iGEM 2025 — Team hnu-china

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{% 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 %}
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## Article Navigation

* [Notebook](#article1)

# Notebook

Note book is magic book.

The following is our [Notebook](https://static.igem.wiki/teams/5883/notebook/notebook.pdf).

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{% block title %}Engineering{% endblock %}
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## Article Navigation

* [Introduction](#article1)

# Engineering

#### Translation and Expansion: Naphthalene Degradation Project in Petroleum

## Introduction

**In petroleum, residual naphthalene poses potential environmental risks due to its persistence and toxicity. To address this issue, our research team initiated a project focused on identifying a gene capable of degrading naphthalene, aiming to develop an efficient biological degradation solution.**

**After an extensive review of relevant literature, databases, and prior research findings, we identified a potential candidate: the nahC gene, which is widely reported to be associated with naphthalene degradation pathways in certain microorganisms. To further study its function, we planned to clone this gene into the pET-30a vector—a commonly used expression vector in prokaryotic systems, known for its high copy number and compatibility with Escherichia coli (E. coli) hosts. Despite dedicated efforts over several days, including optimizing ligation conditions, adjusting transformation protocols (such as heat shock time and temperature), and verifying the integrity of the recombinant vector via gel electrophoresis, we encountered repeated failures in successfully transforming the nahC-containing pET-30a into E. coli. This setback prompted us to reassess our strategy and explore alternative approaches.**

**We then shifted our focus to a novel concept: instead of relying on intracellular expression of the naphthalene-degrading enzyme, could we engineer the enzyme to be expressed on the cell membrane surface of E. coli? This design would allow the enzyme to directly interact with naphthalene in the extracellular environment, potentially enhancing degradation efficiency by eliminating the need for substrate uptake into the cell. To achieve membrane surface expression, we researched and identified inteins—short peptide segments with unique self-splicing and protein ligation capabilities, which can covalently link target proteins to membrane-anchored proteins. Theoretically, fusing the naphthalene-degrading enzyme gene with an intein gene and a membrane anchor gene could enable the enzyme to be displayed on the E. coli cell surface. However, this approach was ultimately abandoned due to two critical challenges: first, the lack of detailed structural information about the naphthalene-degrading enzyme, which made it impossible to predict optimal fusion sites; second, the high complexity of protein conformation—even small changes in the enzyme’s amino acid sequence (from fusion with inteins) could disrupt its active site, rendering it non-functional.**

**Given the difficulties with direct enzyme expression, we decided to first optimize the separation and bioavailability of naphthalene and phenanthrene (another common polycyclic aromatic hydrocarbon, PAH, in petroleum). Through further research, we discovered that rhlAB—a gene cluster encoding enzymes responsible for synthesizing rhamnolipids—could serve as a key auxiliary component. Rhamnolipids are biosurfactants that can reduce the surface tension of aqueous solutions, enhance the solubility and desorption of hydrophobic PAHs (like naphthalene) from environmental matrices (e.g., soil or oil), and thus improve their bioavailability for microbial degradation.**

**To harness the function of rhlAB, we cloned this gene cluster into the pET-28a vector (a sister vector to pET-30a, featuring a kanamycin resistance gene and a His-tag for protein purification) and used the T7 promoter—a strong, inducible promoter that drives high-level gene expression in E. coli strains containing the T7 RNA polymerase gene (e.g., BL21(DE3)). After optimizing induction conditions (including IPTG concentration and induction time), we successfully achieved high-level expression of rhlAB in E. coli, confirmed by detecting rhamnolipid production via thin-layer chromatography (TLC) and measuring its surfactant activity.**

**Next, we sought to expand our PAH degradation capacity to phenanthrene, a more complex PAH than naphthalene. We selected the phnC gene from the phn gene cluster (phn cluster), which is documented to play a crucial role in the upstream steps of phenanthrene degradation. Theoretically, PhnC (the protein encoded by phnC) acts as a transporter or enzyme that initiates the conversion of phenanthrene into catechol—a central intermediate in many aromatic compound degradation pathways, which can be further metabolized by E. coli to produce energy and biomass.**

