iGEM 2025 — Team subcat-greatbay

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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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# NOTEBOOK

![vertical line](https://static.igem.wiki/teams/5736/img/desc/p1-img1.webp)
![卡通图片](https://static.igem.wiki/teams/5736/img/desc/p1-img2.webp)

![shrimp](https://static.igem.wiki/teams/5736/img/desc/p2-nav-image.webp)

[Plasmids and strains construction](#content1)
[Protein expression and purification](#content2)
[Nanoparticle preparation](#content3)
[Performance Testing](#content4)

![shrimp](https://static.igem.wiki/teams/5736/img/desc/p2-middle-image.webp)

### Plasmids and strains construction

### Protein expression and purification

### Nanoparticle preparation

### Performance Testing

![To Top](https://static.igem.wiki/teams/5736/img/home/to-top-2x.webp)

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{% 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 %}
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# ENGINEERING

![vertical line](https://static.igem.wiki/teams/5736/img/desc/p1-img1.webp)
![卡通图片](https://static.igem.wiki/teams/5736/img/desc/p1-img2.webp)

![shrimp](https://static.igem.wiki/teams/5736/img/desc/p2-nav-image.webp)

[Overview](#content1)
[Cycle 1 Preparation of the E. coli-specific recognition module TFP/gp17](#content2)
[Design 1](#design1)
[Build 1](#build1)
[Test 1](#test1)
[Learn 1](#learn1)
[Cycle 2 Preparation of the B. subtilis-specific recognition module BSCBD](#content3)
[Design 2](#design2)
[Build 2](#build2)
[Test 2](#test2)
[Learn 2](#learn2)
[Cycle 3 Synthesis and performance testing of a four-in-one nanoflower system](#content4)
[Design 3](#design3)
[Build 3](#build3)
[Test 3](#test3)
[Learn 3](#learn3)

![shrimp](https://static.igem.wiki/teams/5736/img/desc/p2-middle-image.webp)

### Overview

Our team designed an ​**​integrated four-in-one nanoflower system​​** for ultra-sensitive, rapid, and low-cost bacterial detection. This platform combines ​**​specific recognition, catalytic signal generation, and dual signal amplification​**​ within a single nanostructure, enabling highly efficient pathogen identification. In this project, we conducted three rounds of DBTL (Design-Build-Test-Learn) engineering cycles using Escherichia coli (Gram-negative bacterium) and Bacillus subtilis (Gram-positive bacterium) as detection targets. The cycles included:
1.Preparation of the E. coli-specific recognition module TFP/gp17.
2.Preparation of the B. subtilis-specific recognition module BSCBD.
3.Synthesis and performance testing of a four-in-one nanoflower system.

### Cycle 1 Preparation of the E. coli-specific recognition module TFP/gp17

### **Design 1**

We constructed recombinant expression plasmids pET28a-TFP and pET28a-gp17 to enable soluble expression of TFP (Tail Fiber Protein from Escherichia phage T7) and gp17 (tail fiber protein from Enterobacteria phage T3), and support its application as a bacterial recognition module in nanoflower-based detection systems.
The TFP coding sequence was derived from the template plasmids pUC57-TFP and pUC57-gp17 (synthesized by GenScript), with the original sequence corresponding to NCBI Reference Sequence NP\_042005.1 (553 amino acids) and NP\_523342.1 (558 aa). The vector backbone used for construction was pET28a, a prokaryotic expression vector featuring a T7 promoter (for IPTG-inducible strong expression) and a C-terminal His-tag (for affinity purification).

![Figure 1](https://static.igem.wiki/teams/5736/img/engineering/figure-1.webp)

**Figure 1** Structure diagram of plasmids of (A)pET28a-TFP and (B) pET28a-gp17.

### **Build 1**

The recombinant plasmids pUC-TFP and pUC-gp17 were extracted and purified using a plasmid mini preparation kit. The insert fragments (TFP and gp17) were amplified by PCR from their respective plasmids using gene-specific primers. All samples were separated on a 1% agarose gel stained with YeaGreen nucleic acid dye. Electrophoresis was performed at 180 V for 15 minutes. The gel confirms the successful amplification of target genes. The PCR products for TFP and gp17 are single, sharp bands at the correct sizes, indicating high purity and specificity of amplification (Figure 2).

![Figure 2](https://static.igem.wiki/teams/5736/img/engineering/figure-2.webp)

**Figure 2** Agarose gel electrophoresis of TFP, gp17 and plasmid vector.

The linearized pET28a vector was recombined with the purified TFP or gp17 insert fragments using a homologous recombination cloning kit. The recombination reaction was transformed into E. coli DH5α competent cells. After heat shock recovery, the cells were plated on LB agar plates containing kanamycin (50 µg/mL) and incubated overnight at 37°C. The presence of abundant colonies on both plates demonstrates high recombination efficiency and successful construction of the recombinant plasmids. These colonies were subsequently screened by colony PCR and sequencing to verify correct insertion of the target genes. The colony PCR and sequencing showed that we successfully obtained the correct plasmids (Figure 3).
The sequence-verified recombinant plasmids pET28a-TFP and pET28a-gp17 were then transformed into chemically competent E. coli BL21(DE3) cells via heat shock method. After colony PCR verification, positive transformants were cultured for protein expression.

