iGEM 2025 — Team zqt-china

{% 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

* [Timeline](#section-1)
  + [Experiments Related to Chlorimuron-Ethyl Degradation](#section-2)
    - [IAA (Indole-3-Acetic Acid) Synthesis Experiments](#section-3)
      * [Safety system](#section-4)
        + [Notebook](#section-5)

# Timeline

![Timeline Diagram](https://static.igem.wiki/teams/5887/notebook/img1.avif)

# Experiments Related to Chlorimuron-Ethyl Degradation

|
|  |

Date |

Experiment |

Details |

Notes |

|
|  |

6/18 |

Gene Sequence Retrieval |

Obtain SulE, PnbA, GST sequences from NCBI. |

Successfully retrieved sequences. |

|
|  |

6/19 |

Gene Circuit Design |

Draft gene circuits and plasmid maps for each enzyme. |

Gene circuits designed; minor issue with overlapping restriction sites, resolved by removing redundant sites. |

|
|  |

6/20 |

Plasmid Map Finalization |

Optimize codons for E. coli; remove EcoRI, XbaI, SpeI, PstI, NdeI, XhoI sites. |

Plasmid maps finalized; some codon optimization introduced unintended secondary structure in SulE, fixed manually. |

|
|  |

6/21 |

Gene Synthesis Order |

Order synthesized genes (GST, SulE, PnbA) from Generalbial. |

Genes ordered successfully. |

|
|  |

6/22 |

PCR Amplification Attempt |

Amplify synthesized genes for cloning into pET28a(m). |

SulE amplification initially failed; optimized annealing temperature and succeeded. |

|
|  |

6/23 |

Restriction Digestion & Cloning |

Digest pET28a(m) and PCR fragments; ligate inserts. |

GST and PnbA ligation successful; SulE ligation initially low yield, repeated. |

|
|  |

6/24 |

Transformation into DH5α |

Transform ligated plasmids into DH5α via heat shock (42°C, 1 min). |

Transformation successful for GST and PnbA; SulE required second attempt. |

|
|  |

6/25 |

Screening & Sequencing |

Plate on LB + 100 μg/mL kanamycin; pick clones and sequence. |

Positive clones obtained; SulE clone had single-base mutation, replaced with correct clone. |

|
|  |

6/26 |

Plasmid Extraction |

Extract plasmids from DH5α clones for BL21 transformation. |

Extraction successful. |

|
|  |

6/27 |

BL21 Transformation |

Transform plasmids into BL21 (DE3). |

Transformation successful; SulE BL21 grew slowly, repeated induction later. |

|
|  |

6/28 |

IPTG Induction Trial |

Induce expression of enzymes in BL21 strains. |

GST and PnbA expressed well; SulE expression weak, increased IPTG concentration. |

|
|  |

6/29 |

Crude Enzyme Extraction |

Lyse cells and collect crude enzyme. |

Extraction successful; SulE enzyme partially degraded, adjusted lysis protocol. |

|
|  |

6/30 |

WB Validation |

Verify protein expression via Western blot. |

All three enzymes detected; SulE band faint, required longer exposure. |

|
|  |

7/1 |

Chlorimuron-E Standard Curve |

Prepare serial dilutions of chlorimuron-E; ELISA measurement. |

Standard curve established; minor pipetting errors in first replicate, repeated. |

|
|  |

7/2 |

ELISA Replication |

Repeat ELISA to ensure accuracy. |

Data consistent; curve validated. |

|
|  |

7/3 |

Enzyme Activity Pre-Test |

Test crude enzyme on 20 μg/mL chlorimuron-E for 10 min at 25°C. |

PnbA activity high; SulE and GST low; SulE repeat with higher IPTG improved activity. |

|
|  |

7/4 |

Activity Measurement |

ELISA measurement of degraded chlorimuron-E. |

Results: PnbA 61.2%, SulE 29.8%, GST 32.3%. |

|
|  |

7/5 |

Data Analysis |

Calculate degradation rates and perform Student t-test (n ≥ 3). |

Statistical significance confirmed (p < 0.05). |

|
|  |

7/6 |

Specific Activity Calculation |

Calculate U/μg for each enzyme based on molecular weight and A450 changes. |

PnbA: 0.0038; SulE: 0.0019; GST: 0.0021. |

|
|  |

7/7 |

Temperature Optimization |

Test PnbA activity at 25–40°C. |

Optimal at 30°C; initial 35°C test caused enzyme denaturation. |

|
|  |

7/8 |

Temperature Repeat |

Repeat temp test around 30°C. |

Confirmed 30°C as optimal. |

|
|  |

7/9 |

pH Optimization |

Adjust PBS to pH 5–9; test PnbA activity. |

pH 7 optimal; pH <6 caused reduced activity. |

|
|  |

7/10 |

pH Repeat |

Verify pH effect with replicate tests. |

Results consistent. |

|
|  |

7/11 |

INP Surface Display |

Clone INP-PnbA fusion into BL21. |

Cloning successful; initial surface display low, optimized promoter. |

|
|  |

7/12 |

INP Expression Validation |

Verify INP-PnbA expression on cell surface. |

Expression confirmed; some cells lysed, adjusted induction conditions. |

|
|  |

7/13 |

Whole-Cell Catalysis Trial |

React 20 μg/mL chlorimuron-E with BL21-INP-PnbA for 30 min at 25°C. |

Partial degradation observed; reaction repeated. |

|
|  |

7/14 |

ELISA Measurement |

Measure remaining chlorimuron-E. |

Degraded 28.1 μg/mL; BL21 and BL21-PnbA negligible. |

|
|  |

7/15 |

Replicate Whole-Cell Test |

Confirm degradation efficiency. |

Results consistent. |

|
|  |

7/16–7/17 |

Data Compilation |

Compile all degradation, activity, temperature, and pH data. |

Data consistent; some outlier in SulE temperature test, removed. |

|
|  |

7/18–7/20 |

Analysis & Figures |

Generate figures for publication/presentation. |

Figures completed; some formatting issues corrected. |

|
|  |

7/21–7/25 |

Final Validation |

Repeat key assays for reproducibility; summarize results. |

All major findings validated; minor SulE issues noted but did not affect conclusions. |

