sequencing. | PCR gel showed target bands; selected 2 clones for sequencing. |
| 2025-06-15 | AHL Stock Solution Preparation | Weighing 3OC12-HSL, C10-HSL, 3OHC10-HSL, dissolving in DMSO to prepare 10 mM stock solutions. | All AHL stock solutions were clear; aliquoted and stored at -20°C. |
| 2025-06-16 | Waiting and Preparation | Awaiting sequencing results; preparing media, antibiotics, and utensils needed for testing. | All testing materials prepared and ready. |
| 2025-06-17 | Sequencing Verification and Glycerol Stock Preparation | Analyzing sequencing results, confirming correct clone, performing larger-scale culture, and preparing glycerol stock for cryopreservation. | Sequencing alignment was correct; successfully prepared engineering strain glycerol stock. |
| 2025-06-18 | AHL Biosensor Test: Single Concentration Activation | Activating engineering strain, treating with 10 µM different AHLs for 3 hours, measuring fluorescence and OD600. | Engineering strain showed clear fluorescent response to 3OC12-HSL. |
| 2025-06-19 | Dose-Response Test: Low Concentration Range | Testing 0.001 µM, 0.01 µM, 0.1 µM 3OC12-HSL, measuring fluorescence and OD600. | Observed trend of increasing fluorescence signal with concentration. |
| 2025-06-20 | Dose-Response Test: High Concentration Range | Testing 1 µM, 10 µM 3OC12-HSL, measuring fluorescence and OD600. | Fluorescence signal saturated at 10 µM. |
| 2025-06-21 | Specificity Test: C10-HSL | Testing 0.001 - 10 µM C10-HSL, measuring fluorescence and OD600. | Engineering strain showed weak or no response to C10-HSL. |
| 2025-06-22 | Specificity Test: 3OHC10-HSL | Testing 0.001 - 10 µM 3OHC10-HSL, measuring fluorescence and OD600. | Engineering strain showed no significant response to 3OHC10-HSL. |
| 2025-06-23 | Data Repeats and Gap Filling | Repeating any anomalous data points to ensure at least 3 biological replicates per data point. | Key data points were well reproduced. |
| 2025-06-24 | Data Compilation and Normalization Calculations | Organizing all raw data, subtracting background, calculating normalized fluorescence values (Fluorescence/OD600). | Obtained a complete, clean normalized dataset. |
| 2025-06-25 | Graph Plotting I: Dose-Response Curve | Plotting dose-response curve for 3OC12-HSL (semi-log plot), calculating approximate EC50 value. | Generated clear dose-response curve. |
| 2025-06-26 | Graph Plotting II: Specificity Comparison Chart | Plotting bar chart comparing normalized fluorescence of the three AHLs at 10 µM. | Generated intuitive specificity comparison chart. |
| 2025-06-27 | Statistical Analysis | Performing significance analysis on key data (e.g., t-test). | Confirmed the response to 3OC12-HSL was statistically significant. |
| 2025-06-28 | Lab Notebook & Report Writing I | Organizing all experimental records; drafting the Materials & Methods and Results sections. | Completed the majority of the initial draft. |
| 2025-06-29 | Lab Notebook & Report Writing II | Analyzing experimental results. | Completed the initial report draft. |
| 2025-06-30 | Lab Notebook & Report Writing III | Completing the Discussion and Conclusion sections; integrating all charts and graphs; performing final proofreading. | Formed a complete draft of the experimental report. |
| 2025-07-01 | Project Summary and Archiving | Final data backup; archiving strains and plasmids; lab cleanup; project summary. | All materials archived; project concluded successfully. |

## Lab notebook - system1（Total 34 pages）

Lab Notebook——system1.pdf

1.6MB

[Download](https://static.igem.wiki/teams/5877/notebook/lab-notebook-system1.pdf)

# System 2:Construction and Functional Validation of an AHL-Responsive Indigo Chromogenic Bacterial Strain - 48 pages

This experiment aims to construct an engineered E. coli strain capable of producing visible indigo pigment in response to the key signaling molecule of Ralstonia solanacearum - Acyl-Homoserine Lactone (AHL). Based on the previously constructed AHL biosensor, the reporter gene mRFP is replaced with a polycistronic unit for the indigo synthesis pathway (TnaA-B0034-FMO). The entire project runs from July 2 to July 20, 2025, spanning 19 days.

## Detailed Daily Experiment Plan (July 2, 2025 - July 20, 2025)

| Date | Experiment Title | Key Steps | Experimental Record (Summary) |
| --- | --- | --- | --- |
| 2025-07-02 | Engineering Strain Construction: Cloning Preparation | Sequence verification; XbaI/SpeI double digestion of pSB1A3 vector & synthesized fragment (with TnaA-FMO); Gel purification; Ligation reaction setup. | Digestion successful, ligation reaction established. |
| 2025-07-03 | Transformation and Plating | Heat-shock transformation of ligation products; Plating on Amp+ LB plates; Overnight incubation at 37°C. | Single colonies on experimental plate, no colonies on control plate. |
| 2025-07-04 | Clone Selection and Pre-culture | Picking 12 single colonies, inoculating into LB/Amp+ liquid medium, culturing to OD600=0.6-0.8. | Pre-culture of 12 clones completed, cultures turbid. |
| 2025-07-05 | Colony PCR Verification | PCR using pre-culture as template, agarose gel electrophoresis identification of positive clones. | X/X clones show correct target band. |
| 2025-07-06 | Plasmid Extraction & Sequencing Submission | Plasmid extraction from positive clones; Submission to sequencing company for verification. | Plasmid extraction successful, samples shipped. |
| 2025-07-07 | Sequencing Verification & Glycerol Stock Preparation | Analysis of sequencing results, confirmation of correct clone; Preparation and storage of glycerol stock BL21-HMB at -80°C. | Sequencing error-free, glycerol stock BL21-HMB archived. |
| 2025-07-08 | Indigo Standard Curve Preparation | Preparation of indigo standard solutions at different concentrations; OD620 measurement; Standard curve plotting. | Indigo standard curve established, R² > 0.99. |
| 2025-07-09 | Time-Course Experiment Pilot Run & Medium Optimization | Activation of BL21-HMB; Testing growth and basal indigo production in M9 medium; Optimizing experimental workflow. | Strain grows well in M9 with low background, workflow feasible. |
| 2025-07-10 | Time-Course Experiment: Main Experiment Preparation | Preparation of bulk sterile M9 induction medium, AHL working solutions, and all consumables for multi-timepoint sampling. | All reagents and consumables prepared and ready. |
| 2025-07-11 | Time-Course Experiment: Execution & Sampling (Day 1/Rep1) | Activation and washing of BL21-HMB; Resuspension in M9 induction medium; Addition of 1µM AHLs; Sampling at 0, 2, 4, 6, 8 hours; Centrifugation and supernatant collection. | Completed all timepoint samplings for the first biological replicate. |
| 2025-07-12 | Time-Course Experiment: Execution & Sampling (Day 2/Rep2) | Repeat experiment, performing induction and sampling for the second biological replicate. | Completed all timepoint samplings for the second biological replicate. |
| 2025-07-13 | Time-Course Experiment: Execution & Sampling (Day 3/Rep3) | Repeat experiment, performing induction and sampling for the third biological replicate. | Completed all timepoint samplings for the third biological replicate. |
| 2025-07-14 | Supernatant OD620 Measurement & Data Compilation | Measurement of OD620 for all collected supernatant samples using a plate reader. | OD620 values measured for all samples. |
| 2025-07-15 | Data Analysis I: Kinetic Curve Plotting | Conversion of OD620 data to indigo concentration; Plotting time-course curves of indigo production under induction by three AHLs. | Preliminary kinetic curves generated, showing specific response to 3OC12-HSL. |
| 2025-07-16 | Data Analysis II: Statistical Analysis | Calculation of mean and standard deviation for each group; Performing statistical significance tests (e.g., ANOVA). | Significant difference found between 3OC12-HSL group and controls/other AHL groups. |
| 2025-07-17 | Graph Refinement and Integration | Refining all graphs and integrating them into final report figures. | Final, publication-ready figures generated. |
| 2025-07-18 | Report Drafting I: Introduction & Methods | Writing the project introduction/background and detailed experimental methods section. | Draft of Introduction and Methods sections completed. |
| 2025-07-19 | Report Drafting II: Results & Discussion | Writing the Results section incorporating figures; In-depth analysis and discussion of the results. | Draft of Results and Discussion sections completed. |
| 2025-07-20 | Project Finalization & Archiving | Final review of report; Backing up all data; Archiving strains and experimental records. | Project completed successfully, all materials archived. |