![Figure 3](https://static.igem.wiki/teams/5736/img/engineering/figure-3.webp)

**Figure 3** Transformation of plasmids pET28a-TFP and pET28a-gp17.
(A)Transformation result; (B) Colony PCR verification; (C-D) Sequencing result.

### **Test 1**

The protein expression was induced with 0.1 mM IPTG at 25°C overnight. Cells were harvested by centrifugation, lysed by sonication, and the soluble and insoluble fractions were separated. His-tagged proteins were purified using Ni-NTA affinity chromatography. Samples were analyzed by 12% SDS-PAGE followed by Coomassie Blue staining. The results demonstrate successful expression of both recombinant proteins (TFP: 65.4 kDa; gp17:65.8 kDa). The presence of strong induced bands confirms IPTG-induced expression in the precipitation (Figure 4). The purified recombinant proteins (TFP/gp17) were then subjected to dialysis-assisted refolding to obtain soluble, functional proteins.

![Figure 4](https://static.igem.wiki/teams/5736/img/engineering/figure-4.webp)

**Figure 4** SDS-PAGE Analysis of Recombinant Protein Expression and Purification.
M: Marker, S: Supernatant, P: Precipitate, FL: Flow liquid, W: Wash, E: Elution.

### **Learn 1**

​​In this cycle, we successfully obtained two types of E. coli-binding PBPs, namely TFP and gp17.​​ Both proteins were ​​expressed in the form of insoluble aggregates (inclusion bodies)​​, necessitating ​​additional denaturation and refolding steps​​ to recover them in a soluble and functionally active form. This process involves dissolving the aggregated proteins using strong denaturants (8M urea) followed by gradual removal of the denaturant under controlled buffer conditions to facilitate proper folding. To improve the solubility and yield of functionally folded proteins in future cycles, we propose the following evidence-based strategies:
•**Introduce Fusion Tags to Prevent Aggregation​**​
Fuse the target proteins with ​​solubility-enhancing partners​​ such as ​​MBP (Maltose-Binding Protein)​​ or ​​SUMO (Small Ubiquitin-like Modifier)​​. These tags serve as ​​chaperone-like carriers​​ that improve proper folding, enhance stability, and maintain solubility during expression. MBP is particularly effective due to its high solubility and ability to promote correct folding in fusion constructs, as demonstrated in numerous recombinant protein production systems [1].
•**Optimize Induction Conditions for Proper Folding​**​
Lower induction temperature​​ and ​​reduce IPTG concentration​​ to slow down protein synthesis rates. This minimizes misfolding and aggregation by allowing the cellular chaperone machinery more time to assist in proper folding.
These strategies are widely adopted in recombinant protein production to shift the equilibrium from inclusion body formation toward soluble expression, thereby reducing dependency on costly and inefficient refolding procedures.

### Cycle 2 Preparation of the B. subtilis-specific recognition module BSCBD

### **Design 2**

We constructed a recombinant expression plasmid pET28a-BSCBD to enable expression of endolysin from Bacillus phage B103 (a member of the Beecentumtrevirus genus within the Picovirinae subfamily, Salasmaviridae family), and support its application as a bacterial recognition module in nanoflower-based detection systems.
The BSCBD coding sequence was derived from the template plasmid pUC57-BSCBD (synthesized by GenScript), with the original sequence corresponding to NCBI Reference Sequence NP\_690649.1 (263 aa). The vector backbone used for construction was pET28a, a prokaryotic expression vector featuring a T7 promoter (for IPTG-inducible strong expression) and a C-terminal His-tag (for affinity purification).

![Figure 5](https://static.igem.wiki/teams/5736/img/engineering/figure-5.webp)

**Figure 5** Structure diagram of plasmids of pET28a-BSCBD.

### **Build 2**

The recombinant plasmid pUC-BSCBD was extracted and purified using a plasmid mini preparation kit. The insert fragment (BSCBD) was amplified by PCR from their respective plasmids using gene-specific primers. The agarose gel electrophoresis result showed a clear 804 bp band, while the linearized pET28a vector showed a 5369 bp band, confirming successful amplification of both insert and backbone (Figure 6).

![Figure 6](https://static.igem.wiki/teams/5736/img/engineering/figure-6.webp)

**Figure 6** Agarose gel electrophoresis of BSCBD and plasmid vector.

Using Gibson Assembly, the purified BSCBD amplicon and linearized vector were ligated with 2× CloneExpress Mix in a stoichiometrically optimized system to facilitate homologous recombination. Subsequent colony PCR screening of transformants revealed a 1125 bp amplicon (Figure 7A-B), consistent with the expected size of the BSCBD insert plus partial vector sequence. Finally, Sanger sequencing demonstrated 100% identity between the cloned fragment and the target BSCBD sequence, confirming the accuracy of the plasmid assembly (Figure 7C).
The sequence-verified recombinant plasmids pET28a-BSCBD was then transformed into chemically competent E. coli BL21(DE3) cells via heat shock method. After colony PCR verification, positive transformants were cultured for protein expression.