# IAA (Indole-3-Acetic Acid) Synthesis Experiments

|
|  |

Date |

Experiment |

Method |

Notes/Problems |

|
|  |

7/30 |

Gene Circuit Design |

Draw IAA synthesis gene circuit map |

Circuit design completed |

|
|  |

7/31 |

Gene Sequence Retrieval |

Retrieve iaaM and iaaH sequences from NCBI |

Sequences successfully obtained |

|
|  |

8/1 |

Codon Optimization |

Optimize codons for E. coli; remove EcoRI, XbaI, SpeI, PstI |

IaaH initially had secondary structure issues, resolved after adjustment |

|
|  |

8/2 |

Cloning into Vector |

Clone into pET28a(m) via NdeI/XhoI |

IaaM cloned successfully; IaaH failed initially, repeated and succeeded |

|
|  |

8/3 |

BL21 Transformation |

Transform plasmid into E. coli BL21 |

Transformation successful |

|
|  |

8/4 |

Preliminary Expression Induction |

Induce with IPTG, extract crude enzyme |

IaaH expression weak; IPTG concentration increased |

|
|  |

8/5 |

WB Verification |

Western blot to detect protein expression |

Both enzymes expressed; IaaH band faint |

|
|  |

8/6 |

IAA Standard Curve |

Prepare IAA standards and measure A450 via ELISA |

Standard curve established; pipetting error required repeat |

|
|  |

8/7 |

IAA Curve Re-measurement |

Repeat ELISA for verification |

Data consistent |

|
|  |

8/8 |

Fermentation Induction |

Grow 5 mL LB+kana to OD600=0.6, induce with 0.5 mM IPTG for 12h |

Culture grew well; temperature slightly low, some cells grew slowly |

|
|  |

8/9 |

Supernatant Collection |

Centrifuge 1.5 mL culture, measure IAA via ELISA |

IAA 14.9 μM; wild-type and single-enzyme strains produced almost no IAA |

|
|  |

8/10 |

Production Tracking |

Measure IAA over 24h |

Final IAA 15.09 μM |

|
|  |

8/11 |

Data Organization |

Organize production data |

Data consistent |

|
|  |

8/12 |

Wheat Seed Preparation |

Surface sterilize seeds with 75% ethanol 1 min, rinse 3x with sterile water |

Seeds sterilized; some floated, gently pressed |

|
|  |

8/13 |

Seed Soaking |

Soak seeds in 0 μM (control) or 10 μM IAA for 4–6h |

Soaking uniform; some seeds absorbed insufficiently, extended 30 min |

|
|  |

8/14 |

Sowing |

Place seeds on moist filter paper in Petri dishes, maintain moisture, 25°C |

Seeds evenly placed; filter paper slightly dry, added water |

|
|  |

8/15 |

Germination Observation |

Day 1 germination check |

Low germination; some seeds didn’t sprout, checked water content |

|
|  |

8/16 |

Germination Observation |

Day 2 recording |

Experimental group germination increased; control slower |

|
|  |

8/17 |

Germination Observation |

Day 3 recording |

Data normal; some seeds with black spots removed |

|
|  |

8/18 |

Root & Shoot Measurement |

Day 5 measurement |

Experimental group height 4.17 cm, root 2.43 cm; control height 3.87 cm, root 2.10 cm |

|
|  |

8/19 |

Growth Observation |

Day 6 recording |

Filter paper kept moist; experimental roots slightly curled, monitored water |

|
|  |

8/20 |

Growth Observation |

Day 7 recording |

Root elongation significant; experimental group advantage maintained |

|
|  |

8/21 |

Growth Observation |

Day 8 recording |

Measure shoot and root length; some control roots beginning to shrink |

|
|  |

8/22 |

Growth Observation |

Day 9 recording |

Data recorded normally |

|
|  |

8/23 |

Root & Shoot Measurement |

Day 10 measurement |

Experimental height 10.40 cm, root 9.27 cm; control height 8.90 cm, root 5.07 cm |

|
|  |

8/24 |

Growth Observation |

Day 11 recording |

Filter paper slightly dry, added water |

|
|  |

8/25 |

Growth Observation |

Day 12 recording |

No issues |

|
|  |

8/26 |

Growth Observation |

Day 13 recording |

Experimental leaves slightly yellow; checked nutrient solution |

|
|  |

8/27 |

Growth Observation |

Day 14 recording |

Data normal |

|
|  |

8/28 |

Root & Shoot Measurement |

Day 15 measurement |

Experimental height 17.47 cm, root 15.9 cm; control height 14.83 cm, root 8.23 cm; IAA promoted wheat growth successfully |

# Safety system

|
|  |

Date |

Experiment |

Method |

Notes/Problems |

|
|  |

9/2 |

Low-Temperature Promoter Design |

Design gene circuit with PcspA driving mRFP |

Circuit design completed |

|
|  |

9/3 |

Plasmid Construction |

Clone mRFP under PcspA into plasmid |

Initial ligation failed; repeated successfully |

|
|  |

9/4 |

BL21 Transformation |

Transform plasmid into E. coli BL21 |

Transformation successful |

|
|  |

9/5 |

Preliminary Expression Test |

Induce at 16°C and 37°C, observe mRFP fluorescence |

Fluorescence strong at 16°C; minimal at 37°C |

|
|  |

9/6 |

Fluorescence Measurement |

Quantify mRFP expression |

Data confirmed temperature-dependent expression |

|
|  |

9/7 |

Self-Destruct Gene Design |

Design T4 Holin and T4 Lysozyme gene circuit |

Design completed |

|
|  |

9/8 |

Plasmid Construction |

Clone T4 Holin/Lysozyme into plasmid |

Initial cloning of Lysozyme failed; repeated and succeeded |

|
|  |

9/9 |

BL21 Transformation |

Transform plasmid into E. coli BL21 |

Transformation successful |

|
|  |

9/10 |

Growth Curve Test at 37°C |

Compare wild-type and engineered strain growth |

No significant growth difference; self-destruct not induced at 37°C |

|
|  |

9/11 |

Growth Curve Test at 16°C |

Induce self-destruct system |

Engineered cells gradually died; system functional |

|
|  |

9/12 |

Verification |

Observe cell death under microscope |

Some cells survived longer than expected; increased induction time |

|
|  |

9/13 |

Data Recording |

Record OD600 and cell survival |

Data consistent with expected self-destruction |

|
|  |

9/14 |

Repeat Test |

Confirm reproducibility |

Repeated; minor variation, confirmed function |

|
|  |

9/15 |

Summary & Reporting |

Summarize low-temp promoter and self-destruct system data |

Systems validated; ready for integration with metabolic engineering |

# Notebook

{% 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 %}

Content

* [Overview](#section-1)
  + [Cycle 1: Chlorimuron-ethyl Degradation System](#section-2)
    - [Cycle 1-1: Validation of Degradative Enzymes](#subsection-3)
    - [Cycle 1-2 Cell Surface Display Technology](#subsection-4)
  + [Cycle 2 Plant Nutrient System](#section-5)
    - [Cycle 3 Safety System](#section-6)
      * [Cycle 3-1 Cold-Inducible Promoter Test](#subsection-7)
      * [Cycle 3-2 Suicide System](#subsection-8)
    - [Summary](#section-9)

# Overview

Chlorimuron-ethyl is a sulfonylurea herbicide widely used in the soybean–wheat rotation system. It inhibits acetohydroxyacid synthase (AHAS/ALS) to block the synthesis of branched-chain amino acids, thereby suppressing crop growth. However, its strong soil persistence may cause rooting disorders in subsequent crops, soil microbiome imbalance, and water pollution, posing risks to agricultural production and the ecosystem.

To address this, we designed and constructed a chlorimuron-ethyl degradation system. We screened three degradative enzymes from different sources—SulE (esterase) from H. zhihuaiae, PnbA (carboxylesterase) from strain CHL1, and GST (glutathione S-transferase) from K. jilinsis 2N3—and achieved heterologous expression and validation in E. coli. ELISA quantification showed that PnbA performed best, degrading 61.2% of chlorimuron-ethyl within 10 min, with optimal conditions at 30 ℃ and pH 7.

We further applied a cell surface display strategy to anchor PnbA on the host surface, enabling whole-cell catalysis and improving degradation efficiency. Meanwhile, to ensure controllability and biosafety in environmental applications, we designed an cold-inducible suicide system, allowing on-demand self-lysis to mitigate risks of gene and strain release. Altogether, from enzyme validation and whole-cell catalysis to safety control, we provide a complete degradation solution, offering a new avenue for bioremediation of chlorimuron-ethyl in farmland.

# Cycle 1: Chlorimuron-ethyl Degradation System

## Cycle 1-1: Validation of Degradative Enzymes

### Design

Chlorimuron-ethyl, a sulfonylurea herbicide commonly used in soybean–wheat rotations for its low cost and rapid action, inhibits AHAS/ALS, thereby blocking the synthesis of valine, leucine, and isoleucine. This disrupts protein/enzyme production, inhibits cell division in meristematic tissues, and ultimately causes plant death.

However, due to its long soil persistence (≥6 months), chronic use impairs rooting and growth of subsequent crops, disrupts soil microbial communities and enzyme activities, and reduces soil fertility. Its high water solubility facilitates entry into aquatic systems, interfering with aquatic plant growth and potentially causing DNA damage in aquatic/amphibian species—threatening ecosystems and biodiversity. It also suppresses chlorophyll and superoxide dismutase, reducing photosynthesis and nutrient uptake.