**We cloned the phnC gene into a suitable expression vector, transformed it into E. coli, and verified the successful construction of the phnC-engineered strain via PCR and DNA sequencing. To maximize PAH degradation efficiency, we then combined this phnC-containing strain with the previously constructed rhlAB-expressing strain in a co-culture system. In this system, the rhlAB strain produces rhamnolipids to enhance the desorption and solubility of naphthalene and phenanthrene, while the phnC strain converts phenanthrene into catechol for subsequent degradation.**

**To evaluate the performance of this co-culture system, we conducted soil-based degradation experiments. Soil samples contaminated with naphthalene and phenanthrene were treated with the co-culture, and after a set incubation period, we used a UV-visible spectrophotometer to measure the residual PAH concentrations (based on the characteristic absorption peaks of naphthalene and phenanthrene in the UV range). The results clearly showed a significant reduction in the total PAH content in the treated soil compared to the untreated control group, confirming the effectiveness of our engineered strain co-culture in degrading petroleum-derived PAHs. This research provides a promising biological strategy for the remediation of PAH-contaminated environments.**

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## Article Navigation

* [I
  Ligation, Transformation, and Verification of the Degradation Cassette](#article1)

  + [Principle](#1-1)
  + [Conclusion](#1-2)
  + [Experimental Plan](#1-4)
  + [Procedure](#1-5)+ [Functional Verification](#1-6)
* [II
  Ligation, Transformation, and Verification of PhnSR](#article2)

  + [Principle](#2-1)
  + [Procedure](#2-2)
  + [Functional Verification](#2-3)
* [III
  Ligation, Transformation, and Preliminary Verification of rhlAB](#article3)

  + [Principle](#3-1)
  + [Procedure](#3-2)
  + [Functional Detection](#3-3)

# Experiments

## I. Ligation, Transformation, and Verification of the Degradation Cassette Ligation of NahABFEDG with nahC and phnC:

### Principle:

  The A-G genes of the Nah cluster are responsible for converting naphthalene to catechol (Figure 2A-II) and can theoretically also convert phenanthrene. If phenanthrene undergoes this conversion, the product is 1,2-dihydroxynaphthalene, which is an intermediate in naphthalene degradation (Figure 1A-III) and can enter the naphthalene degradation pathway. Precisely because of this, organisms possessing the nah cluster also have a limited ability to degrade phenanthrene. The reason for this weak activity is likely that nahC has a significantly lower affinity for 1,2-dihydroxyphenanthrene (Figure 1B-III) than for the naphthalene-derived intermediate (Figure 1A-III). In contrast, the phnC gene from the phn cluster can act on 1,2-dihydroxyphenanthrene (B-III), but its downstream pathway (Figure 2B1) differs from the naphthalene degradation pathway of the nah cluster and is specific for phenanthrene degradation.

### Conclusion:

  Introducing phnC into cells containing nahA-G will likely enable them to convert both phenanthrene and naphthalene to catechol.

![111](https://static.igem.wiki/teams/5883/experiments/111.webp)

![222](https://static.igem.wiki/teams/5883/experiments/222.webp)

#### Figures:

  Figure 1A to Figure 2A: Degradation of naphthalene by the nah cluster.

  Figure 1B to Figure 2B: Degradation of phenanthrene by the phn cluster.

### Experimental Plan:

  Parallel experiments are designed comparing the modified nah cluster (introducing phnC) and the native nah cluster (using nahC). Three fragments were ordered: nahA-G (with nahC excised), nahC, and phnC, all supplied in the pET30a+ vector. Using the NdeI & EcoRI sites located after nahA-G in the vector, nahC and phnC will be ligated separately into the backbone, requiring only a single ligation step for each. This will yield the recombinant plasmids pET30a+-nahABFEDG-nahC and pET30a+-nahABFEDG-phnC. These will then be transformed separately, protein expression checked, and degradation capability tested.