## Lab notebook - system2（Total 48 pages）

Lab Notebook——system2.pdf

2.1MB

[Download](https://static.igem.wiki/teams/5877/notebook/lab-notebook-system2.pdf)

# System 3:Construction of the Salicylic Acid Overexpression Engineered Strain and Production Experiment - 32 pages

This experiment involved constructing an engineered E. coli strain for enhanced salicylic acid production. The salicylic acid synthesis genes (ICS and IPL) were cloned into a pET28a vector, transformed into E. coli BL21(DE3), and verified to create the BL21-ICS-IPL strain. For production testing, the engineered strain and a control strain were cultured in M9Y medium with IPTG induction. Over 24 hours, samples were collected every 4 hours to measure cell density (OD₆₀₀) and salicylic acid concentration using ELISA. The results demonstrated that the engineered BL21-ICS-IPL strain successfully produced significantly higher levels of salicylic acid compared to the control.

## Detailed Daily Experiment Plan (July 24, 2025 - August 18, 2025)

| ate | Experiment Title | Key Steps | Experimental Record (Summary) |
| --- | --- | --- | --- |
| 2025-07-24 | Plasmid Construction | Digest, Ligate | Completed double digestion and ligation of vector and insert. |
| 2025-07-25 | Transformation | Heat-shock, Plate | Transformed ligation product into competent cells and plated. |
| 2025-07-26 | Clone Screening | Pick colonies, Colony PCR | Picked single colonies and performed colony PCR screening. |
| 2025-07-27 | Sequencing | Submit samples, Prepare media | Sent positive clones for sequencing. Prepared production media. |
| 2025-07-28 | Awaiting Results | Data organization | Organized data while awaiting sequencing results. |
| 2025-07-29 | Strain Storage | Analyze sequence, Make glycerol stocks | Sequence confirmed. Prepared glycerol stocks of BL21-HMB and control strains. |
| 2025-07-30 | Production - Activation | Overnight culture | Activated seed cultures of BL21-HMB and control strains. |
| 2025-07-31 | Production - Induction/Sampling | Induce, Sample every 4h | Induced with IPTG. Sampled every 4 hours for 24 hours. |
| 2025-08-01 | ELISA Assay | Prepare standard curve, Test samples | Measured salicylic acid concentrations in all samples using ELISA kit. |
| 2025-08-02 | Data Analysis | Calculate concentrations, Plot data | Calculated sample concentrations from standard curve and generated plots. |
| 2025-08-03 | Reporting | Analyze results, Write report | Analyzed data and drafted experimental report. |
| 2025-08-04 to 2025-08-18 | Project Wrap-up | Final review | Conducted final review of report and backed up data. |

## Lab notebook - system3（Total 32 pages）

Lab Notebook——system3.pdf

1.5MB

[Download](https://static.igem.wiki/teams/5877/notebook/lab-notebook-system3.pdf)

{% endblock %}

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

Content

* [Abstract](#section-1)
  + [Cycle 1 —— Construction of AHL Biosensor Engineered Bacteria](#section-2)
    - [Design](#subsection-3)
    - [Build](#subsection-4)
    - [Test](#subsection-5)
    - [Learn](#subsection-6)
  + [Cycle 2 —— Construction of AHL-Inducible indigo Biosynthesis Engineered Bacteria](#section-7)
    - [Design](#subsection-8)
    - [Build](#subsection-9)
    - [Test](#subsection-10)
    - [Learn](#subsection-11)
  + [Cycle 3 —— Construction of Salicylic Acid Synthesis Engineered Bacteria](#section-12)
    - [Design](#subsection-13)
    - [Build](#subsection-14)
    - [Test](#subsection-15)
    - [Learn](#subsection-16)
  + [References](#section-17)

# Abstract

To address the serious threat of bacterial wilt caused by *Ralstonia solanacearum* in crops, we successfully developed a synthetic biology-based "detection-visualization-treatment" system through three complete "Design-Build-Test-Learn" (DBTL) cycles. This system consists of three core modules: the detection module constructs an AHL-responsive biosensor strain that can accurately report quorum sensing signals via red fluorescent protein (mRFP); the visualization module, through iterative improvement, successfully converts the signal into a visible blue color reaction; the treatment module, through rational design, constructs an engineered strain capable of efficiently synthesizing salicylic acid (SA), expected to activate the plant immune system and enhance resistance to bacterial wilt. After continuous optimization through three DBTL cycles, we successfully obtained all functional strains with stable and reliable performance, laying a solid foundation for the practical field application of the system.

![Abstract Diagram](https://static.igem.wiki/teams/5877/engineering/img1.webp)

# Cycle 1 —— Construction of AHL Biosensor Engineered Bacteria

## Design

The goal was to construct an engineered *E. coli* strain capable of specifically detecting acyl-homoserine lactone (AHL) quorum sensing signal molecules. Through literature review, we selected the quorum sensing regulatory protein QscR from *Pseudomonas aeruginosa* PAO1 and its native regulatory promoter PQscR as the core sensing elements. When QscR binds to AHL, it activates the expression of the downstream reporter gene mRFP, enabling quantitative detection of AHL presence and concentration through fluorescence intensity.

![Design Diagram](https://static.igem.wiki/teams/5877/engineering/img2.webp)

Figure 1 AHL Sensor Genetic Circuit

## Build

We first performed *E. coli* codon optimization on the QscR gene from *P. aeruginosa* PAO1 and eliminated internal XbaI, SpeI, EcoRI, and PstI restriction sites to ensure compliance with the BioBrick standard. The optimized gene sequence was synthesized by a professional biology company. Subsequently, the complete J23100-B0034-QscR-B0015-PQscR-mRFP expression cassette was precisely cloned into the standard pSB1A3 vector using XbaI and SpeI restriction enzymes, constructing the recombinant plasmid. This was transformed into *E. coli* BL21(DE3) cells, positive clones were verified by colony PCR, and finally the stable engineered strain named BL21-mRFP was obtained.