Therefore, we selected three degradative enzymes: SulE from H. zhihuaiae, PnbA from strain CHL1, and GST from K. jilinsis 2N3.

![Cycle 1-1: Validation of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/engineering/img1.webp)

Figure 1. Principle of chlorimuron-ethyl–degrading enzymes.

### Build

To obtain strains capable of efficiently degrading chlorimuron-ethyl, we designed a dedicated genetic circuit comprising the chlorimuron-ethyl–degrading enzymes. We selected E. coli BL21 (DE3) as the chassis organism and, using standard molecular cloning procedures, inserted the coding sequences of SulE (esterase from H. zhihuaiae), PnbA (carboxylesterase from strain CHL1), and GST (glutathione S-transferase from K. jilinsis 2N3) into the pET28a(m) expression vector. The resulting recombinant plasmids were then transformed into E. coli BL21 (DE3) to establish the engineered degradation strains (see Figure 2).

![Cycle 1-1: Validation of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/engineering/img2.webp)

Figure 2. Gene circuit and plasmid maps for the degradation system.

### Testing

Coding sequences for SulE, PnbA, and GST were obtained from NCBI, codon-optimized for E. coli, and edited to remove EcoRI, XbaI, SpeI, PstI, NdeI, XhoI to meet RFC#10 and pET28a(m) requirements, then synthesized (Generalbial, China). Inserts were cloned via NdeI/XhoI into pET28a(m). After PCR amplification and gel verification (Figure 3), heat-shock transformation (42 ℃, 1 min) into E. coli DH5α was performed. Kan (100 μg/mL) LB agar (1.5% agar) was used to select positives, which were sequenced (Tsingke, Beijing). Plasmids were purified (DP103, Tiangen) and transformed into E. coli BL21(DE3) to yield expression strains.

![Cycle 1-1: Validation of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/engineering/img3.webp)

Figure 3. Agarose gel confirming insert amplification.

For expression, strains were IPTG-induced, crude lysates prepared, and Western blot (WB) confirmed protein production for all three enzymes (Figure 4).

![Cycle 1-1: Validation of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/engineering/img4.webp)

Figure 4. Western blot validation of SulE, PnbA, and GST expression.

To evaluate the enzyme activity and degradation efficiency, we first established a standard calibration curve for chlorimuron-ethyl. The log10 of chlorimuron-ethyl concentration was used as the independent variable, and the absorbance at 450 nm (A450) measured by ELISA served as the dependent variable.

A regression analysis was performed to generate the standard curve correlating concentration and absorbance (Figure 5).

![Cycle 1-1: Validation of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/engineering/img5.webp)

Figure 5. Standard curve of chlorimuron-ethyl. (A) Relationship between chlorimuron-ethyl concentration and A450 absorbance determined by ELISA.(B) Linear correlation between log10 (chlorimuron-ethyl concentration) and A450 absorbance.

To verify the catalytic efficiency of the three degradation enzymes, we tested the ability of their crude enzyme extracts to degrade chlorimuron-ethyl. A total of 1 mL of enzyme solution (1 μg/mL) was added to 20 μg/mL chlorimuron-ethyl, and the reaction was carried out at 25 ℃ for 10 minutes. Afterward, 50 μL of each sample was diluted tenfold, and the remaining chlorimuron-ethyl concentration was determined using an ELISA kit (ml103804, Mlbio).

For data analysis, Student’s t-test was applied to compare two groups, with p < 0.05 considered statistically significant. All experiments were performed with at least three independent biological replicates (n ≥ 3). The results demonstrated that PnbA showed the highest degradation capacity, with an absorbance at 450 nm (A450) of 0.57, compared to 0.39 for SulE and 0.40 for GST (Figure 6).

![Cycle 1-1: Validation of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/engineering/img6.webp)

Figure 6. ELISA analysis of chlorimuron-ethyl degradation by different enzymes.

Based on the standard curve and the measured A450 values, we calculated the residual chlorimuron-ethyl concentration and corresponding degradation rates (%). After a 10-minute reaction at 25 ℃, PnbA achieved a 61.2% degradation rate, while SulE and GST showed 29.8% and 32.3%, respectively (Figure 7).

![Cycle 1-1: Validation of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/engineering/img7.webp)

Figure 7. Degradation efficiency of SulE, PnbA, and GST after 10-minute reactions at 25 ℃.

Next, we defined one enzyme activity unit (U) as the amount of enzyme required to degrade 1 μmol of chlorimuron-ethyl per minute at 25 ℃. Specific activity (U/μg) was calculated based on the change in chlorimuron-ethyl concentration and its molecular weight (357.76 g/mol). The results showed that PnbA exhibited the highest specific activity of 0.0038 U/μg, followed by GST (0.0021 U/μg) and SulE (0.0019 U/μg) (Figure 8).

![Cycle 1-1: Validation of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/engineering/img8.webp)

Figure 8. Comparison of specific enzyme activities of SulE, PnbA, and GST.

We further investigated the optimal reaction conditions of PnbA. By adjusting the incubation temperature, we determined the optimal temperature based on relative activity, calculated as a percentage of the maximum specific activity (100%). The results indicated that PnbA exhibited its highest catalytic activity at 30 ℃ (Figure 9A). To determine the optimal pH, the pH of PBS buffer was adjusted using 1 M NaOH and 1 M HCl, and enzyme activity was measured accordingly. The results confirmed that PnbA performed best at pH = 7 (Figure 9B).

![Cycle 1-1: Validation of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/engineering/img9.webp)

Figure 9. Optimal reaction conditions for PnbA. (A) Temperature-dependent enzyme activity assay. (B) pH-dependent enzyme activity assay.

### Learn

We successfully evaluated the catalytic performance of the three chlorimuron-ethyl–degrading enzymes and identified PnbA as the most effective enzyme.

At 25 ℃ for 10 minutes, PnbA degraded 61.2% of chlorimuron-ethyl with a specific activity of 0.0038 U/μg. Furthermore, the optimal catalytic conditions of PnbA were determined to be 30 ℃ and pH = 7, providing a solid foundation for subsequent whole-cell catalysis experiments.

## Cycle 1-2 Cell Surface Display Technology

### Design

Cell surface display enables the presentation of peptides or proteins on the surface of microbial cells by fusing them with an anchoring motif. The fusion between the target protein and anchoring protein can occur through N-terminal fusion, C-terminal fusion, or sandwich fusion, depending on the structural requirements. In this project, we selected ice nucleation protein (INP) as the anchoring carrier. INP is a well-characterized outer membrane protein originating from ice-nucleating bacteria associated with plants, known for its ability to induce ice crystal formation in supercooled water. Due to its robust anchoring efficiency, INP has been widely applied in E. coli surface display systems. Therefore, we designed a fusion construct where INP was fused to the N-terminus of PnbA, enabling the anchoring of PnbA on the cell surface to facilitate whole-cell catalysis of chlorimuron-ethyl degradation (Figure 10).

![Cycle 1-2  Cell Surface Display Technology Diagram](https://static.igem.wiki/teams/5887/engineering/img10.webp)

Figure 10. Schematic diagram of PnbA surface display using INP as an anchoring protein.

### Build

To enable whole-cell catalysis, we utilized ice nucleation protein (INP) as a carrier for anchoring PnbA to the outer membrane of *E. coli*. The INP sequence was introduced into the BL21–PnbA strain, generating the engineered strain BL21–INP–PnbA (Figure 11).

![Cycle 1-2  Cell Surface Display Technology Diagram](https://static.igem.wiki/teams/5887/engineering/img11.webp)

Figure 11. Construction of the BL21–INP–PnbA strain for whole-cell degradation of chlorimuron-ethyl.