![333](https://static.igem.wiki/teams/5883/experiments/333/333.webp)

### Procedure:

  1. Culture bacteria containing pET-30a(+)-nahC, pET-30a(+)-phnC, and pET30a+-nahABFEDG in preparation for plasmid extraction.

  2. Amplify nahC and phnC fragments via colony PCR from revived cultures of pET-30a(+)-nahC and pET-30a(+)-phnC, respectively. Use a 50μL PCR system with ordered primers. These primers have the required restriction enzyme site sequences incorporated internally, flanked by 5' protective bases. Run 5μL of the PCR product on a gel to verify nucleic acid size, followed by PCR product purification.

  NahC: 906bp, Annealing Temp: 54°C
  phnC: 825bp, Annealing Temp: 59°C

#### Ordered Primer Sequences

  nahC-EcoRI-F: ATAT**GAATTC**GCCTGACTCAGTTTTACATC
  nahC-R: ATAT**GCTAGC**GAATCAGGAGGAAACTAT
  phnC-EcoRI-F: ATAT**GAATTC**GGACCCGCCACCTTCATTTC
  phnC-R: ATAT**GCTAGC**GAGAGAGGAGGACAAGAT

  3. After culturing pET30a+-nahABFEDG bacteria for 16 hours, extract the plasmid. Use DL5000 marker as reference, and run the gel using 0.8% agarose, with gel preparation ratio according to protocol. The full length of the pET30a+-nahABFEDG plasmid is 14.3 kbp.

  4. Perform double digestion on pET30a+-nahABFEDG, nahC PCR product, and phnC PCR product using EcoRI & NheI. Use the system specified in the protocol, with a digestion duration of 4 hours. After digestion of pET30a+-nahABFEDG, perform gel extraction (or adjust centrifugation speed during product purification) to remove the small fragment between the two restriction sites on the plasmid, preventing self-ligation. This is necessary because the size of the final ligation product is close to that of the original pET30a+-nahABFEDG, making them difficult to distinguish even by gel electrophoresis. Ligate to obtain the two recombinant plasmids: pET30a+-nahABFEDG-nahC and pET30a+-nahABFEDG-phnC.

  5. Transform into E. coli DH5α. Use the recombinant plasmid transformation procedure as per the protocol. After transformation, plate 20μl/200μl onto LA (Kan+) plates. Incubate at 37°C in a constant temperature incubator for 16 hours. Select successfully transformed colonies (those able to grow on LA (Kan+) plates) and send for sequencing. Use T7 and T7 terminator primers for sequencing.

  6. Compare sequencing results with the designed sequences and check using NCBI's BLAST function. After confirming successful transformation, extract the plasmids and transform them into the final chassis – the expression strain BL21(DE3). Our degradation cassette at this stage relies on the T7 promoter of pET30a(+) for activation. The T7 promoter of the pET series does not function in DH5α due to the lack of T7 RNA polymerase. After transformation, plate 20μl/200μl onto LA (Kan+) plates. Incubate at 37°C in a constant temperature incubator for 16 hours. Select successfully transformed colonies. Detect three tags via Western Blot: FLAG tag on NahAc, HA tag on NahE, and 6\*His tag on NahC/phnC.

### Functional Verification of the Degradation Cassette:

  An experiment is designed to compare the residual phenanthrene content after engineered bacteria are cultured in PHE-containing medium for specific time intervals.

  (1) PHE Standard Curve: Prepare a stock solution of 1mg PHE / ml DMSO.
  PHE Final Concentration (μg/ml): 2, 4, 6, 8, 10
  Stock Solution Added (μl/ml medium): 2, 4, 6, 8, 10
  Use a microplate reader to characterize the phenanthrene concentration in the solution. Program: Absorbance Monochromator - 251nm, because PHE has an absorption peak at 251nm. Establish the correlation between the microplate reader reading and the PHE final concentration.