![Figure 7](https://static.igem.wiki/teams/5736/img/engineering/figure-7.webp)

**Figure 7** Transformation of plasmids pET28a-BSCBD.
(B)Transformation result; (B) Colony PCR verification; (C-D) Sequencing result.

### **Test 2**

The protein expression was induced with 0.1 mM IPTG at 25°C overnight. Cells were harvested by centrifugation, lysed by sonication, and the soluble and insoluble fractions were separated. His-tagged proteins were purified using Ni-NTA affinity chromatography. Samples were analyzed by 12% SDS-PAGE followed by Coomassie Blue staining. However, the SDS-PAGE result showed that there was no target band observed (Theoretical molecular weight: 33.1 kDa).

![Figure 8](https://static.igem.wiki/teams/5736/img/engineering/figure-8.webp)

**Figure 8** SDS-PAGE Analysis of Recombinant Protein Expression and Purification.
M: Marker, S: Supernatant, P: Precipitate, FL: Flow liquid, W: Wash, E: Elution.

### **Learn 2**

The failure of BSCBD expression in E. coli is likely attributable to ​​lysin-induced host cell toxicity​​ and ​​insufficient control of premature expression​​ during the pre-induction phase. Lysins, such as BSCBD, are designed to hydrolyze bacterial cell walls. When produced intracellularly in E. coli, even low levels of leakage expression can compromise cell wall integrity, leading to reduced viability, poor growth, and ultimately failed protein production [2].
To address this, we propose employing ​​specialized E. coli strains equipped with tighter transcriptional control mechanisms​​, such as ​​BL21(DE3) pLysS or BL21(DE3) pLysE​​. These strains carry the ​​pLys plasmid​​ encoding T7 lysozyme, which inhibits basal expression by suppressing T7 RNA polymerase activity prior to induction. This minimizes premature lysin production, thereby reducing host toxicity and improving cell survival until induction. Additionally, optimizing ​​induction conditions​​—such as lowering induction temperature, reducing inducer (IPTG) concentration, and delaying induction until high cell density is achieved—can further mitigate metabolic burden and enhance functional protein folding.

### Cycle 3 Synthesis and performance testing of a four-in-one nanoflower system

### **Design 3**

After the first two rounds of DBTL, we successfully obtained the TFP and gp17 proteins targeting E. coli. Next, we will prepare four-in-one nano-flowers for subsequent bacterial detection. This four-in-one nano flower is mainly composed of the following components:
■ Phage-derived Binding Proteins (PBPs): Engineered TFP and gp17 proteins provide species-specific bacterial recognition.
■ Horseradish Peroxidase (HRP): Catalyzes 3,3',5,5'-Tetramethylbenzidine (TMB) oxidation for visible signal generation.
■ Gold-Platinum Nanozyme (AuPt): Enhances catalytic activity with peroxidase-like properties.
■ Calcium Phosphate Matrix (CaHPO4): Forms 3D nanoflowers structure providing high surface area.

![Figure 9](https://static.igem.wiki/teams/5736/img/results/figure-5.webp)

**Figure 9** The preparation process and detection principle of the four-in-one nano flower [3].

### **Build 3**

The preparation of the four-in-one nanoflower mainly consists of three steps. The first step is to synthesize AuPt nanoparticles. The second step is to prepare a calcium-based nano-flower scaffold (i.e., a three-in-one nanoflower). The third step is to adsorb AuPt nanoparticles onto the nanoized scaffold through electrostatic adsorption, thereby obtaining a four-in-one nano-flower. (For the detailed preparation method, please refer to the [Experiment](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page.)
The synthesized ​​four-in-one nanoflowers​​ were subjected to detailed microstructural and compositional characterization using ​​transmission electron microscopy (TEM)​​. TEM analysis confirmed the successful formation and uniform dispersion of ​​gold-platinum (AuPt) nanozyme particles across the nanoflower scaffold. These metallic nanoparticles were consistently anchored across the calcium phosphate-based matrix via ​​electrostatic adsorption and surface complexation. This homogeneous distribution is critical for maximizing catalytic accessibility and enhancing signal amplification efficiency in subsequent biosensing applications.

![Figure 10](https://static.igem.wiki/teams/5736/img/engineering/figure-10.webp)

**Figure 10** Transmission Electron Microscopy Characterization of HRP-PBP-CaHPO₄@AuPt.

### **Test 3**

After successfully obtaining the four-in-one nanoflowers, we measured and compared their catalytic performance. The experimental results indicated that, compared to individual AuPt nanoparticles and the three-in-one nanoflowers (HRP-PBP-CaHPO₄), the four-in-one nanoflowers (HRP-PBP-CaHPO₄@AuPt) exhibited the highest catalytic performance (Figure 11). This is attributed to the surface of the four-in-one nanoflowers being able to adsorb a large number of AuPt nanoparticles, endowing them with a dual signal amplification function. This also suggests that we can potentially use trace amounts of the four-in-one nanoflowers to achieve significant detection results.