### Test

To evaluate the catalytic performance of the surface-displayed system, we collected the cell pellets from the cultured strains and resuspended them in a 20 μg/mL chlorimuron-ethyl solution (C109992, Aladdin).The mixtures were incubated at 25 ℃ for 30 minutes.

After incubation, the remaining chlorimuron-ethyl in the supernatant was quantified using a competitive ELISA assay. We compared the degradation capacities of wild-type BL21, BL21–PnbA, and BL21–INP–PnbA. The results showed that BL21 and BL21–PnbA exhibited negligible degradation activity, whereas BL21–INP–PnbA efficiently degraded 28.1 μg/mL of chlorimuron-ethyl within 30 minutes (Figure 12).

![Cycle 1-2  Cell Surface Display Technology Diagram](https://static.igem.wiki/teams/5887/engineering/img12.webp)

Figure 12. Degradation of chlorimuron-ethyl by whole-cell catalysis after 30 minutes of reaction at 25 ℃.The engineered strain BL21–INP–PnbA demonstrated significantly higher degradation efficiency compared to control strains.

### Learn

We successfully utilized cell surface display technology to express PnbA on the outer surface of E. coli*,* enabling whole-cell catalysis. The engineered strain BL21–INP–PnbA effectively degraded 28.1 μg/mL of chlorimuron-ethyl after 30 minutes at 25 ℃, confirming that surface anchoring markedly enhances degradation performance.

# Cycle 2 Plant Nutrient System

### Design

To address the issue of soil fertility depletion caused by long-term crop rotation, which limits plant growth and development, our team designed an indole-3-acetic acid (IAA) expression system after consultation with agricultural experts. The goal of this system is to reduce the negative impact of herbicide residues while simultaneously enriching the soil with plant growth–promoting substances, thereby enhancing seed germination and plant development. IAA, a naturally occurring auxin, is synthesized through the tryptophan–indole-3-acetamide (IAM) pathway, which involves two enzymatic reactions. In the first step, tryptophan is converted to IAM by the enzyme tryptophan-2-monooxygenase (IaaM) encoded by the *iaaM* gene. In the second step, IAM is hydrolyzed to IAA by IAM hydrolase (IaaH) encoded by the *iaaH* gene (Figure 13).

![Cycle 2  Plant Nutrient System Diagram](https://static.igem.wiki/teams/5887/engineering/img13.webp)

Figure 13. Schematic diagram of the heterologous IAA biosynthesis pathway in *E. coli*

### Build

To construct the IAA biosynthesis strain, we first designed a gene circuit for IAA synthesis. The genes *iaaM* and *iaaH* were retrieved from the NCBI database, codon-optimized for E. coli, and all restriction sites including EcoRI, XbaI, SpeI, and PstI were removed to comply with the RFC#10 standard and pET28a(m) cloning requirements. The optimized genes were cloned into the pET28a(m) expression vector via NdeI and XhoI restriction sites, and the verified constructs were then transformed into E. coli BL21 (Figure 14).

![Cycle 2  Plant Nutrient System Diagram](https://static.igem.wiki/teams/5887/engineering/img14.webp)

Figure 14. Gene circuit and related genetic elements for IAA biosynthesis in *E. coli*.

### Test

To quantify IAA production, we first established a standard calibration curve using an indole-3-acetic acid ELISA kit, measuring absorbance at 450 nm (Figure 15).

![Cycle 2  Plant Nutrient System Diagram](https://static.igem.wiki/teams/5887/engineering/img15.webp)

Figure 15. Standard curve of indole-3-acetic acid (IAA).(A) Relationship between IAA concentration and A450 absorbance determined by ELISA. (B) Linear correlation between log10 (IAA concentration) and A450 absorbance.

The engineered strains were cultured in 5 mL of LB medium containing kanamycin, and when the culture reached OD₆₀₀ = 0.6, 0.5 mM IPTG was added for induction. After 12 hours of shaking incubation at 180 rpm and 30℃, 1.5 mL of culture was centrifuged, and the supernatant was collected for ELISA measurement. Absorbance readings at 450 nm were compared to the standard curve to calculate IAA concentrations. The results showed that the engineered strain produced 14.9 μM of IAA after 12 hours of induction at 30℃, while the wild-type *E. coli* BL21 and the strain expressing only *iaaM* exhibited negligible IAA production (Figure 16).

![Cycle 2  Plant Nutrient System Diagram](https://static.igem.wiki/teams/5887/engineering/img16.webp)

Figure 16. Comparison of IAA concentrations between the engineered strain and wild-type *E. coli* BL21 after 12 hours of induction at 30℃.

To assess production stability, we measured IAA accumulation over a 24-hour period, and the results demonstrated that the engineered strain could produce 15.09 μM of IAA within 24 hours (Figure 17).

![Cycle 2  Plant Nutrient System Diagram](https://static.igem.wiki/teams/5887/engineering/img17.webp)

Figure 17. IAA production levels of the engineered strain at different time points within 24 hours.

Table 1. Effect of IAA on the growth and development of wheat seedlings

![Cycle 2  Plant Nutrient System Diagram](https://static.igem.wiki/teams/5887/engineering/img18.webp)

To evaluate the effect of microbially synthesized IAA on plant development, we conducted a wheat seed germination and growth assay. Healthy, intact wheat seeds were surface sterilized with 75% ethanol for 1 minute and rinsed three times with sterile water. The seeds were soaked in either 0 μM IAA (control) or 10 μM IAA (treatment) for 4–6 hours, then placed evenly on moist filter paper in Petri dishes. The dishes were kept at 25℃ under constant humidity, and germination and growth were observed daily. Root length and shoot height were measured on day 5, day 10, and day 15, and the results are summarized in Table 1.

![Cycle 2  Plant Nutrient System Diagram](https://static.igem.wiki/teams/5887/engineering/img19.webp)

Figure 18. Comparison of wheat seedling growth between the control group (0 μM IAA) and the experimental group (10 μM IAA) on day 15.

On day 5, the treatment group exhibited an average shoot height of 4.17 cm and root length of 2.43 cm, compared to 3.87 cm and 2.10 cm in the control group. On day 10, the treatment group measured 10.40 cm and 9.27 cm, while the control group measured 8.90 cm and 5.07 cm. On day 15, the treated seedlings reached 17.47 cm in height and 15.9 cm in root length, whereas the control group measured 14.83 cm and 8.23 cm, respectively. These results indicate that IAA significantly enhances seed germination and promotes the growth of wheat seedlings (Figure 18).

### Learn

In summary, we successfully constructed an IAA-producing E. coli strain capable of synthesizing 14.9 μM of IAA within 12 hours at 30℃. Furthermore, the addition of 10 μM IAA significantly promoted wheat germination and growth, with the experimental group exhibiting greater vigor and development than the control group by day 15. These results demonstrate that our engineered strain can effectively synthesize plant auxin and promote crop growth, providing a potential biological solution for soil fertility enhancement and sustainable agriculture.

# Cycle 3 Safety System

## Cycle 3-1 Cold-Inducible Promoter Test

### Design

To ensure biosafety under field conditions, we designed a temperature-responsive regulatory system tailored to the soybean–wheat crop rotation widely practiced in northern China. This agricultural cycle typically occurs from September to October, when daytime temperatures range from 23℃ to 28℃, and nighttime temperatures drop to 12℃–19℃. Therefore, a cold-inducible promoter was chosen to control gene expression in a temperature-dependent manner—activating degradation genes during the warmer daytime to efficiently break down chlorimuron-ethyl, while triggering self-lysis at lower nighttime temperatures, ensuring that the engineered bacteria do not persist or spread in the environment.

### Build

To test the functionality of the cold-inducible promoter, we constructed an mRFP reporter system under the control of the PcspA promoter (Figure 16).