  (2) IPTG Concentration Gradient: Prepare a 1M IPTG aqueous solution.
  IPTG Final Concentration (mM): 0, 1, 2
  Stock Solution Added (μl/ml medium): 0, 1, 2

  (3) Control: Use DMSO instead of the PHE DMSO solution to assess the effect of DMSO on absorbance.

  (4) Control: Use BL21(DE3) without the target genes instead of the engineered bacteria, to verify that the degradation capability is conferred by the target genes and not by other IPTG-induced pathways.

## II. Ligation, Transformation, and Verification of PhnSR

### Principle:

  PhnSR is the component within the phn operon that senses PHE. PhnR encodes a repressor protein that normally interacts with a sequence upstream of the phnS promoter, inhibiting the expression of downstream genes. When PhnR binds salicylic acid, an intermediate of PHE degradation, this repression is lifted. We designed a system where EGFP expression is controlled by phnSR to verify whether phnSR can function in E. coli.

![444](https://static.igem.wiki/teams/5883/experiments/444/444.webp)

### Procedure:

  1. Culture bacteria containing pET-30a(+)-phnSR in preparation for plasmid extraction. After culturing pET-30a(+)-phnSR for 16 hours, extract the plasmid. Use DL5000 marker as reference, and run the gel using 0.8% agarose, with gel preparation ratio according to protocol. The full length of the plasmid is 8.9 kbp.

  2. PCR amplify the EGFP gene from the lab-stocked plasmid pEGFP-6X Myc-EGFP. Run the PCR product on a gel. EGFP: 714bp, Annealing Temp: 53°C.

#### Ordered Primers:

  EGFP-F-AvrII: ATAT**CCTAGG**ATGGTGAGCAAGGGCGAG
  EGFP-R-HindIII: ATAT**AAGCTT**TCACTTGTACAGCTCGTC

  3. Digest both the EGFP PCR product and pET-30a(+)-phnSR separately with AvrII & HindIII. Use the system specified in the protocol, with a digestion duration of 4 hours. Ligate to obtain pET-30a(+)-phnSR-EGFP.

  4. Transform into E. coli DH5α. Use the recombinant plasmid transformation procedure as per the protocol. After transformation, plate 20μl/200μl onto LA (Kan+) plates. Incubate at 37°C in a constant temperature incubator for 16 hours. Select successfully transformed colonies (those able to grow on LA (Kan+) plates) and send for sequencing. Use T7 and T7 terminator primers for sequencing.

### Functional Verification of the Sensing Cassette:

  (1) Use the microplate reader's Fluorescence - 507nm setting to characterize the fluorescence intensity resulting from EGFP expression. Measure hourly for three hours under inducing conditions during continuous culture.

  (2) SA Concentration Gradient: Prepare a 1M SA stock solution.
  SA Final Concentration (mM): 0, 0.1, 0.5, 1, 5

  Stock Solution Added (μl per ml of culture? Note: text says \*10^-1μl/ml, might need verification): 0, 1, 5, 10, 50

Based on the time gradient and inducer concentration gradient, the most direct indicator in this experiment is whether the fluorescence intensity increases with higher inducer dose and longer time.

## III. Ligation, Transformation, and Preliminary Verification of rhlAB

### Principle:

  Pseudomonas bacteria secrete a biosurfactant, rhamnolipids, to desorb weakly polar organic compounds from soil particles and facilitate their entry into the cell membrane. We will transfer pET28a(+) containing inserted rhlAB into E. coli to enable it to produce rhamnolipids, thereby enhancing the adsorption of PHE by the engineered bacteria. (A figure can be added here.)

![555](https://static.igem.wiki/teams/5883/experiments/555/555.webp)

### Procedure:

  1. pET28a(+) is available in the lab. Pre-culture DH5α containing pET28a(+). Incubate at 37°C, 220 rpm for 16 hours, then extract the plasmid.
  2. Amplify rhlAB via PCR from a lab-prepared lysate of Pseudomonas aeruginosa. Note: Pseudomonas aeruginosa is not on the white list; directly handling live cells may pose a safety risk to the project, hence we use the lysate. Run the PCR product on a gel and purify. RhlAB: 2234bp, Annealing Temp: [Value missing from original text].