![Figure 11](https://static.igem.wiki/teams/5736/img/engineering/figure-11.webp)

**Figure 11** Catalytic performance of AuPt NPs, Three-in-one NFs and Four-in-one NFs of (B) TFP and (C) gp17.

•**Optimization of reaction conditions**
The catalytic activity of the synthesized TFP-nanoflowers and gp17-nanoflowers was then assessed under varying conditions to determine optimal performance. For time optimization, reactions were conducted at fixed temperature (25°C) with TMB/H₂O₂ substrate, and absorbance at 650 nm was measured at different time points (5, 10, 15, 20, 25, 30 min). For temperature optimization, reactions were carried out for a fixed duration (30min) at different temperatures (4°C, 25°C, 30°C, 37°C, 50°C). All measurements were performed in triplicate. The results demonstrate that both nanoflower constructs maintain optimal catalytic activity at physiological temperature (37°C) with reaction completion within 20-30 minutes (Figure 12). These optimized conditions will be applied in subsequent bacterial detection assays to ensure maximum sensitivity and reliability.
![Figure 12](https://static.igem.wiki/teams/5736/img/engineering/figure-12.webp)

**Figure 12** Optimization of reaction conditions of (A, B) HRP-TFP-CaHPO4@AuPt, and (C, D) HRP-gp17-CaHPO4@AuPt

•**Bacterial detection performance test**
Subsequently, serial dilutions of E. coli (ranging from 10¹ to 104CFU/mL) were prepared and detected using four different detection systems for each protein construct (TFP and gp17). The detection results showed that the four-component nanoflower systems (HRP-PBP-CaHPO₄-AuPt NPs) demonstrate significantly enhanced detection sensitivity compared to three-component and control systems. The incorporation of AuPt nanoparticles provides synergistic catalytic amplification, greatly improving the detection limit and dynamic range. The performance trends of TFP and gp17 are similar, indicating that this nano-flower-like structure has extremely high sensitivity in bacterial detection (Figure 13). Compared with the four-component nano-flower of gp17, TFP has a higher detection signal value. We have verified this on the
[Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) page. The results show that TFP has a higher affinity than gp17, enabling it to bind to more E. coli during the detection process, thereby achieving a better detection outcome.

![Figure 13](https://static.igem.wiki/teams/5736/img/results/figure-9-new.webp)

**Figure 13** Bacterial Test Results of (A) HRP-TFP-CaHPO4@AuPt, and (B) HRP-gp17-CaHPO4@AuPt

We performed linear regression analysis on bacterial concentration versus B/R ratio and found a good linear relationship for both quadruple-combination nanoflowers within the detection range of 10¹–10⁴ CFU/mL (R² values of 0.9434 and 0.9286, respectively), as shown in Figure 14. These standard curves can serve as a reference for us in the subsequent detection of unknown concentrations of bacteria.
![Figure 14](https://static.igem.wiki/teams/5736/img/engineering/figure-14.webp)

**Figure 14** The linear curve between B/R ratio and E. coli concentration of (A) HRP-TFP-CaHPO4@AuPt, and (B) HRP-gp17-CaHPO4@AuPt

Additionally, we calculated the LOD and RSD values, with results shown in the table below. The LOD values for both quadruple-combination nanoflowers ranged from 10.1 to 19.4 CFU/mL. Within the detection range of 10¹ to 10⁴ CFU/mL, the RSD values were 3.07–5.23% and 3.54–6.51%, respectively, indicating satisfactory reproducibility and repeatability (Table 1).

**Table 1** The calculation results of LOD and RSD.

|  | LOD (CFU/mL) | RSD (%) |
| --- | --- | --- |
| HRP-TFP-CaHPO₄@AuPt | 19.4 | 3.07-5.23 |
| HRP-gp17-CaHPO₄@AuPt | 10.1 | 3.54-6.51 |

•**Specificity Test**
To test the specificity of the four-in-one nano-flower, we introduced different types of strains (B. subtilis and E. coli). Test results indicated that both types of quadruple-combination nanoflowers produce only weak signal values (B/R < 1) for B. subtilis, while exhibiting high signal values (B/R = 3~4) for E. coli (Figure 15). Furthermore, in a mixed culture of B. subtilis and E. coli, the nanoflowers maintain high signal values, demonstrating that their detection performance remains unaffected in the presence of different bacterial species.
![Figure 15](https://static.igem.wiki/teams/5736/img/engineering/figure-15.webp)

**Figure 15** Detection specificity of (A) HRP-TFP-CaHPO4@AuPt and (B) HRP-gp17-CaHPO4@AuPt.
BS: B. subtilis; EC: E. coli; EC+BS: E. coli and B. subtilis mixture

•**Detection of real samples**
In order to test whether the four-in-one nano flowers can perform detection in complex environments, we conducted tests on real samples. We employed HRP-TFP-CaHPO₄@AuPt to detect E. coli in real samples. The results demonstrated successful detection of bacteria in milk (milky white) and tea (light brown). Signal intensity increased proportionally with bacterial concentration, indicating that our detection method reliably avoids interference from sample coloration and exhibits both reliability and universality (Figure 16).
![Figure 16](https://static.igem.wiki/teams/5736/img/engineering/figure-16.webp)