![Build Diagram](https://static.igem.wiki/teams/5877/engineering/img3.webp)

Figure 2 Construction of the AHL Biosensor Engineered Bacteria.

(A) AHL sensor recombinant plasmid map. (B) Agarose gel electrophoresis of QscR. (C) Agarose gel electrophoresis of mRFP.

## Test

### Response of Biosensor Strain to Different AHL Molecules

The sequence-verified engineered strain was inoculated into LB medium and activated overnight, then subcultured at a 1:100 ratio into fresh LB medium. When the OD₆₀₀ reached 0.3 at 37°C, 1 mL of bacterial culture was transferred to a 24-well plate. AHL molecules including 10 μM 3OC12-HSL, C10-HSL, and 3OHC10-HSL were added separately. After 3 hours of induction at 37°C, the OD₆₀₀ value and mRFP fluorescence intensity at 584/607 nm were simultaneously detected using a microplate reader. After subtracting the background value, the normalized fluorescence value (fluorescence intensity/OD₆₀₀) for each sample was calculated, and the dose-response characteristics of the sensor were tested using different AHL concentrations.

The 3OC12-HSL induced group had the highest normalized fluorescence intensity (11.40 ± 0.52 RFU/OD), followed by the C10-HSL group (7.64 ± 0.31 RFU/OD), and the 3OHC10-HSL group had the weakest response (5.33 ± 0.16 RFU/OD).

![Test Diagram](https://static.igem.wiki/teams/5877/engineering/img4.webp)

Figure 3 Relative mRFP fluorescence intensity after 3h induction with different AHLs

### Dose Response of Biosensor Strain to Different AHL Molecules

The three AHL molecules showed weak responses in the 0.001-0.01 μM concentration range, a sharp increase in fluorescence intensity at 0.1 μM, and reached a response saturation plateau in the 1-10 μM concentration range. The sensor had the highest sensitivity to 3OC12-HSL (11.13 ± 0.53 RFU/OD), followed by C10-HSL (8.22 ± 0.49 RFU/OD) and 3OHC10-HSL (5.26 ± 0.45 RFU/OD).

![Test Diagram](https://static.igem.wiki/teams/5877/engineering/img5.webp)

Figure 4 Analysis of Biosensor Strain Sensitivity and Dose-Response Relationship

(A) Relative mRFP fluorescence intensity after 3h induction with different concentrations of 3OC12-HSL. (B) Relative mRFP fluorescence intensity after 3h induction with different concentrations of C10-HSL. (C) Relative mRFP fluorescence intensity after 3h induction with different concentrations of 3OHC10-HSL.

## Learn

The experiment successfully verified that the QscR protein from *P. aeruginosa* PAO1 and its regulatory element PQscR can be efficiently assembled and function in the *E. coli* system. The constructed AHL biosensor engineered bacteria possess highly specific recognition capability for characteristic AHL molecules of *R. solanacearum* (3OC12-HSL, C10-HSL, 3OHC10-HSL), with the highest response sensitivity to 3OC12-HSL. However, preliminary application revealed that although the fluorescence-based reporter system performs excellently under laboratory conditions, its reliance on fluorescence detection equipment limits its promotion and application in field environments, making it particularly unsuitable for farmers lacking professional equipment.

Based on this finding, we systematically optimized the system by replacing the fluorescent reporter gene with the indigo synthesis pathway (TnaA and FMO), achieving a functional upgrade from instrument detection to visual observation. This improvement allows detection results to be directly interpreted via obvious blue precipitation, greatly enhancing the field applicability and user-friendliness of the system, laying a solid foundation for developing disease detection tools truly suitable for field environments.

# Cycle 2 —— Construction of AHL-Inducible indigo Biosynthesis Engineered Bacteria

## Design

To achieve visualization of detection, we upgraded the fluorescent reporter system in the already constructed AHL biosensor to a visible colorimetric output system. By replacing the reporter gene from mRFP with the indigo biosynthesis pathway genes (TnaA and FMO), we aimed to construct an engineered strain capable of producing blue indigo precipitate in response to AHL signals, enabling rapid, on-site visual detection without complex instruments.

![Design Diagram](https://static.igem.wiki/teams/5877/engineering/img6.webp)

Figure 5 indigo Colorimetric Biosensor Genetic Circuit

## Build

We first cloned the codon-optimized TnaA and FMO genes into the pSB1A3 vector using XbaI and SpeI restriction enzyme sites, replacing the original mRFP reporter gene, to construct the recombinant plasmid. The insertion of the TnaA and FMO genes was verified by colony PCR, and clones with correct PCR verification were sent for sequencing to confirm sequence accuracy. The sequenced, error-free engineered strain BL21-TnaA-FMO was cultured in LB/Amp⁺ medium to OD₆₀₀ ≈ 0.6, mixed with an equal volume of 50% glycerol, and stored at -80°C for backup.

![Build Diagram](https://static.igem.wiki/teams/5877/engineering/img7.webp)

Figure 6 Construction of AHL-Inducible indigo Biosynthesis Engineered Bacteria.

(A) indigo colorimetric biosensor plasmid map. (B) Agarose gel electrophoresis of TnaA. (C) Agarose gel electrophoresis of FMO.

## Test

The verified engineered strain was inoculated into M9 medium containing 0.4% glucose, and 1μM of 3OC12-HSL, C10-HSL, or 3OHC10-HSL was added to induce indigo synthesis. Then, 1 mL of bacterial culture was collected every 2 hours, centrifuged at 10,000 × g for 1 minute, and the supernatant was collected. The absorbance at 620 nm was measured using a microplate reader, using a 10 mM indigo solution dissolved in DMSO for calibration. A standard curve was prepared using different concentrations of indigo (0 mM, 0.25 mM, 0.5 mM, 1 mM, 1.5 mM, 3 mM). Each experimental group was performed in triplicate. indigo yield was quantitatively analyzed by comparison with the indigo standard curve. The experimental results showed that all three AHL molecules could effectively induce indigo synthesis. Within 0-2 hours, the indigo yield was very low; it began to accumulate significantly after 4 hours; explosive growth occurred at 6 hours; and within the 8-hour observation period, the yield reached its highest level. Among them, 3OC12-HSL had the strongest induction capability, with an 8-hour yield of 2.14 ± 0.14 mM, significantly higher than C10-HSL and 3OHC10-HSL. This time course indicates that to ensure reliable detection results, the observation time in field applications should not be less than 6 hours, with the optimal interpretation window being 6-8 hours.

![Test Diagram](https://static.igem.wiki/teams/5877/engineering/img8.webp)

Figure 7 Colorimetric Analysis of the indigo Biosensor.