#### Ordered Primers:

  rhlAB-R-SalI: ATAT**GTCGAC**GGACGCAGCCTTCAG
  rhlAB-F-NdeI: ATAT**CATATG**CGGCGCGAAAGTCTG

  3. Perform double digestion separately on the pET28a(+) plasmid and the rhlAB PCR product using NdeI & SalI. Use the system specified in the protocol, with a digestion duration of 4 hours. Ligate to obtain pET-28a(+)-rhlAB.

  4. Transform into E. coli DH5α. Use the recombinant plasmid transformation procedure as per the protocol. After transformation, plate 20μl/200μl onto LA (Kan+) plates. Incubate at 37°C in a constant temperature incubator for 16 hours. Select successfully transformed colonies (those able to grow on LA (Kan+) plates) and send for sequencing. Use primers rhlAB-R-SalI and rhlAB-F-NdeI for sequencing.

### Functional Detection:

  This experiment utilizes CTAB-Methylene Blue plates for detection. The cationic surfactant CTAB (cetyltrimethylammonium bromide) combined with the indicator methylene blue gives a highly specific reaction for rhamnolipid production. When engineered bacteria secrete rhamnolipids (an anionic surfactant), a clear zone or dark blue halo will form around the colonies. This occurs because rhamnolipids form an insoluble blue complex with CTAB-methylene blue, causing precipitation and resulting in a clear hydrolysis zone against the dark blue background. Streak the engineered bacteria to be tested, along with a negative control E. coli (non-rhamnolipid producing), onto the plates. Incubate inverted at 37°C for 16 hours. Colonies of engineered bacteria capable of secreting rhamnolipids will be surrounded by an opaque dark blue halo or a clear hydrolysis zone.

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{% block title %}Results{% endblock %}
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## Article Navigation

* [I
  Degradation Module](#article1)
  + [Cloning](#1-1)
  + [Validation](#1-2)
* [II
  Sensing Module](#article2)
  + [Cloning](#2-1)
  + [Validation](#2-2)

# Results

## I. Degradation Module

### 1. Cloning

**We constructed two plasmids, pET30a(+)-nahABFEDG-phnC and pET30a(+)-nahABFEDG-nahC, and transformed them into E. coli DH5α. After transformation, the cells were plated on LB (Kan+) agar plates. Since pET30a(+) carries a Kanamycin resistance (Kanr) gene, successfully transformed engineered bacteria should form colonies. The results after 16 hours of plate culture are shown below.**

![科普展图片1](https://static.igem.wiki/teams/5883/result/aabb/aa.avif)

![科普展图片3](https://static.igem.wiki/teams/5883/result/aabb/bb.avif)

**Colonies presumed to contain the plasmid were picked and inoculated into LB (Kan+) broth for expansion culture. PCR results after 8 hours are shown below . The primers used were:**

  nahC-EcoRI-F: ATAT**GAATTC**GCCTGACTCAGTTTTACATC

  nahC-R: ATAT**GCTAGC**GAATCAGGAGGAAACTAT

  phnC-EcoRI-F: ATAT**GAATTC**GGACCCGCCACCTTCATTTC

  phnC-R: ATAT**GCTAGC**GAGAGAGGAGGACAAGAT

![CC](https://static.igem.wiki/teams/5883/result/cc.avif)

**It can be observed that colonies 1, 2, and 3 from the pET30a(+)-nahABFEDG-nahC transformation and colonies 1 and 2 from the pET30a(+)-nahABFEDG-phnC transformation showed positive bands, suggesting the presence of the target coding sequence. To conclusively confirm successful transformation, the plasmids were sent for sequencing. The sequencing primers used were T7 and T7ter. The sequencing results were aligned with the target gene sequence obtained from NCBI. The results indicated that the engineered bacteria containing pET30a(+)-nahABFEDG-phnC were successfully transformed, while the nah series did not yield conclusive results.**