**Figure 16** Real sample testing of HRP-TFP-CaHPO4@AuPt. (A) milk, (B) tea.

•**Storage stability test**
Finally, we conducted an investigation into the storage stability of the four-in-one nano flowers. The findings indicated that the reduction in enzyme activity of the HRP-PBP-CaHPO4@AuPt nanoflowers was significantly less in comparison to that of free HRP and HRP-PBP-CaHPO4 nanoflowers (Figure 17). These results suggested that the prepared HRP-PBP-CaHPO4@AuPt nanoflowers exhibited great resistance to high temperature and storage ability at room temperature because of the exceptional stability of AuPt.
![Figure 17](https://static.igem.wiki/teams/5736/img/engineering/figure-17.webp)

**Figure 17** Storage stability in PBS at room temperature of HRP-PBP-CaHPO4@AuPt nanoflowers.

### **Learn 3**

In this round of testing, we successfully obtained four-in-one nanoflowers and conducted a detailed study on its detection performance. Compared with the traditional colorimetric method, this method has the following advantages (Table 2):
•Short detection time: The entire detection process (bacterial binding + display) takes no more than 30 minutes.
•High detection sensitivity: Even at low concentrations (as low as 10 CFU/mL), it can detect the signal value.
•High detection specificity: It can also identify the target bacteria in complex matrices, avoiding false positives caused by environmental factors.
•User-friendly: This detection method can be combined with a mini program in the future. Just by taking a photo with a mobile phone, it can intelligently analyze the RGB values and output the bacterial concentration.

**Table 2** Different type of colorimetric methods for E. coli detection.

| Methods | Device Structure | LOD (CFU/mL) | Time | Linear range | Ref |
| --- | --- | --- | --- | --- | --- |
| Phage-activated DNAzyme hydrogel sensor | Naked-eye detection, Smartphone | 10 CFU/mL | Not mentioned | 10¹–10⁷ CFU/mL | [4] |
| Phage lysis of β-gal/pH-CuO₂ nanoenzyme cascade | Smartphone, Microplate reader | 15 CFU/mL | >30 min | 10¹–10⁷ CFU/mL | [5] |
| Click-chemistry-mediated nanozyme colorimetric method | Microplate reader | 5 CFU/mL | 20 min | 5–10⁶ CFU/mL | [6] |
| Loop-mediated isothermal amplification colorimetric dual DNAzyme reaction | Naked-eye detection, Smartphone, LAMP | 100 CFU/mL | 1.5 h | 10¹–10⁹ CFU/mL | [7] |
| LAMP colorimetric method based on FTA card | Naked-eye detection, LAMP, FTA Card | 25 CFU/mL | 35 min | Not mentioned | [8] |
| Four-in-one Dual-Signal Amplifying Nanoflowers | Naked-eye detection, Smartphone | 10-20 CFU/mL | 30 min | 10¹–104 CFU/mL | Our project |
| LAMP: A colorimetric loop-mediated isothermal amplification | | | | | |

**Reference**
[1] Raran-Kurussi S, Waugh DS. The ability to enhance the solubility of its fusion partners is an intrinsic property of maltose-binding protein but their folding is either spontaneous or chaperone-mediated. PLoS One. 2012;7(11): e49589.
[2] Love, M. J., Abeysekera, G. S., Muscroft-Taylor, A. C., Billington, C., & Dobson, R. C. J. (2020). On the catalytic mechanism of bacteriophage endolysins: Opportunities for engineering. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1868(1), 140302.
[3] Hong, B., Qin, T., Wang, W., Luo, L., Li, Y., Ma, Y., & Wang, J. (2025). Rapid and ultrasensitive detection of Salmonella typhimurium based on dual signal amplification of four-in-one AuPt nanozyme coated enzyme-antibiotic-inorganic nanoflowers. Sensors and Actuators B: Chemical, 426, 137097.
[4] H. Mann, S. Khan, A. Prasad, F. Bayat, J. Gu, K. Jackson, Y. Li, Z. Hosseinidoust, T. F. Didar, C. D. M. Filipe, Bacteriophage-Activated DNAzyme Hydrogels Combined with Machine Learning Enable Point-of-Use Colorimetric Detection of Escherichia coli. Adv. Mater. 2024, 37, 2411173.
[5] Zeng, Q., Deng, T., Yang, Y., Wu, W., Jiang, Z., Wu, H., Deng, C. (2025). pH-Adaptable CuO2 photo-responsive oxidase with phage-lysed β-galactosidase based cascade reaction for colorimetric detection of Escherichia coli in drinking water with high specificity and sensitivity. Journal of Hazardous Materials, 492, 138295.
[6] Alzahrani A. A novel strategy for Escherichia coli detection in raw beef in combination with click chemistry. NPJ Sci Food. 2025 Apr 24;9(1):59.
[7] Sewid AH, Ramos JH, Dylewski HC, Castro GI, D’Souza DH, et al. (2025) Colorimetric dual DNAzyme reaction triggered by loop-mediated isothermal amplification for the visual detection of Shiga toxin-producing Escherichia coli in food matrices. PLOS ONE 20(4): e0320393.
[8] Fumin Chen, Junyu Wang and Weiguang Li et al. Visual and Rapid Detection of Escherichia coli O157:H7 in Stool Samples by FTA Card-based Loop-mediated Isothermal Amplification. Zoonoses. 2023. Vol. 3(1).