(A) indigo standard curve. (B) Physical images of indigo production induced by different AHLs. (C) indigo content after treatment with 1μM 3OC12-HSL for different times. (D) indigo content after treatment with 1μM C10-HSL for different times. (E) indigo content after treatment with 1μM 3OHC10-HSL for different times.

## Learn

This phase of research successfully achieved the technical upgrade of the AHL biosensor from fluorescence detection to visual detection. Through rational design, we replaced the reporter system from mRFP with the indigo biosynthesis pathway (TnaA-B0034-FMO), successfully constructing an engineered strain capable of producing visible blue precipitate in response to AHL signals. Based on achieving visual detection of AHL, a salicylic acid (SA) treatment system can be introduced to generate a visual detection signal (indigo precipitate) during the pathogen recognition stage, while continuously secreting salicylic acid, a plant systemic resistance activator, during the control stage, enhancing plant resistance to bacterial wilt.

# Cycle 3 —— Construction of Salicylic Acid Synthesis Engineered Bacteria

## Design

Our goal was to construct an engineered strain capable of efficiently synthesizing salicylic acid (SA). We selected key genes for salicylic acid synthesis from plants and bacteria - isochorismate synthase gene (ICS) and isochorismate pyruvate lyase gene (IPL) - and ensured their efficient expression in *E. coli* through codon optimization. This engineered bacteria aims to enhance plant innate immunity against bacterial wilt through sustainable production of salicylic acid as a bioelicitor, providing a material basis for developing new biological control strategies.

![Design Diagram](https://static.igem.wiki/teams/5877/engineering/img9.webp)

Figure 8 Salicylic Acid Synthesis Engineered Bacteria Genetic Circuit

## Build

First, codon optimization for *E. coli* was performed on the key salicylic acid synthesis genes ICS and IPL, and the full genes were synthesized by a biology company. The optimized ICS-IPL fusion gene fragment was cloned into the corresponding multiple cloning site of the pET28a expression vector via NdeI and XhoI double digestion, constructing the recombinant expression plasmid pET28a-ICS-IPL. The verified correct recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells for positive clone screening. Single colonies were picked, the insertion of the target gene was verified by colony PCR, and after DNA sequencing confirmed the correct reading frame and absence of mutations, the recombinant engineered strain BL21/pET28a-ICS-IPL was obtained.

![Build Diagram](https://static.igem.wiki/teams/5877/engineering/img10.webp)

Figure 9 Salicylic Acid Synthesis Engineered Bacteria Plasmid Map

## Test

The verified engineered strain was inoculated into M9Y medium. When the OD₆₀₀ reached 0.6, 0.5 mM IPTG was added to induce protein expression. Cultivation continued, and 1 mL of bacterial culture was collected every 4 hours. After centrifugation at 10,000 × g for 1 minute, the supernatant was collected. The salicylic acid concentration in the supernatant was quantitatively analyzed using a salicylic acid ELISA detection kit, and the normalized yield was calculated. The results showed that after induction with 0.5 mM IPTG, the SA yield at 4h was 5.61 ± 1.55 mg/L, at 8h was 23.11 ± 2.91 mg/L, at 12h was 52.82 ± 3.31 mg/L, at 16h was 65.35 ± 6.29 mg/L, at 20h was 79.48 ± 6.67 mg/L, and at 24h was 84.02 ± 4.66 mg/L. After 24 hours of induction, the blank control group (BL21 empty strain) produced almost no salicylic acid (content close to 0 mg/L), while the engineered bacteria group (BL21-ICS-IPL) showed a significantly increased salicylic acid yield, exceeding 80 mg/L.

![Test Diagram](https://static.igem.wiki/teams/5877/engineering/img11.webp)

Figure 10 Salicylic Acid Production Test.

(A) Salicylic acid ELISA standard curve (A450). (B) Log10 values of salicylic acid concentration and A450 absorbance from ELISA analysis. (C) Salicylic acid production over time. (D) Salicylic acid content after 24h induction in different engineered strains.

## Learn

This phase of research successfully constructed an engineered strain capable of efficiently synthesizing salicylic acid. Experimental verification showed that after IPTG induction, this strain can efficiently convert endogenous chorismate to salicylic acid, with a yield significantly higher than the blank control. The construction of this engineered bacteria provides a stable biosynthesis platform for studying the application of salicylic acid as a plant immune elicitor, achieving the functional expansion of the bacterial wilt control system from "detection" to "control".

# References

[1] WANG B, BARAHONA M, BUCK M. Engineering modular and tunable genetic amplifiers for scaling transcriptional signals in cascaded gene networks[J]. Nucleic Acids Res, 2018, 46(3): 1174-1189.

[2] MUKHERJEE S, BASSLER B L. Bacterial quorum sensing in complex and dynamically changing environments[J]. Nat Rev Microbiol, 2019, 17(6): 371-382.

[3] SAEIDI N, WONG C K, LO T M, et al. Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen[J]. Mol Syst Biol, 2011, 7: 521.

[4] WILLIAMS T C, PRETORIUS I S, PAULSEN I T. Synthetic biology for the rapid, precise and compliant detection of microbes[J]. Curr Opin Biotechnol, 2016, 38: 201-206.

[5]A B Flavier, S J Clough, M A Schell.Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum[J].Mol Microbiol,1997,26(2):251-9.

[6] Shuting Hu, Guangbao Zhang, Xiaoqiang Jia. Improvement of a highly sensitive and specific whole-cell biosensor by adding a positive feedback amplifier[J]. Synthetic and Systems Biotechnology, 2023, 8: 292-299.

[7] HAN G H, SHIN H J, KIM S W. Optimization of bio-indigo production by recombinant E. coli harboring fmo gene[J]. Enzyme Microb Technol, 2008, 42(7): 617-623.

[8] MA Q, ZHANG X, QU Y. Biodegradation and biotransformation of indole: advances and perspectives[J]. Front Microbiol, 2018, 9: 2625.

[9] LIN Y, SUN X, YUAN Q, et al. Extending shikimate pathway for the production of muconic acid and its precursor salicylic acid in Escherichia coli[J]. Metab Eng, 2014, 23: 62-69.

[10] VERMA S, SINGH A, MISHRA A. Salicylic acid biosynthesis and metabolism: a divergent pathway for plants and bacteria[J]. Biomolecules, 2021, 11(5): 705.

[11] YAN F, LIU X, ZHANG S, et al. Metabolic engineering of Escherichia coli for salicylic acid production using the heterologous EntC-PchB pathway[J]. Biotechnol Bioeng, 2022, 119(5): 1225-1237.