![Cycle 3-1  Cold-Inducible Promoter Test Diagram](https://static.igem.wiki/teams/5887/engineering/img20.webp)

Figure 19. Construction of the cold-inducible promoter validation system, in which the PcspA promoter drives mRFP expression as a visible reporter.

### Test

Experimental results demonstrated that after incubation for 12 hours at different temperatures (16℃, 25℃, and 37℃), the measured fluorescence/OD₆₀₀ values were 221.32, 202.08, and 38.92, respectively. These results indicate that the promoter exhibited the strongest activity at 16℃, confirming its low-temperature inducibility (Figure 17).

![Cycle 3-1  Cold-Inducible Promoter Test Diagram](https://static.igem.wiki/teams/5887/engineering/img21.webp)

Figure 20. Performance of the cold-inducible promoter (PcspA) at different temperatures. The fluorescence/OD₆₀₀ ratio is highest at 16℃, indicating maximal induction of mRFP expression.

### Learn

We successfully validated the function of the cold-inducible promoter in *E. coli*, confirming that PcspA can effectively induce the expression of mRFP at 16℃, providing a reliable temperature-sensitive regulatory element for subsequent biosafety control.

## Cycle 3-2 Suicide System

### Design

To further enhance biosafety, we designed a temperature-dependent suicide system regulated by the CspA promoter, enabling the engineered bacteria to undergo self-lysis when exposed to low temperatures. The system is based on two lytic proteins derived from T4 bacteriophage—T4 Holin and T4 Lysozyme. T4 Holin is a small membrane protein containing two transmembrane domains, with its N-terminus located in the cytoplasm and the C-terminus in the periplasmic space. This structure allows Holin to form pores in the bacterial membrane, disrupting membrane integrity. T4 Lysozyme, a small globular protein consisting of 164 amino acids, binds to the bacterial cell wall, where it cleaves the glycosidic bonds between N-acetylglucosamine and N-acetylmuramic acid. This action compromises the cell wall’s structural stability and disrupts osmotic balance, ultimately leading to cell lysis and death.

### Build

We constructed a plasmid in which the pCspA promoter drives the co-expression of T4 Holin and T4 Lysozyme, enabling temperature-controlled activation of the suicide system (Figure 18).

![Cycle 3-2  Suicide System Diagram](https://static.igem.wiki/teams/5887/engineering/img22.webp)

Figure 21. Design and fragment verification of the temperature-inducible suicide system driven by the CspA promoter, including T4 Holin and T4 Lysozyme modules.

### Test

To evaluate the system, we compared the growth curves of the engineered strain and wild-type *E. coli* at 37℃ and 16℃. At 37℃, both strains exhibited similar growth patterns, indicating that the suicide system remained inactive under normal cultivation conditions. However, when the temperature was lowered to 16℃, the engineered strain underwent continuous cell death, validating the functional activation of the suicide gene circuit (Figure 19).

![Cycle 3-2  Suicide System Diagram](https://static.igem.wiki/teams/5887/engineering/img23.webp)

Figure 22. Functional validation of the temperature-inducible suicide system at 37℃ and 16℃.

### Learn

We successfully constructed and validated the temperature-inducible suicide system, confirming that it can effectively trigger self-lysis at 16℃, while remaining inactive at 37℃. This ensures environmental safety by preventing the uncontrolled proliferation of engineered bacteria outside the intended application context.

# Summary

In this study, we successfully constructed and validated an integrated chlorimuron-ethyl degradation system with embedded safety controls. Among the three degradation enzymes tested, PnbA was identified as the optimal candidate, exhibiting the highest degradation rate and specific activity. By combining cell surface display technology, the engineered bacteria achieved efficient degradation of chlorimuron-ethyl within a short time frame, significantly enhancing its application potential. Furthermore, the introduction of a cold-inducible suicide system ensures precise control and environmental containment of the engineered strain. Collectively, this work provides an effective biological solution for the remediation of chlorimuron-ethyl pollution, while establishing a biosafe framework for engineered microorganisms in environmental and agricultural applications.

{% 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 Chlorimuron-ethyl-Degrading Enzyme Overexpression Strains](#section-1)
  + [Gene Expression and Crude Enzyme Preparation](#section-2)
    - [Enzyme Assay and Biochemical Characterization](#section-3)
      * [Construction of Surface Display System](#section-4)
        + [Analysis of Chlorimuron-ethyl Degradation by Whole-Cell Biocatalysts](#section-5)
          - [Construction of Indole-3-Acetic Acid (IAA) Producing Strains](#section-6)
            * [Determination of Indole-3-Acetic Acid (IAA) Production in Engineered Strains](#section-7)
              + [Testing of Cold-Inducible Reporter Strains](#section-8)
                - [Construction of Cold-Inducible Lysis System](#section-9)
                  * [Testing of Cold-Inducible Lysis System](#section-10)
                    + [IAA-Mediated Promotion of Plant Rooting](#section-11)
                      - [Statistical Analysis](#section-12)

# Construction of Chlorimuron-ethyl-Degrading Enzyme Overexpression Strains

The genes encoding GST, SulE, and PnbA were synthesized (Generalbial, China), codon-optimized for *E. coli*, and modified to remove EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards and the cloning requirements of the pET28a(m) vector, after which the fragments were inserted into the pET28a(a) backbone using NdeI and XhoI restriction sites. Competent *E. coli* BL21(DE3) (D1009S) and DH5α (D0351) cells were purchased from Beyotime (China), and the recombinant plasmids were transformed into *E. coli* DH5α via heat shock at 42 °C for 1 min, followed by selection on LB agar plates (1.5% agar) containing 100 μg/mL kanamycin, with positive clones verified by sequencing (Qingke, Beijing) to obtain DH5α recombinant strains. Plasmids were extracted using a plasmid extraction kit (DP103, Tiangen) and subsequently transformed into *E. coli* BL21(DE3) to generate engineered expression strains, which were cryopreserved at –80 °C with 25% (v/v) glycerol as a cryoprotectant. The engineered strains were cultured at 37 °C, 150 rpm in LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL kanamycin for inoculation and scale-up.

# Gene Expression and Crude Enzyme Preparation

The engineered strains BL21-GST, BL21-SulE, and BL21-PnbA were inoculated into LB medium supplemented with 100 μg/mL kanamycin at a 1:100 ratio and activated overnight at 37 °C, 150 rpm. Subsequently, 100 μL of the overnight culture was transferred into 20 mL of LB medium containing 100 μg/mL kanamycin in a sterile 50 mL centrifuge tube and cultured at 37 °C, 180 rpm for 3–4 h until the OD600 reached 0.6. Protein expression was induced by adding 0.5 mM IPTG (ST098-1g, Beyotime) and lowering the incubation temperature to 16 °C for an additional 16 h. Cells were harvested by centrifugation at 10,000 × g for 5 min, and the pellet was resuspended in 5 mL of pre-chilled PBS (pH 7.2) at 4 °C. Cell disruption was performed on ice using an ultrasonic cell disruptor (Jingxin, Shanghai, China) under the conditions of 70 W power, 1 s on/3 s off, for a total of 20 min. The lysate was centrifuged at 13,000 × g for 20 min at 4 °C, and the supernatant was collected as the crude enzyme extract. The supernatant was filtered through a 0.22 μm PES hydrophilic membrane (BS-PES-45, Biosharp), then concentrated to ~1 mL using a 10 kDa ultrafiltration tube (BS-UFC-150-010, Biosharp) by centrifugation at 2,500 × g for 30–40 min at 4 °C. Protein concentration was determined at 595 nm using the Bradford assay (P0006, Beyotime) with BSA as the standard. Finally, the crude enzyme extract was aliquoted in 100 μL portions and stored at –80 °C for further use.