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{% 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 %}
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# EXPERIMENTS

![vertical line](https://static.igem.wiki/teams/5736/img/desc/p1-img1.webp)
![卡通图片](https://static.igem.wiki/teams/5736/img/desc/p1-img2.webp)

![shrimp](https://static.igem.wiki/teams/5736/img/desc/p2-nav-image.webp)

[Construction of Plasmids and Strains](#content1)
[Protein expression and purification](#content2)
[Preparation and test of Nanoparticle](#content3)

![shrimp](https://static.igem.wiki/teams/5736/img/desc/p2-middle-image.webp)

### Construction of Plasmids and Strains

**1.Preparation and Dispensing of LB Solid Medium**

| LB fluid medium | LB solid medium |
| --- | --- |
| Tryptone (10 g/L) | Tryptone (10 g/L) |
| Yeast extract (5 g/L) | Yeast extract (5 g/L) |
| NaCl (10 g/L) | NaCl (10 g/L) |
| - | Agar (15 g/L) |
| ddH2O: Make up to 1000 mL | |

**Apparatus and materials:**
Electronic balance, Weighing paper, Erlenmeyer flask, Measuring cylinder, Magnetic stirrer. Autoclave, Sterile petri dishes, Sealing film and plastic wrap, rubber bands, clean bench, Heat-resistant gloves.
**Medium Preparation Procedure**
(1)Turn on the electronic balance and weigh: 10 g Tryptone, 5 g Yeast extract, 10 g NaCl, (If solid medium is needed) 15 g Agar
(2)Add the weighed solid reagents into the 1000 mL Erlenmeyer flask.
(3)Add approximately 800 mL of ddH2O and gently swirl or use the magnetic stirrer until the reagents dissolve completely.
(4)Add ddH2O to bring the total volume up to 1000 mL and mix well.
**Sterilization**
(1)Seal the Erlenmeyer flask with aluminum foil or sealing film and secure it with a rubber band.
(2)Place the flask into the autoclave, set at 121 °C, and sterilize for 20 min.
(3)After sterilization, allow the pressure to return to normal before carefully removing the flask.
**Dispensing into Petri Dishes (Performing under sterile conditions)**
(1)Cool the sterilized medium to around 55-60 °C (it should feel warm to the touch but not too hot).
(2)Place sterile petri dishes on the sterile workbench.
(3)Open the petri dishes and, using the alcohol lamp for sterilization, pour the medium into the dishes to fill them to about half their volume.
(4)Gently shake the petri dishes to distribute the medium evenly.
(5)Once the medium solidifies, cover the petri dishes and seal them (using sealing film or tape).
(6)Label the petri dishes (including information such as medium type, date, and the person who prepared them).
(7)Store the sealed petri dishes upside down (to prevent condensation on the surface) in the refrigerator at 4 °C (It is recommended to use them within 1-2 weeks).

**2.Cultivation of *E. coli* with Plasmids in Liquid Medium**
**Apparatus and materials:**
Plasmid-carrying *E. coli* DH5α: pET28a, pUC57-BSCBD, pUC57-gp17, pUC57-TFP, Kanamycin, LB medium, Pipette and sterile pipettes.

**Preparation of Bacterial Cultures**
(1)Retrieve the prepared *E. coli* plates carrying the plasmids pET28a, pUC57-BSCBD, pUC57-gp17, and pUC57-TFP from the refrigerator.
(2)Ensure that these plates are stored at 4°C and are not older than a week for optimal bacterial viability.
(3)Take 8 sterile culture tubes and add 2 mL of LB medium to each container using a pipette.
(4)Using a separate pipette, add kanamycin to each container to achieve a final concentration of 0.2 μg/mL.
(5)Using a pipette, carefully pick a single colony from the plate of *E. coli* strain carrying the plasmid of interest (pET28a, pUC57-BSCBD, pUC57-gp17, or pUC57-TFP).
(6)Gently dip the pipette tip into the colony and transfer the bacterial sample into the appropriate 2 mL medium container.
(7)Repeat the process for each plasmid-containing strain, making sure to keep the strains separated and correctly labeled.
(8)Incubate overnight in a culture incubator at 37°C and 220 rpm.