{% 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 %}

Content

* [Construction of Bacterial Wilt Detection Engineered Bacteria](#section-1)
  + [Culture and Preservation of Bacterial Wilt Detection Engineered Bacteria](#section-2)
    - [AHL Biosensor Testing](#section-3)
      * [AHL Biosensor Dose-Response Testing](#section-4)
        + [Construction of Indigo-Producing Strain](#section-5)
          - [Testing of Indigo-Producing Strain](#section-6)
            * [Construction of Salicylic Acid Overexpression Engineered Bacteria](#section-7)
              + [Salicylic Acid Production Test](#section-8)
                - [Statistical Analysis](#section-9)
                  * [Appendix: Salicylic Acid ELISA Kit](#section-10)

# Construction of Bacterial Wilt Detection Engineered Bacteria

Based on the codon preference of *E. coli*, the QscR gene derived from *Pseudomonas aeruginosa* PAO1 was codon-optimized. Restriction sites (EcoRI, XbaI, SpeI, PstI) were eliminated to comply with BioBricks™ standard RFC#10 (verified via SnapGene® software). The complete genetic circuit, containing the J23100 constitutive promoter, B0034 ribosome binding site, optimized QscR gene, B0015 terminator, PQscR promoter, and mRFP reporter gene, was synthesized by Generalbial Company. This sequence was cloned into the pSB1A3 vector using XbaI and SpeI and transformed into *E. coli* BL21(DE3) competent cells (purchased from Beyotime) via heat shock (42°C, 1 min). Positive clones were selected on LB plates containing 50 µg/mL ampicillin. Single colonies were picked and inoculated into LB/Amp liquid medium, followed by shaking culture at 37°C until OD₆₀₀ reached 0.6-0.8. Preliminary verification was performed by colony PCR (using Vazyme 2×Taq Master Mix), and confirmation was obtained by sequencing (conducted by Tsingke Biotechnology Co., Beijing), ultimately yielding the engineered strain BL21-mRFP.

# Culture and Preservation of Bacterial Wilt Detection Engineered Bacteria

The sequence-verified engineered strain BL21-mRFP was inoculated into LB/Amp⁺ medium and cultured to mid-log phase (OD₆₀₀ ≈ 0.6). It was then mixed with an equal volume of 50% glycerol and stored at -20°C for future use.

# AHL Biosensor Testing

3OC12-HSL (O9139), C10-HSL (07028), and 3OHC10-HSL (68873) purchased from Sigma-Aldrich were dissolved in DMSO to prepare 10 mM stock solutions. The constructed AHL biosensor engineered bacteria were inoculated at a 1:100 ratio into 5 mL of LB medium containing 50 µg/mL ampicillin and activated overnight at 37°C, 150 rpm. Then, 100 µL of the culture was transferred to 5 mL of fresh LB/Amp⁺ medium and cultured at 37°C, 180 rpm until OD₆₀₀ reached 0.3. Then, 1 mL of the bacterial culture was aliquoted into a 24-well plate, and 10 µM of 3OC12-HSL, C10-HSL, or 3OHC10-HSL was added separately. After continued cultivation for 3 hours at 37°C, 180 rpm, the absorbance at 600 nm (OD₆₀₀) and mRFP fluorescence intensity (excitation 584 nm/emission 607 nm) were simultaneously measured using a Molecular Devices FlexStation 3 microplate reader. The normalized fluorescence value (fluorescence intensity/OD₆₀₀) was calculated after subtracting the background value.

# AHL Biosensor Dose-Response Testing

To test the dose-response effect, the AHL biosensor engineered bacteria were exposed to different concentrations of AHL molecules under the same culture conditions. The mRFP fluorescence response across different concentration gradients was detected using the Molecular Devices FlexStation 3 microplate reader to establish a dose-response curve.

# Construction of Indigo-Producing Strain

The Indigo synthesis genes TnaA and FMO, codon-optimized to meet the RFC#10 standard, were synthesized. Based on the AHL biosensor, the mRFP reporter gene was replaced with the TnaA-B0034-FMO polycistronic unit. The J23100-B0034-QscR-B0015-PQscR-TnaA-B0034-FMO sequence was cloned into the pSB1A3 vector using XbaI and SpeI. The recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells (Beyotime) via heat shock. Positive clones were selected on LB plates containing 50 µg/mL ampicillin. The correct engineered strain was obtained after verification by colony PCR and sequencing (Tsingke, Beijing).

# Testing of Indigo-Producing Strain

A single colony of the verified Indigo-producing engineered strain was inoculated into 5 mL of LB/Amp⁺ medium and activated overnight at 37°C. After centrifugation and washing, it was subcultured at a 1:100 ratio into 5 mL of M9 medium (Sangon Biotech) containing 50 µg/mL ampicillin, 0.4% glucose, 1 mM MgSO₄, and 50 µM CaCl₂. Different AHLs (3OC12-HSL, C10-HSL, or 3OHC10-HSL) were added to induce Indigo production. Then, 1 mL of bacterial culture was collected every 2 hours, centrifuged, and the supernatant was resuspended for absorbance measurement at 620 nm. A standard curve was prepared using Indigo standard (SM4168, Beyotime) for quantification. All experiments were performed with three replicates.

# Construction of Salicylic Acid Overexpression Engineered Bacteria

The salicylic acid synthesis genes ICS (derived from *E. coli* EntC) and IPL (derived from *Pseudomonas fluorescens* PchB), codon-optimized to meet the RFC#10 standard, were synthesized. The ICS-B0034-IPL sequence was cloned into the pET28a(m) vector using NdeI and XhoI restriction sites. The recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells (Beyotime). Positive clones were selected on LB plates containing 50 µg/mL kanamycin. The correct engineered strain was obtained after verification by colony PCR and sequencing (Tsingke, Beijing).

# Salicylic Acid Production Test

The verified salicylic acid engineered strain was inoculated at a 1:100 ratio into LB medium containing kanamycin and activated overnight at 37°C. Then, 100 µL of the culture was transferred to M9Y medium (M9 basal medium supplemented with 1 g/L yeast extract, 2 g/L MOPS, 10 g/L glycerol, 2.5 g/L glucose, 1 mM MgSO₄, and 50 µM CaCl₂) containing 50 µg/mL kanamycin. When the OD₆₀₀ reached 0.6 during cultivation at 37°C, 180 rpm, 0.5 mM IPTG was added to induce expression. Samples of 1 mL were taken every 24 hours, centrifuged, and the supernatant was collected. The salicylic acid concentration was detected using an ELISA kit, and the normalized yield (salicylic acid concentration/OD₆₀₀) was calculated.

# Statistical Analysis

Data analysis and graphing were performed using GraphPad Prism software. Data are presented as mean ± standard deviation (SD). For multiple group comparisons, one-way ANOVA with Tukey's post hoc test was used for difference analysis. For comparisons between two groups, Student's t-test was used. A p-value less than 0.05 was considered statistically significant.