P0006 Bradford Protein Concentration Assay Kit.pdf

0.4MB

[Download](https://static.igem.wiki/teams/5887/experiments/p0006-bradford-protein-concentration-assay-kit.pdf)

# Enzyme Assay and Biochemical Characterization

The crude enzyme extract was diluted in PBS to a final concentration of 1 μg/mL, and 20 μg/mL chlorimuron-ethyl (C109992, Aladdin) was added. After incubation at 25 °C for 10 min, a 50 μL sample was taken, diluted 10-fold, and analyzed using a chlorimuron-ethyl ELISA kit (ml103804, mlbio) to determine the residual chlorimuron-ethyl content. A standard curve was generated by plotting the log₁₀-transformed chlorimuron-ethyl concentration as the independent variable against the absorbance at 450 nm (A₄₅₀) as the dependent variable through regression analysis. Based on the standard curve and the measured A₄₅₀ values, the remaining chlorimuron-ethyl concentration and degradation rate (%) were calculated. One unit of enzyme activity (U) was defined as the amount of enzyme required to degrade 1 μmol of chlorimuron-ethyl per minute at 25 °C, and the specific activity was calculated according to the change in chlorimuron-ethyl concentration and its molecular weight (357.76 g/mol). To determine the optimal pH, PBS was adjusted with 1 M NaOH or 1 M HCl, and enzyme activity was measured under different conditions. Similarly, the optimal temperature was determined by incubating the reactions at varying constant temperatures. Relative enzyme activity was calculated by normalizing each condition to the maximum specific activity, which was set as 100%.

Chlorimuron-ethyl Detection Kit.pdf

0.7MB

[Download](https://static.igem.wiki/teams/5887/experiments/sulfosulfuron-detection-kit.pdf)

# Construction of Surface Display System

The INP-PnbA gene was synthesized and codon-optimized to comply with RFC#10 standards, then cloned into the pET28a(m) vector via NdeI and XhoI restriction sites. A total of 100 μg of the recombinant plasmid was transformed into *E. coli* BL21(DE3) (D1009S, Beyotime) using the heat-shock method. Transformants were selected on LB agar plates containing 100 μg/mL kanamycin, and positive clones were verified by colony PCR. Correct clones were subsequently inoculated into 5 mL of LB medium containing 100 μg/mL kanamycin and cultured overnight at 37 °C, 180 rpm. For cryopreservation, 1 mL of the BL21-INP-PnbA culture was mixed with 1 mL of 50% (v/v) glycerol and stored at –80 °C for future use.

# Analysis of Chlorimuron-ethyl Degradation by Whole-Cell Biocatalysts

Engineered strains and control strains were inoculated into LB medium containing 100 μg/mL kanamycin at a 1:100 ratio and cultured overnight at 37 °C, 150 rpm. Subsequently, 100 μL of the overnight culture was transferred into 5 mL of LB medium containing 100 μg/mL kanamycin in a sterile 50 mL centrifuge tube and incubated at 37 °C, 180 rpm for 3–4 h until the OD600 reached 0.6. Protein expression was induced by adding 0.5 mM IPTG (ST098-1g, Beyotime) and lowering the temperature to 16 °C for an additional 16 h. Cells were harvested by centrifugation at 10,000 × g for 5 min, washed three times with PBS (pH 7.2), and resuspended in 1 mL PBS, adjusting the OD600 to 1. Chlorimuron-ethyl (C109992, Aladdin) was added to a final concentration of 20 μg/mL. The reaction was carried out at 25 °C for 30 min, after which samples were centrifuged at 10,000 × g for 1 min. The pellet was discarded, and 50 μL of the supernatant was collected for analysis. Chlorimuron-ethyl concentration was determined using a chlorimuron-ethyl ELISA kit (ml103804, mlbio), and the residual concentration was calculated based on the standard curve and absorbance at 450 nm (A₄₅₀).

# Construction of Indole-3-Acetic Acid (IAA) Producing Strains

The *iaaM* and *iaaH* genes of the IAM pathway were synthesized (Generalbial, China) and assembled into a bicistronic sequence with the RBS B0034 between the two genes. The sequences were codon-optimized for *E. coli* and modified to remove EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards and the cloning requirements of the pET28a(m) vector. The constructs were cloned into the pET28a(a) backbone using NdeI and XhoI restriction sites. After sequence verification, the recombinant plasmids were transformed into *E. coli* BL21(DE3) (D1009S, Beyotime). Positive clones were selected on LB agar plates (1.5% agar) supplemented with 100 μg/mL kanamycin. Strains were preserved at –80 °C using 25% (v/v) glycerol as a cryoprotectant.

# Determination of Indole-3-Acetic Acid (IAA) Production in Engineered Strains

The IAA-producing engineered strains were inoculated into 5 mL of LB medium containing kanamycin and cultured overnight (12–16 h) at 37 °C, 180 rpm in a shaking incubator for activation. A 100 μL aliquot of the activated culture was transferred into 5 mL of fresh LB medium in a 25 mL Erlenmeyer flask and incubated at 37 °C, 180 rpm for 3–4 h. The optical density of the culture was measured at 600 nm (OD₆₀₀) using a microplate reader (FlexStation 3, Molecular Devices, USA). When OD₆₀₀ reached 0.6, protein expression was induced by adding 0.5 mM IPTG, and the incubation temperature was adjusted to 30 °C for an additional 12 h at 180 rpm. Throughout cultivation, the flasks were wrapped in aluminum foil to prevent IAA degradation caused by light exposure. After induction, 1.5 mL of culture was collected into a 1.5 mL microcentrifuge tube and centrifuged at 10,000 × g for 1 min. From the supernatant, 1 mL was collected, and 10 μL was mixed with 900 μL of blank LB medium to prepare a 100-fold diluted sample, which was thoroughly mixed and used for analysis. The IAA concentration was determined using an indole-3-acetic acid ELISA kit (YJ147100, YanjuBio). A standard curve was generated with known IAA concentrations, and absorbance (OD) was measured at 450 nm using a microplate reader (FlexStation 3, Molecular Devices, USA) to quantify IAA levels in the bacterial culture.

Plant-IAA.pdf

0.2MB

[Download](https://static.igem.wiki/teams/5887/experiments/plant-iaa.pdf)

# Testing of Cold-Inducible Reporter Strains

Overnight-activated cold-inducible reporter strains were inoculated into LB medium at a 1:100 ratio and cultured at different temperatures for 12 h under 180 rpm shaking conditions. A 200 μL sample of the culture was taken, and fluorescence (excitation wavelength: 584 nm; emission wavelength: 607 nm) as well as OD600 were measured using a microplate reader (Thermo Fisher Scientific, USA). The normalized fluorescence ratio (Fluorescence/OD600) was then calculated.

# Construction of Cold-Inducible Lysis System

The cold-inducible promoter *PcspA* and *E. coli* lethal genes T4 Holin–T4 Lysozyme were synthesized, with the T4 Holin–T4 Lysozyme operon placed downstream of *PcspA*. The T4 Holin and T4 Lysozyme sequences were codon-optimized for *E. coli*, and EcoRI, XbaI, SpeI, and PstI restriction sites were removed to comply with RFC#10 standards. The construct PcspA–T4 Lysis was cloned into the pSB1A3 vector between the XbaI and SpeI sites. Recombinant plasmids were transformed into *E. coli* BL21, and positive clones were screened on LB agar plates containing 100 μg/mL ampicillin (Amp) and verified by sequencing (Tsingke, China), yielding engineered strains. Engineered strains were cultured at 37 °C in LB broth supplemented with ampicillin, and bacterial growth was monitored by measuring OD600 using a microplate reader (FlexStation 3, Molecular Devices, USA).