![DD](https://static.igem.wiki/teams/5883/result/dd.avif)

![EE](https://static.igem.wiki/teams/5883/result/eeffgghhii/ee.avif)

**We extracted the plasmid from the transformed DH5α cells and performed another transformation into the expression host strain BL21(DE3). The cells were plated on LB (Kan+) plates, and colonies were picked, resulting in the chassis BL21(DE3) strain containing pET30a(+)-nahABFEDG-phnC.**

### 2. Validation

**We established a fluorescence standard curve for PHE at an emission wavelength of 345 nm to facilitate the evaluation of the engineered bacteria's ability to degrade PHE. The 345 nm wavelength corresponds to one of the characteristic emission peaks of PHE.**

**A 1 mM stock solution of PHE was prepared using DMSO as the solvent. Aliquots of 1, 5, 10, 20, and 50 μL of this stock solution were added to LB medium to achieve final PHE concentrations of 1, 5, 10, 20, and 50 μg/mL in the systems, respectively. LB medium was used as a control. All samples were diluted tenfold before being added to a 96-well plate (without dilution, our standard curves consistently showed very flat responses). The standard curve obtained using the microplate reader at an emission wavelength of 365 nm is presented below.**

![JJ](https://static.igem.wiki/teams/5883/result/jklmn/jj.avif)

**After applying linear fitting, we found the R² value to be relatively low. We recognized that while fluorescence intensity is directly proportional to the concentration of the sample solution at low concentrations, it may deviate from linearity at higher concentrations due to factors such as the quenching effect. Subsequently, we employed quadratic fitting, which yielded satisfactory results.**

**Using this standard curve, we proceeded with the PHE degradation experiment involving the engineered bacteria containing pET30a(+)-nahABFEDG-phnC.**

**We prepared a 1 M IPTG stock solution using water as the solvent. We designed three experimental systems along with their respective blank controls:**

**Group 1: No bacterial inoculation.**

**Group 2: Inoculated with DH5α lacking any foreign plasmid.**

**Group 3: Inoculated with an equal volume of the engineered bacteria containing pET30a(+)-nahABFEDG-phnC.**

**After 8 hours of culture, we added 1 µL of the IPTG stock solution to each system. Then, 50 µL of the PHE stock solution in DMSO was added to the sample groups, while the blank groups received no PHE. Samples of 100 µL were taken at 0.5, 1.0, and 1.5 hours, and their fluorescence at an emission wavelength of 345 nm was measured using a microplate reader. The results, calculated by referencing the standard curve, are shown in the accompanying figure.**

![KK](https://static.igem.wiki/teams/5883/result/jklmn/kk.avif)

![LL](https://static.igem.wiki/teams/5883/result/jklmn/ll.avif)

**The results from the graph indicate that in the system without any bacterial inoculation, the PHE concentration remained largely stable during the first 0.5 hours, followed by a slight decrease after 1 hour. This reduction is likely attributed to precipitation due to the relatively high concentration.**

**In systems inoculated with either DH5α or the engineered bacteria, PHE concentration decreased within the first 0.5 hours, with the rate of decline slowing after 1 hour. The PHE concentration in the system with the engineered bacteria was significantly lower than that in the system with plasmid-free DH5α, indicating preliminary degradation capability of the engineered bacteria toward PHE.**

**The faster decrease in PHE concentration in the control group inoculated with DH5α compared to the mock group (no bacteria) may be due to bacterial absorption of PHE.**

**The results in the graph indicate that in the system without any bacterial inoculation, the PHE concentration remained essentially unchanged within the first 0.5 hours, but showed a slight decrease after 1 hour, which is presumed to be due to precipitation caused by the relatively high concentration.**