# Appendix: Salicylic Acid ELISA Kit

植物水杨酸（SA）酶联免疫分析(ELISA).pdf

0.3MB

[Download](https://static.igem.wiki/teams/5877/experiment/sa-elisa.pdf)

{% endblock %}

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

Content

* [Overview](#section-1)
  + [Construction of the AHL Biosensor Engineered Bacteria](#section-2)
    - [Objective](#subsection-3)
    - [Methods](#subsection-4)
    - [Results](#subsection-5)
    - [Conclusion](#subsection-6)
  + [Analysis of Response Specificity of the Biosensor Strain to Various AHL Molecules](#section-7)
    - [Objective](#subsection-8)
    - [Methods](#subsection-9)
    - [Results](#subsection-10)
    - [Conclusion](#subsection-11)
  + [Analysis of Biosensor Strain Sensitivity and Dose-Response Relationship](#section-12)
    - [Objective](#subsection-13)
    - [Methods](#subsection-14)
    - [Results](#subsection-15)
    - [Conclusion](#subsection-16)
  + [Construction of AHL-Inducible Indigo Biosynthesis Engineered Bacteria](#section-17)
    - [Objective](#subsection-18)
    - [Methods](#subsection-19)
    - [Results](#subsection-20)
    - [Conclusion](#subsection-21)
  + [Colorimetric Analysis of the Indigo Biosensor](#section-22)
    - [Objective](#subsection-23)
    - [Methods](#subsection-24)
    - [Results](#subsection-25)
    - [Conclusion](#subsection-26)
  + [Construction and Yield Evaluation of Salicylic Acid Synthesis Engineered Bacteria](#section-27)
    - [Objective](#subsection-28)
    - [Methods](#subsection-29)
    - [Results](#subsection-30)
    - [Conclusion](#subsection-31)
  + [Summary](#section-32)
    - [References](#section-33)

# Overview

This project successfully constructed and validated an engineered bacterial system for the detection and control of bacterial wilt. The biosensor, based on an AHL-sensing component, can specifically recognize signal molecules released by the *Ralstonia solanacearum*, enabling early pathogen detection. The visualization module successfully converted the signal into a visible blue colorimetric reaction, confirming its feasibility for rapid on-site detection. The therapeutic module's engineered bacteria can effectively synthesize salicylic acid, enhancing disease resistance by activating plant immune responses. The integrated system achieves the goal from pathogen detection to immune intervention, providing a new technical approach to address the challenge of bacterial wilt control.

![Overview Diagram](https://static.igem.wiki/teams/5877/results/img1.webp)

# Construction of the AHL Biosensor Engineered Bacteria

## Objective

To construct an engineered *E. coli* strain capable of specifically detecting AHL quorum-sensing signal molecules. This strain constitutively expresses the AHL receptor protein QscR. Upon QscR binding to AHL, it activates the expression of the downstream reporter gene mRFP, allowing quantitative detection of AHL presence and concentration via fluorescence intensity.

![Objective Diagram](https://static.igem.wiki/teams/5877/results/img2.webp)

Figure 1 AHL Sensor Genetic Circuit

## Methods

The codon-optimized QscR gene (from *Pseudomonas aeruginosa* PAO1) and its promoter PQscR were first cloned into the pSB1A3 vector [1]. Specifically, the J23100-B0034-QscR-B0015-PQscR-mRFP expression cassette was inserted into the vector using XbaI and SpeI restriction enzymes, constructing a recombinant plasmid compliant with BioBricks™ standard RFC#10 [2][3]. The recombinant plasmid was transformed into *E. coli* BL21(DE3) super competent cells via heat shock (42°C, 1 min) and plated on LB agar plates containing 50 µg/mL ampicillin, followed by incubation at 37°C for 16 h. Single colonies were picked and inoculated into 5 mL of LB/Amp⁺ liquid medium, shaken at 37°C, 180 rpm until OD600 reached 0.6-0.8. Positive clones were verified by colony PCR (using 2×Taq Master Mix) and sent for sequencing validation [4]. The sequence-verified engineered strain was inoculated into 5 mL of LB/Amp⁺ medium, cultured at 37°C, 180 rpm to OD600 ≈ 0.6. Then, 1 mL of the culture was mixed with 1 mL of 50% glycerol and stored at -20°C for backup.

![Methods Diagram](https://static.igem.wiki/teams/5877/results/img3.webp)

Figure 2 AHL Sensor Recombinant Plasmid Map

## Results

As shown in the Figure 3, clear bands were observed at the expected sizes in the PCR amplification products. The QscR gene amplification band was located at 711 bp, and the mRFP gene amplification band was at 678 bp, completely consistent with the theoretical design sizes, with no non-specific amplification bands present.

![Results Diagram](https://static.igem.wiki/teams/5877/results/img4.webp)

Figure 3 Agarose gel electrophoresis of QscR and mRFP. (A) Agarose gel electrophoresis of QscR. (B) Agarose gel electrophoresis of mRFP.

## Conclusion

These results successfully verified the correctness of the codon-optimized QscR gene and the mRFP fragment, indicating accurate gene synthesis and cloning processes. This provides complete core functional components for the subsequent construction of the AHL biosensor engineered bacteria.

# Analysis of Response Specificity of the Biosensor Strain to Various AHL Molecules

## Objective

To validate the specific recognition capability of the constructed biosensor engineered bacteria for characteristic AHL molecules of *R. solanacearum* (3OC12-HSL, C10-HSL, 3OHC10-HSL), confirming its ability to specifically respond to key signal molecules associated with bacterial wilt, laying the foundation for subsequent applications.

## Methods

The sequence-verified engineered strain was inoculated at a 1:100 ratio into 5 mL of LB medium containing 50 µg/mL ampicillin and activated overnight at 37°C, 150 rpm. Then, 100 µL of the culture was re-inoculated into a 50 mL sterile centrifuge tube containing 5 mL of Amp⁺ LB medium and cultured at 37°C until OD600 reached 0.3. Then, 1 mL of the culture was transferred to a 24-well plate, and 10 µM of 3OC12-HSL, C10-HSL, or 3OHC10-HSL was added respectively. After 3 hours of induction at 37°C, the OD600 and mRFP fluorescence intensity at 584/607 nm were measured simultaneously using a microplate reader. After subtracting the LB medium background, the normalized fluorescence value (fluorescence intensity/OD600) for each sample was calculated.

![Methods Diagram](https://static.igem.wiki/teams/5877/results/img5.webp)

Figure 4 Experimental procedure for testing AHL sensor response to three AHL molecules

## Results

As shown in the figure 5, after induction with 10 µM different AHL molecules, the engineered strain showed significantly different fluorescence responses. The 3OC12-HSL induced group had the highest normalized fluorescence intensity (11.40 ± 0.52 RFU/OD), followed by the C10-HSL group (7.64 ± 0.31 RFU/OD), and the 3OHC10-HSL group had the weakest response (5.33 ± 0.16 RFU/OD). Statistical analysis indicated extremely significant differences among the three groups (*P* < 0.001), demonstrating the sensor's ability to distinguish between different types of AHL molecules.

![Results Diagram](https://static.igem.wiki/teams/5877/results/img6.webp)

Figure 5 Relative mRFP fluorescence intensity after 3h induction with different AHLs

## Conclusion

The successfully constructed AHL biosensor possesses specific recognition capability for all three characteristic AHL molecules of *R. solanacearum*, with the strongest response to 3OC12-HSL. This sensor can effectively distinguish AHL molecules with different structures, laying the foundation for the specific detection of *R. solanacearum*.

# Analysis of Biosensor Strain Sensitivity and Dose-Response Relationship

## Objective

To determine the detection sensitivity of the constructed AHL biosensor for characteristic AHL molecules of *R. solanacearum*, determine its minimum effective detection threshold, and verify whether the system can be effectively activated within the expected field AHL concentration range produced during *R. solanacearum* infection (literature reports are typically at the nanomolar level [5]), thereby evaluating its practical feasibility for field early warning.