**3.Plasmid extraction**
**Apparatus and materials:**
Column tube CP3, Collection tube, 1.5 mL centrifuge tube, 2 mL centrifuge tube, Centrifuge, Vortex mixer, Dry bath incubator, Buffer P1, Buffer P2, Buffer P3, Buffer PWT, Deionized distilled water, Inoculated bacterial culture (2 mL, containing target plasmid)
**Procedure**
(1)Harvest Bacterial Cells: Transfer 2 mL of the target plasmid-containing bacterial culture into a 2 mL centrifuge tube. Centrifuge at 8000 × g for 2 min. Discard the supernatant completely.
(2)Resuspension: Add 250 μL Buffer SP1 to the cell pellet. Resuspend thoroughly by vortexing or pipetting until the pellet is completely suspended.
(3)Lysis: Add 250 μL Buffer SP2. Immediately invert the tube gently 8 times to mix completely. Incubate at room temperature for 2-4 min (do not exceed 5 min).
(4)Neutralization: Add 350 μL Buffer SP3. Gently invert the tube up and down until a white flocculent precipitate forms and the solution below becomes clear.
(5)Supernatant Transfer: Centrifuge at 12000 × g for 3 min. Carefully transfer the clear supernatant to a CP3 column tube (adsorption column) placed in a collection tube.
(6)Binding: Centrifuge the column at 12000 × g for 1 min. Discard the flow-through from the collection tube.
(7)Wash: Add 500 μL Buffer PWT to the column. Centrifuge at 9000 × g for 1 min. Discard the flow-through. Repeat this wash step once more for a total of two washes.
(8)Drying the Column: Centrifuge the empty column at 9000 × g for 1 min to remove residual buffer.
(9)Elution: Place the CP3 column into a clean 1.5 mL centrifuge tube. Add 50-100 μL elution buffer or deionized distilled water to the center of the adsorption membrane. Incubate at room temperature for 1 min.
(10)Collection of Plasmid DNA: Centrifuge at 12000 × g for 1 min. The eluted plasmid DNA is now in the 1.5 mL tube. Store the plasmid DNA at -20 °C.

**4.PCR Amplification**
**Apparatus and Materials:**
PCR thermocycler, PCR tubes (0.2 mL), Vortex mixer, Microcentrifuge, 2 × Hieff PCR Master Mix, Template DNA, Forward and reverse primers, ddH2O
**Procedure**
(1)Preparation of PCR Reaction Mixture

| Reagent | Volume (μL) |
| --- | --- |
| 2 × Hieff PCR Master Mix | 25 |
| Primer F (10 μM) | 2 |
| Primer R (10 μM) | 2 |
| DNA template | 1 (10 ng) |
| ddH2O | 20 |
| Total | 50 |

(2)PCR Thermocycler Setup

| Step | Temperature | Time |
| --- | --- | --- |
| 1 | 94 ℃ | 5 min |
| 2 | 94 ℃ | 30 s |
| 3 | 55 ℃ | 30 s |
| 4 | 72 ℃ | 30 s/Kb |
| 5 | Go to step 2 | 30 Cycles |
| 6 | 72 ℃ | 4 min |
| 7 | 4 ℃ | infinite hold |

(3)Place the PCR tubes in the thermocycler.
(4)Start the PCR program with the optimized annealing temperature and extension time.
(5)After completion, store samples at 4°C for immediate analysis or at -20°C for long-term storage.

**5.Agarose Gel Electrophoresis**
**Apparatus and materials:**
Casting/gel tray, casting stand, well combs, voltage source, gel box, microwave oven, pipette and pipette tips, 1 × TAE buffer, agarose, 10,000 × nucleic acid dye (YeaGreen), UV transilluminator.
**Procedure**
(1)Gel Preparation: Prepare agarose solution: Add 1 g agarose to 100 mL of 1 × TAE buffer in an Erlenmeyer flask (1:100 ratio). Gently swirl the flask to mix the agarose and buffer.
(2)Dissolving Agarose: Heat the Erlenmeyer flask in a microwave oven until the solution begins to boil. Swirl the flask to mix the solution thoroughly. Repeat heating and swirling until the solution is completely clear with no visible undissolved agarose particles.
(3)Adding DNA Stain: Allow the agarose solution to cool slightly. Add 10 μL of 10,000 × YeaGreen nucleic acid dye to the flask (final dilution 1:10,000). Swirl the flask gently to ensure thorough mixing of the dye.
(4)Casting the Gel: Pour the agarose solution into the prepared gel casting tray fitted with well combs.
(5)Remove any air bubbles by gently tapping the tray. Allow the gel to solidify at room temperature for approximately 20 min.
(6)Setting up Electrophoresis: Once solidified, carefully remove the well combs. Place the gel casting tray into the electrophoresis tank. Add 1× TAE buffer to the tank until the gel surface is completely submerged (approximately 2-3 mm above the gel).
(7)Sample Loading: Starting from the left well, load 5 μL of DNA molecular weight marker. Load 10 μL DNA samples into the remaining wells.
(8)Running Electrophoresis: Place the lid on the electrophoresis tank and ensure proper electrode connections. Set the power supply parameters (Voltage: 180 V, Current: 300 mA, Time: 30 min). Press the start button to begin electrophoresis.
(9)Visualization: After the run is complete (approximately 20-30 min), turn off the power supply. Carefully remove the gel from the electrophoresis tank. Visualize DNA bands using UV light. Document results by photography if needed.