**In systems inoculated with either DH5α or the engineered bacteria, a decrease in PHE concentration was observed within the first 0.5 hours, followed by a slowdown in the rate of decrease after 1 hour. The PHE concentration in the system inoculated with the engineered bacteria was significantly lower than that in the system with DH5α (without the plasmid), indicating that the engineered bacteria preliminarily demonstrated the ability to degrade PHE.**

**The faster decrease in PHE concentration in the control group inoculated with DH5α compared to the mock group (no bacteria) is possibly because the bacteria themselves absorbed PHE.**

## II. Sensing Module

### 1. Cloning

**We constructed the plasmid pET30a(+)-phnR-P-phnS-EGFP and transformed it into E. coli DH5α. After transformation, the cells were plated on LB (Kan+) agar plates. The results after 16 hours of plate culture are shown below.**

![FF](https://static.igem.wiki/teams/5883/result/eeffgghhii/ff.avif)

**Colonies presumed to contain the plasmid were picked and inoculated into LB (Kan+) broth for expansion culture. PCR results after 8 hours are shown below . The primers used were:**

  EGFP-F-AvrII: ATAT**CCTAGG**ATGGTGAGCAAGGGCGAG

  EGFP-R-HindIII: ATAT**AAGCTT**TCACTTGTACAGCTCGTC

![GG](https://static.igem.wiki/teams/5883/result/eeffgghhii/gg.avif)

**Due to researcher error, the agarose gel was not completely dissolved during preparation for this electrophoresis, causing band smearing. Nevertheless, it can be inferred that the first and second samples contain the target sequence. To conclusively confirm successful transformation, the plasmids were sent for sequencing. The sequencing primers used were EGFP-F-AvrII and EGFP-R-HindIII. The sequencing results were aligned with the target gene sequence obtained from NCBI.**

![HH](https://static.igem.wiki/teams/5883/result/eeffgghhii/hh.avif)

![II](https://static.igem.wiki/teams/5883/result/eeffgghhii/ii.avif)

**The results confirmed the successful transformation of the engineered bacteria containing pET30a(+)-phnR-P-phnS-EGFP. The plasmid was extracted and then transformed into BL21(DE3). The cells were plated on LB (Kan+) plates, and colonies were picked, resulting in the chassis BL21(DE3) strain containing pET30a(+)-phnR-P-phnS-EGFP.**

### 2. Validation

**According to our design, adding SA (salicylic acid), an intermediate metabolite of PHE, to the solution should activate the sensing module phnSR, leading to the expression of the downstream EGFP gene. The fluorescence emitted by EGFP was detected using a microplate reader.**

**We prepared a 1 M stock solution of SA in anhydrous ethanol and established a concentration gradient to test the sensitivity of phnSR. Eight-hour cultures of the engineered bacteria were divided into six tubes and treated as follows:**

**Control:** Added 50 µL of anhydrous ethanol to account for any potential effects of the solvent itself.
  **Blank:**No addition of anhydrous ethanol or SA.
  **Group 1:** Added 1 µL of 10⁻¹ M SA stock solution.
  **Group 2:**Added 5 µL of 10⁻¹ M SA stock solution.
  **Group 3:**Added 10 µL of 10⁻¹ M SA stock solution.
  **Group 4:**Added 50 µL of 10⁻¹ M SA stock solution.

**Samples were taken at the 1-hour, 2-hour, and 3-hour time points. The OD₆₀₀ was measured using the microplate reader, and fluorescence was observed under 300 nm illumination using a gel imager. Based on the results, it appears that EGFP was not expressed in any of the groups. Although the microplate reader detected fluorescence in the experimental groups, this fluorescence did not change over time. Furthermore, the control and blank groups also exhibited varying levels of fluorescence. Critically, in the experimental groups, the fluorescence intensity remained constant over time, whereas it should have increased if expression was occurring.**

**Currently, we hypothesize that although the transformation was successful, the phnS component may not function correctly in E. coli, or phnR cannot be activated by SA under the current conditions. This part might require changing the chassis organism.**

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