## Methods

Concentration gradient experiments were performed using three AHL molecules: 3OC12-HSL, C10-HSL, and 3OHC10-HSL (0.001, 0.01, 0.1, 1, 10 µM). The strain culture and induction conditions were the same as before. After induction, fluorescence signals were detected using a microplate reader, and normalized fluorescence values were calculated.

## Results

The concentration gradient experiment results (Figure 6) showed that all three AHL molecules exhibited typical dose-dependent response trends:

* Weak response in the 0.001-0.01 µM
* Sharp increase in fluorescence intensity at 0.1 µM
* Response saturation plateau in the 1-10 µM
* The sensor had the highest sensitivity to 3OC\_\_\_SUB\_\_\_12\_\_\_-HSL (11.13 ± 0.53 RFU/OD), followed by C\_\_\_SUB\_\_\_10\_\_\_-HSL (8.22 ± 0.49 RFU/OD) and 3OHC\_\_\_SUB\_\_\_10\_\_\_-HSL (5.26 ± 0.45 RFU/OD).

![Results Diagram](https://static.igem.wiki/teams/5877/results/img7.webp)

Figure 6 Analysis of biosensor strain sensitivity and dose-response relationship

(A) Relative mRFP fluorescence intensity after 3h induction with different concentrations of 3OC12-HSL. (B) Relative mRFP fluorescence intensity after 3h induction with different concentrations of C10-HSL. (C) Relative mRFP fluorescence intensity after 3h induction with different concentrations of 3OHC10-HSL.

## Conclusion

The constructed AHL biosensor has high detection sensitivity, with a detection limit for 3OC12-HSL reaching 0.01 µM. The sensor shows a good linear response characteristic in the 0.01-1 µM, meeting the quantitative detection requirements for *R. solanacearum* quorum sensing signal molecules.

# Construction of AHL-Inducible Indigo Biosynthesis Engineered Bacteria

## Objective

Based on the AHL biosensor, replace the fluorescent reporter gene with the indigo synthesis pathway (TnaA and FMO) to construct a colorimetric sensor that produces a visible blue precipitate (indigo) for rapid on-site detection of AHL.

![Objective Diagram](https://static.igem.wiki/teams/5877/results/img8.webp)

Figure 7 Indigo colorimetric sensor genetic circuit

## Methods

First, the mRFP reporter gene in the AHL biosensor was replaced with TnaA and FMO. The J23100-B0034-QscR-B0015-PQscR-TnaA-B0034-FMO sequence was cloned into the pSB1A3 vector using XbaI and SpeI [6][7]. Subsequently, the recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells via heat shock (42°C, 1 min) and plated on LB agar plates containing 50 µg/mL ampicillin (Amp⁺) (pH 7.0), followed by incubation at 37°C for 16 h. Single colonies were picked and inoculated into 5 mL of LB/Amp⁺ liquid medium, shaken at 37°C, 180 rpm until OD600 reached 0.6-0.8. Positive clones were verified by colony PCR (2×Taq Master Mix, Vazyme). The sequence-verified engineered strain was inoculated into 5 mL of LB/Amp⁺ medium and cultured at 37°C, 180 rpm. The growth curve was monitored using a FlexStation 3 (Molecular Devices, USA) until the mid-log phase (OD600 ≈ 0.6). Then, 1 mL of the culture was mixed with 1 mL of 50% glycerol and stored at -20°C for backup.

![Methods Diagram](https://static.igem.wiki/teams/5877/results/img9.webp)

Figure 8 Indigo colorimetric biosensor plasmid map

## Results

Agarose gel electrophoresis results showed clear bands for the PCR amplification products of the TnaA and FMO genes, with sizes at 1422 bp and 1368 bp respectively, completely consistent with the expected fragment sizes.

![Results Diagram](https://static.igem.wiki/teams/5877/results/img10.webp)

Figure 9 Construction of AHL-inducible indigo biosynthesis engineered bacteria. (A) Agarose gel electrophoresis of TnaA. (B)Agarose gel electrophoresis of FMO.

## Conclusion

The AHL-inducible indigo biosynthesis engineered bacteria were successfully constructed. Gene cloning and sequencing results indicated that the indigo synthesis pathway genes TnaA and FMO were accurately integrated into the expression vector, providing reliable engineered bacterial material for subsequent colorimetric analysis.

# Colorimetric Analysis of the Indigo Biosensor

## Objective

To evaluate the response characteristics of the indigo biosensor to characteristic AHL molecules of *R. solanacearum*, determine the minimum detection time required for the sensor to produce visible blue color by measuring indigo yield changes at different time points, and analyze its detection sensitivity and dynamic response range, providing an experimental basis for determining the optimal observation time window in field applications.

## Methods

The verified engineered strain was inoculated into 5 mL of LB broth containing 50 µg/mL ampicillin and activated overnight in a shaking incubator at 37°C and 180 rpm. The engineered bacteria were then inoculated at a 1:100 ratio into M9 medium containing 0.4% glucose, and 1 µM of 3OC12-HSL, C10-HSL, or 3OHC10-HSL was added to induce indigo synthesis [8]. Then, 1 mL of the culture was collected every 2 hours, centrifuged at 10,000 × g for 1 minute, and the supernatant was collected. The absorbance at 620 nm was measured using a microplate reader. A 10 mM indigo solution was prepared using DMSO. A standard curve was prepared using different concentrations of indigo (0 mM, 0.25 mM, 0.5 mM, 1 mM, 1.5 mM, 3 mM). Each experimental group was performed in triplicate. indigo yield was quantitatively analyzed by comparing it with the indigo standard curve.

![Methods Diagram](https://static.igem.wiki/teams/5877/results/img11.webp)

Figure 10 Operation procedure for colorimetric analysis of the indigo biosensor

## Results

Time-course monitoring results (Figure 11) showed that all three AHL molecules could induce indigo synthesis, exhibiting a similar time-dependent accumulation pattern:

* 0-2 hours: Very low indigo yield (<0.30 mM), lag phase.
* 4 hours: Significant accumulation began (0.48-0.56 mM), entering the logarithmic growth phase.
* 6 hours: Explosive growth occurred (1.11-1.40 mM).
* 8 hours: Peak yield was reached within the observation period.

Comparison of induction efficiency showed that 3OC12-HSL had the strongest induction capability (yield at 8 hours: 2.14 ± 0.14 mM), significantly higher than C10-HSL (1.70 ± 0.06 mM) and 3OHC10-HSL (1.42 ± 0.09 mM) (*P* < 0.01). Visible blue precipitate formation was observed with the naked eye, and the color intensity positively correlated with the AHL concentration.

![Results Diagram](https://static.igem.wiki/teams/5877/results/img12.webp)

Figure 11 Colorimetric analysis of the indigo biosensor.

(A) indigo standard curve. (B) Physical images of indigo production induced by different AHLs. (C) indigo content after treatment with 1 µM 3OC12-HSL for different times. (D) indigo content after treatment with 1 µM C10-HSL for different times. (E) indigo content after treatment with 1 µM 3OHC10-HSL for different times.