# Testing of Cold-Inducible Lysis System

Overnight-activated cold-inducible lysis strains were inoculated at a 1:100 ratio into 5 mL of Amp+LB medium and cultured at 16 °C or 37 °C under 180 rpm shaking conditions for 20 h. At regular intervals, 200 μL culture samples were taken, and OD600 was measured using a microplate reader (FlexStation 3, Molecular Devices, USA) to generate bacterial growth curves.

# IAA-Mediated Promotion of Plant Rooting

Healthy and intact wheat seeds were surface-sterilized with 75% ethanol for 1 min, followed by three rinses with sterile water. The seeds were then soaked in either 0 μM (control) or 10 μM IAA solution for 4–6 h. After soaking, seeds were evenly placed onto Petri dishes lined with moistened filter paper, which was kept hydrated throughout cultivation at ~25 °C under constant conditions. Seed germination was monitored daily, and root length and shoot height were measured on days 5, 10, and 15 to evaluate the effect of IAA on wheat germination and growth.

# Statistical Analysis

Data analysis and graphing were performed using GraphPad Prism software. Results were presented as mean ± standard deviation (SD). For multiple group comparisons, one-way ANOVA followed by Tukey’s post-hoc test was used. For comparisons between two groups, Student’s t-test was applied. A p-value less than 0.05 was considered statistically significant. All experiments were performed with at least three independent biological replicates (n ≥ 3).

{% 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)
  + [Strain Construction](#section-2)
    - [Expression and Assay of Degradative Enzymes](#section-3)
      * [Indole-3-Acetic Acid (IAA) Biosynthesis and Characterization](#section-4)
        + [Biosafety System Construction and Validation](#section-5)
          - [Conclusion](#section-6)

# Overview

This study systematically engineered and validated multiple strains along two axes: chlorimuron-ethyl degradation and indole-3-acetic acid (IAA) biosynthesis. We retrieved the gene sequences of three degradative enzymes—SulE, PnbA, and GST—from the literature and NCBI, designed the gene circuits and plasmid maps, and cloned them into the pET28a(m) vector before transforming E. coli BL21(DE3) to obtain herbicide-degrading strains. Western blot (WB) and ELISA confirmed successful expression and catalytic activity of all three enzymes; PnbA showed the best performance for chlorimuron-ethyl degradation. We further optimized reaction temperature and pH and achieved cell-surface display of PnbA to enhance whole-cell catalysis. For IAA biosynthesis, we constructed a dual-enzyme strain (iaaM + iaaH) and quantified IAA via ELISA, showing stable production within 12–24 h. A wheat germination assay verified the growth-promoting effects of IAA. To address biosafety, we built a cold-inducible promoter system and demonstrated its feasibility at 16 °C, indicating potential for environmental containment in field applications.

# Strain Construction

#### Construction of Gene Circuits

To engineer strains capable of degrading chlorimuron-ethyl, we selected three enzymes: SulE (esterase) from Hansschlegelia zhihuaiae, PnbA (carboxylesterase) from strain CHL1, and GST (glutathione S-transferase) from Klebsiella jilinsis 2N3. We retrieved the corresponding gene sequences from NCBI and created the gene circuit diagrams and plasmid maps for each (see Figure 1).

![Strain Construction Diagram](https://static.igem.wiki/teams/5887/results/img1.webp)

Figure 1. Gene circuit designs and plasmid maps for the three chlorimuron-ethyl–degrading enzymes.

#### Overexpression Strains for Degradative Enzymes

We synthesized GST, SulE, and PnbA coding sequences (Generalbial, China), performed codon optimization for E. coli, and removed EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI sites to comply with RFC#10 and pET28a(m) cloning requirements. Inserts were cloned into pET28a(m) via NdeI/XhoI. After PCR amplification, target bands were verified (Figure 2), and the recombinant plasmids were transformed into E. coli DH5α by heat shock (42 °C, 1 min). Kanamycin (100 μg/mL) LB agar (1.5% agar) was used to select positive colonies, which were sequence-verified (Tsingke, Beijing). Plasmids were purified (DP103, Tiangen) and transformed into E. coli BL21(DE3) to generate the expression strains.

![Strain Construction Diagram](https://static.igem.wiki/teams/5887/results/img2.webp)

Figure 2. Agarose gel electrophoresis confirming insert amplification

# Expression and Assay of Degradative Enzymes

#### Expression and Assay of Degradative Enzymes

To confirm expression, IPTG induction was performed for all three strains, crude lysates were prepared, and Western blot (WB) verified protein production. Results indicated successful expression of SulE, PnbA, and GST (see Figure 3).

![Expression and Assay of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/results/img3.webp)

Figure 3. Western blot verification of the three degradative enzymes

#### Functional Validation: Standard Curve

We first established a standard curve for chlorimuron-ethyl by regressing A450 values against the log10 concentration of the herbicide (Figure 4).

![Expression and Assay of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/results/img4.webp)

Figure 4. Chlorimuron-ethyl calibration: (A) Concentration vs. A450; (B) log10(concentration) vs. A450.

To compare catalytic activities, 1 mL of crude enzyme (1 μg/mL) was incubated with 20 μg/mL chlorimuron-ethyl at 25 °C for 10 min. Samples (50 μL) were 10× diluted and analyzed using a competitive ELISA kit (ml103804, mlbio). Student’s t-test was used (two groups; p < 0.05 considered significant), with ≥3 biological replicates. PnbA performed best, yielding A450 = 0.57, versus 0.39 for SulE and 0.40 for GST (see Figure 5).

![Expression and Assay of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/results/img5.webp)

Figure 5. ELISA readouts after treatment with the three enzymes

Using the calibration curve, residual herbicide and degradation rates were calculated from A450 values. After 10 min at 25 °C, PnbA achieved 61.2% degradation, versus 29.8% (SulE) and 32.3% (GST) (Figure 6).

![Expression and Assay of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/results/img6.webp)

Figure 6. Degradation percentages after 10 min at 25 °C

Specific activity (U/μg) was defined as enzyme required to degrade 1 μmol of chlorimuron-ethyl per minute at 25 °C (MW = 357.76 g/mol). Measured values: PnbA = 0.0038 U/μg, SulE = 0.0019 U/μg, GST = 0.0021 U/μg (Figure 7).

![Expression and Assay of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/results/img7.webp)

Figure 7. Specific activities of PnbA, SulE, and GST

#### Optimal Conditions for PnbA

First, we investigated the catalytic capacity of PnbA at different temperatures by adjusting the incubator to fixed setpoints and testing the crude enzyme for its optimal reaction temperature. Taking the highest specific activity as 100%, we calculated the relative activity of each group. The results showed that PnbA exhibited optimal catalytic performance at 30 °C (Figure 8).

![Expression and Assay of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/results/img8.webp)

Figure 8. Effect of temperature on PnbA activity

Next, to investigate the effect of pH on the catalytic efficiency of PnbA, we adjusted the pH of PBS using 1 M NaOH and 1 M HCl, and tested the crude enzyme for its optimal pH. The experimental results demonstrated that PnbA showed the highest catalytic activity at pH = 7 (Figure 9).

![Expression and Assay of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/results/img9.webp)

Figure 9. Effect of pH on PnbA activity

#### Analysis of Chlorimuron-ethyl Degradation by Whole-cell Catalysts

To evaluate whole-cell catalytic degradation of chlorimuron-ethyl, we used ice nucleation protein (INP) as an anchoring carrier to express PnbA on the bacterial cell surface. Specifically, we introduced the INP sequence into the BL21-PnbA strain to obtain the BL21-INP-PnbA engineered strain, and collected the cultured cell pellets.

A 20 μg/mL chlorimuron-ethyl solution (C109992, Aladdin) was added, and the reaction was carried out at 25 °C for 30 minutes. The competitive ELISA assay was then performed to measure the degradation capacity of wild-type BL21, BL21-PnbA, and BL21-INP-PnbA.

The results showed that BL21 and BL21-PnbA exhibited negligible degradation, whereas BL21-INP-PnbA effectively degraded 28.1 μg/mL of chlorimuron-ethyl within 30 minutes (Figure 10).

![Expression and Assay of Degradative Enzymes Diagram](https://static.igem.wiki/teams/5887/results/img10.webp)

Figure 10. Analysis of chlorimuron-ethyl degradation by different whole-cell catalysts.(A) Genetic circuit diagram of PnbA surface display; (B) PCR verification of the INP fragment;(C) Whole-cell catalytic degradation of chlorimuron-ethyl after 30 minutes of reaction

# Indole-3-Acetic Acid (IAA) Biosynthesis and Characterization

#### Construction of IAA-producing Strains

First, we designed the genetic circuit for IAA biosynthesis and obtained the iaaM and iaaH genes—key components of the IAA pathway—from the NCBI database. These genes were codon-optimized for E. coli and modified to remove EcoRI, XbaI, SpeI, and PstI restriction sites to comply with the RFC#10 assembly standard and pET28a(m) cloning requirements. The optimized gene fragments were cloned into the pET28a(m) vector via the NdeI and XhoI restriction sites. Finally, the verified constructs were transformed into E. coli BL21 (DE3) to obtain the engineered IAA-producing strains (Figure 11).

![Indole-3-Acetic Acid (IAA) Biosynthesis and Characterization Diagram](https://static.igem.wiki/teams/5887/results/img11.webp)

Figure 11. Schematic diagram of the IAA biosynthetic pathway and related genes

#### Evaluation of IAA Production in Engineered Strains

First, the standard curve of indole-3-acetic acid (IAA) was established using a commercial ELISA assay kit by measuring absorbance at 450 nm (Figure 12).

![Indole-3-Acetic Acid (IAA) Biosynthesis and Characterization Diagram](https://static.igem.wiki/teams/5887/results/img12.webp)

Figure 12. Standard curve of indole-3-acetic acid (IAA). (A) Relationship between IAA concentration and A450 absorbance in ELISA assay.(B) Linear regression of log10(IAA concentration) versus A450 absorbance

Next, the engineered strain was cultured in 5 mL of LB medium supplemented with kanamycin. When the OD₆₀₀ reached 0.6, 0.5 mM IPTG was added to induce expression, and the culture was shaken at 180 rpm for 12 hours. After induction, 1.5 mL of bacterial culture was centrifuged, and the supernatant was analyzed using the IAA ELISA kit, measuring absorbance at 450 nm. IAA concentrations were calculated by comparison with the standard curve. The results showed that the IAA-producing engineered strain synthesized 14.9 μM of IAA after 12 hours of induction at 30°C, whereas wild-type BL21 and the strain expressing only IaaM produced negligible amounts of IAA (Figure 13).

![Indole-3-Acetic Acid (IAA) Biosynthesis and Characterization Diagram](https://static.igem.wiki/teams/5887/results/img13.webp)

Figure 13. Comparison of IAA concentrations between engineered strains and wild-type BL21 after 12-hour induction at 30°C

Finally, we quantified the IAA production over a 24-hour period, and the results demonstrated that the engineered strain continuously produced IAA, reaching a final concentration of 15.09 μM within 24 hours (Figure 14).

![Indole-3-Acetic Acid (IAA) Biosynthesis and Characterization Diagram](https://static.igem.wiki/teams/5887/results/img14.webp)

Figure 14. Time-course of IAA production in engineered *E. coli* within 24 hours

#### IIAA Promotion of Plant Growth

Table 1. Effect of IAA on the growth and development of wheat seedlings

![Indole-3-Acetic Acid (IAA) Biosynthesis and Characterization Diagram](https://static.igem.wiki/teams/5887/results/img15.webp)

To verify whether IAA promotes plant growth, we selected healthy and intact wheat seeds, disinfected them with 75% ethanol for 1 minute, and rinsed them three times with sterile water. The seeds were then soaked in 0 μM IAA solution (control) and 10 μM IAA solution (treatment) for 4–6 hours. After soaking, the seeds were evenly placed on moist filter paper in Petri dishes, which were maintained at approximately 25°C in a constant-temperature incubator. The filter paper was kept moist throughout the experiment. Germination and growth were observed daily, and root length and plant height were measured on days 5, 10, and 15 to assess the growth-promoting effect of IAA (Table 1).

![Indole-3-Acetic Acid (IAA) Biosynthesis and Characterization Diagram](https://static.igem.wiki/teams/5887/results/img16.webp)

Figure 15. Growth comparison of control and IAA-treated wheat seedlings on day 15

Experimental results showed that:

* On day 5, the average plant height and root length of the treatment group were 4.17 cm and 2.43 cm, compared to 3.87 cm and 2.10 cm in the control group.
* On day 10, the treatment group averaged 10.40 cm in height and 9.27 cm in root length, while the control group measured 8.90 cm and 5.07 cm, respectively.
* On day 15, the treatment group reached 17.47 cm in height and 15.9 cm in root length, compared to 14.83 cm and 8.23 cm in the control.

Overall, IAA-treated wheat seedlings exhibited significantly enhanced growth, confirming that IAA effectively promotes seed germination and seedling development (Figure 15).

# Biosafety System Construction and Validation

#### Validation of the Cold-Inducible Promoter Function

Given that our application scenario occurs in September, we selected a cold-inducible promoter to prevent potential release of the engineered bacteria into the environment. To evaluate its function, we constructed an mRFP reporter system driven by the PcspA promoter (Figure 16A, B). Experimental results showed that at 16°C, the engineered bacteria expressed red fluorescent protein (RFP) efficiently, whereas expression at 37°C (the optimal growth temperature for *E. coli*) was significantly lower (Figure 16C).

![Biosafety System Construction and Validation Diagram](https://static.igem.wiki/teams/5887/results/img17.webp)

Figure 16. Verification of the cold-inducible promoter system. (A) Design of the PcspA–mRFP genetic circuit. (B) Verification of PcspA and mRFP fragments. (C) Fluorescence analysis under low-temperature induction.

#### Construction and Verification of the Suicide System

Next, we cloned T4 phage-derived lysis genes—T4 holin and T4 lysozyme—into plasmids to test their functionality. At 37°C, both wild-type and engineered strains showed similar growth curves, with no observable cell lysis. However, when the temperature was reduced to 16°C, the engineered strain exhibited progressive cell death, confirming the successful activation and function of the suicide gene system (Figure 17).

![Biosafety System Construction and Validation Diagram](https://static.igem.wiki/teams/5887/results/img18.webp)

Figure 17. Verification of the temperature-dependent suicide system. (A) Design of the suicide gene circuit. (B) Validation of T4 holin and T4 lysozyme fragments. (C) Growth curve at 37°C (no induction). (D) Growth curve at 16°C, showing temperature-triggered self-lysis.

# Conclusion

This study successfully constructed and validated multiple functional engineered strains, achieving the following:

* Chlorimuron-ethyl–degrading strain:All three enzymes showed degradation activity, with PnbA performing best (61.2% degradation rate, specific activity 0.0038 U/μg). Under optimized conditions, PnbA displayed excellent catalytic efficiency, and surface expression via INP further enhanced its degradation performance.
* IAA-producing strain: The engineered strain stably synthesized 14.9–15.09 μM IAA within 12–24 hours, effectively promoting wheat germination and growth.
* Cold-inducible biosafety system: The PcspA promoter successfully triggered gene expression at 16°C, demonstrating potential for environmental containment and biosafety assurance.

In summary, this work establishes an efficient microbial catalytic system for chlorimuron-ethyl degradation, constructs an IAA-producing strain that stimulates plant growth, and integrates a cold-inducible safety mechanism for biocontainment.

Together, these advances provide a feasible and controllable synthetic biological strategy for agricultural pollutant remediation and crop growth enhancement.

{% endblock %}