## Conclusion

The constructed indigo colorimetric biosensor exhibited good time-dependent response characteristics to all three AHL molecules, with the highest sensitivity to 3OC12-HSL. Experimental results indicate that the minimum effective detection time for the sensor should not be less than 6 hours: rapid color development begins 4 hours after induction, and the ideal detection effect is achieved within 6-8 hours, forming a clearly distinguishable blue precipitate visible to the naked eye. Therefore, it is recommended to set the detection time within the 6-8 hour range in practical applications. This ensures sufficient completion of the colorimetric reaction while meeting the timeliness requirements of rapid on-site detection, providing a clear basis for judgment and a reliable time window for field diagnosis.

# Construction and Yield Evaluation of Salicylic Acid Synthesis Engineered Bacteria

## Objective

To construct an engineered bacterial strain capable of efficiently synthesizing salicylic acid (SA). Salicylic acid is a key hormone in plant disease resistance signaling pathways. This engineered bacteria can be used to study the feasibility of enhancing plant resistance to bacterial wilt through the application of exogenous SA elicitors.

![Objective Diagram](https://static.igem.wiki/teams/5877/results/img13.webp)

Figure 12 Salicylic acid synthesis genetic circuit

## Methods

First, the codon-optimized salicylic acid synthesis genes ICS and IPL were cloned into the pET28a vector [9][10] and transformed into *E. coli* BL21(DE3). Positive clones were screened on LB plates containing kanamycin and verified for correctness by colony PCR and sequencing. The verified engineered strain was inoculated into M9Y medium. When the OD600 reached 0.6, 0.5 mM IPTG was added to induce protein expression. Cultivation continued, and 1 mL of the culture was collected every 4 hours. After centrifugation at 10,000 × g for 1 minute, the supernatant was collected. The salicylic acid concentration in the supernatant was quantitatively analyzed using a salicylic acid ELISA detection kit, and the normalized yield was calculated [11].

![Methods Diagram](https://static.igem.wiki/teams/5877/results/img14.webp)

Figure 13 Salicylic acid synthesis plasmid map

## Results

As shown in Figures 14A and 14B, this study established a salicylic acid standard curve using the ELISA method. Figure 14A illustrates the direct relationship between salicylic acid concentration and A450 absorbance, while Figure 14B displays the linear relationship between the logarithm (Log10) of salicylic acid concentration and A450 absorbance, verifying the reliability of the quantitative detection.

Regarding salicylic acid production, the time-course curve (Figure 14C) demonstrates that after induction with 0.5 mM IPTG, the engineered strain exhibited a time-dependent accumulation of salicylic acid over 24 hours. Specifically, the yield increased continuously from 5.61 ± 1.55 mg/L at 4 hours to 84.02 ± 4.66 mg/L at 24 hours.

The between-group comparison conducted after 24 hours of induction (Figure 14D) further revealed that the salicylic acid yield of the engineered strain group (BL21-ICS-IPL) exceeded 80 mg/L, while the yield from the blank control group (BL21 empty strain) was nearly negligible. The difference between the two groups was highly significant (P < 0.0001), conclusively demonstrating that the constructed engineered strain possesses the capability for efficient salicylic acid synthesis.

![Results Diagram](https://static.igem.wiki/teams/5877/results/img15.webp)

Figure 14 Salicylic acid production test.

(A) Salicylic acid ELISA standard curve (A450). (B) Log10 values of salicylic acid concentration and A450 absorbance from ELISA analysis. (C) Salicylic acid production over time. (D) Salicylic acid content after 24h induction in different engineered strains.

## Conclusion

The experiment successfully constructed the engineered strain BL21-ICS-IPL capable of efficiently synthesizing salicylic acid. The combination of heterologously expressed ICS and IPL genes endowed the engineered bacteria with a strong salicylic acid synthesis capacity, and the yield was significantly higher than that of the original strain. This proves the effectiveness of this genetic design strategy and provides an efficient biosynthesis platform for the subsequent development of salicylic acid-based plant immune activators.

# Summary

The experimental results of this project systematically validate the feasibility of a synthetic biology-based strategy for the detection and control of bacterial wilt. Through the constructed AHL biosensor, we have not only achieved nM-level high-sensitivity detection of the characteristic signal molecules of Ralstonia solanacearum but also successfully transformed the detection signal into a visible blue colorimetric reaction, establishing a 6-8 hour optimal detection window. Crucially, all experimental data were validated within the natural secretion range of AHL by R. solanacearum, which fully demonstrates that the system possesses the sensitivity foundation for field application.

Particularly noteworthy is that our modular design enabled the transition from laboratory instrument-based detection to field visual interpretation. Research confirmed that replacing the fluorescent reporter system with the indigoidine synthesis pathway not only maintained detection sensitivity but also significantly enhanced the practicality and promotion potential of the technology. This design approach effectively addresses the key limitations of traditional molecular detection technologies, namely their strong dependence on equipment and operational complexity.

We believe that this technology not only represents a novel approach to disease detection but also heralds the future direction of precision agriculture and green plant protection.

# References

[1] WANG B, BARAHONA M, BUCK M. Engineering modular and tunable genetic amplifiers for scaling transcriptional signals in cascaded gene networks[J]. Nucleic Acids Res, 2018, 46(3): 1174-1189.

[2] MUKHERJEE S, BASSLER B L. Bacterial quorum sensing in complex and dynamically changing environments[J]. Nat Rev Microbiol, 2019, 17(6): 371-382.

[3] SAEIDI N, WONG C K, LO T M, et al. Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen[J]. Mol Syst Biol, 2011, 7: 521.

[4] WILLIAMS T C, PRETORIUS I S, PAULSEN I T. Synthetic biology for the rapid, precise and compliant detection of microbes[J]. Curr Opin Biotechnol, 2016, 38: 201-206.

[5]A B Flavier, S J Clough, M A Schell.Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum[J].Mol Microbiol,1997,26(2):251-9.

[6] Shuting Hu, Guangbao Zhang, Xiaoqiang Jia. Improvement of a highly sensitive and specific whole-cell biosensor by adding a positive feedback amplifier[J]. Synthetic and Systems Biotechnology, 2023, 8: 292-299.

[7] HAN G H, SHIN H J, KIM S W. Optimization of bio-indigo production by recombinant E. coli harboring fmo gene[J]. Enzyme Microb Technol, 2008, 42(7): 617-623.

[8] MA Q, ZHANG X, QU Y. Biodegradation and biotransformation of indole: advances and perspectives[J]. Front Microbiol, 2018, 9: 2625.

[9] LIN Y, SUN X, YUAN Q, et al. Extending shikimate pathway for the production of muconic acid and its precursor salicylic acid in Escherichia coli[J]. Metab Eng, 2014, 23: 62-69.

[10] VERMA S, SINGH A, MISHRA A. Salicylic acid biosynthesis and metabolism: a divergent pathway for plants and bacteria[J]. Biomolecules, 2021, 11(5): 705.

[11] YAN F, LIU X, ZHANG S, et al. Metabolic engineering of Escherichia coli for salicylic acid production using the heterologous EntC-PchB pathway[J]. Biotechnol Bioeng, 2022, 119(5): 1225-1237.

{% endblock %}