**6.Gel Extraction**
**Apparatus and Materials:**
Analytical balance, dry bath incubator (50°C), centrifuge, razor blade, Buffer B2, Wash Solution, Elution Buffer
**Procedure**
(1)Gel Slice Excision: Using a clean scalpel or razor blade, carefully excise the agarose gel slice containing the DNA fragment of interest under UV or blue light illumination. Transfer the gel slice to a pre-weighed 1.5 mL microcentrifuge tube. Weigh the tube with the gel slice and calculate the gel weight by subtraction.
(2)Gel Solubilization: Add Buffer B2 to the gel slice at a ratio of 3 times the gel weight. Incubate the mixture at 50°C in a dry bath incubator. Vortex the tube every 2-3 min to facilitate dissolution. Continue heating until the gel slice is completely dissolved.
(3)DNA Binding: Transfer the entire mixture to a spin column placed in a collection tube. Centrifuge at 8,000 × g for 30 s. Discard the flow-through from the collection tube. Place the column back into the same collection tube.
(4)Washing: Add 500 μL Wash Solution to the column. Centrifuge at 9,000 × g for 30 s.
(5)Discard the flow-through from the collection tube. Repeat this washing step once more for a total of two washes.
(6)Column Drying: Place the column into a new collection tube. Centrifuge at 9,000 × g for 1 min to remove residual wash solution.
(7)DNA Elution: Place the column into a new 1.5 mL microcentrifuge tube. Add 50 μL of Elution Buffer to the center of the column membrane. Incubate at room temperature for 1 min to allow the buffer to penetrate the membrane. Centrifuge at 12,000 × g for 1 min. The purified DNA is now in the 1.5 mL tube and ready for downstream applications.
(8)Storage: Store the purified DNA at 4°C for short-term use or at -20°C for long-term storage.

**7.DNA Concentration Measurement**
**Apparatus and Materials:**
NanoDrop spectrophotometer, DNA samples, ddH2O, Pipettes and pipette tips, Lint-free wipes.
**Procedure**
(1)Instrument Preparation: Turn on the NanoDrop spectrophotometer and allow it to warm up according to manufacturer's instructions. Launch the NanoDrop software on the connected computer. Select "Nucleic Acid" measurement mode.
(2)Blanking: Clean pedestal with ddH2O. Pipette 2 μL ddH2O onto pedestal. Click "Blank" to establish the baseline measurement. Lift the sampling arm and clean pedestal with a lint-free wipe.
(3)Sample Measurement: Pipette 2 μL of the linearized vector sample onto the lower pedestal. Lower the sampling arm and click "Measure". Record the concentration (ng/μL). Clean the pedestals after measurement. Repeat the same process for the insert fragment sample.

**8.Gibson Assembly for Seamless DNA Cloning**
**Apparatus and Materials:**
Thermalcycler, Microcentrifuge, 2× CloneExpress Mix, Insert DNA fragment, Linearized vector DNA, ddH₂O, 0.2 mL PCR tubes, Pipettes and pipette tips
**Procedure**
(1)Prepare the Gibson Assembly reaction in a 0.2 mL PCR tube (Gibson Assembly Master Mix, Insert DNA, Vector DNA, ddH2O up to 10 μL).
(2)Mix gently by pipetting and briefly centrifuge to collect contents at the bottom.

| Reagent | Volume (μL) |
| --- | --- |
| 2 × Clonexpress mix | 5 |
| Target Gene Insert | 0.02 × Insert bp\* |
| pET28a Vector | 0.04 × Vector bp\* |
| ddH2O | Up to 10 |
| Total | 10 |

\*bp = base pairs of the DNA fragment
Vector DNA Calculation: Volume (μL) = 0.04 × Vector length (bp)
Insert DNA Calculation: Volume (μL) = 0.02 × Insert length (bp)

(3)Place the reaction tube in a heating block. Incubate at 50℃ for 10 min.

| Step | Temperature | Time |
| --- | --- | --- |
| 1 | 50 ℃ | 30 min |
| 2 | 10 ℃ | infinite hold |

**9.Transformation of DNA into *E. coli* DH5α Using the Heat Shock Method**
**Apparatus and Materials:**
Ice bucket, Water bath, Shaking incubator, centrifuge, Competent *E. coli* DH5α cells, Gibson Assembly product, LB liquid medium, LB agar plates with antibiotic, 1.5 mL microcentrifuge tubes, Pipettes and sterile pipette tips, Sterile spreader.
**Procedure**
(1)Thawing and Sample Addition: Remove *E. coli* DH5α competent cells from the -80°C freezer. Thaw the competent cells on ice for 5 min. Add 10 μL of the Gibson Assembly product to the thawed competent cells. Mix gently by tapping the tube. Incubate the mixture on ice for 30 min to allow DNA uptake preparation.
(2)Heat Shock and Recovery: Quickly transfer the tubes to a 42°C water bath incubate for 90 s. Immediately transfer the tubes back to ice and incubate for 3-5 min. Add 700 μL of sterile LB liquid medium to the transformation mixture. Transfer to a shaking incubator set at 37°C, 200 rpm, incubate for 60 min.
(3)Centrifugation and Plating: Centrifuge the tubes at 5,000 rpm for 2 min to pellet the cells. Carefully discard the supernatant, leaving approximately 100 μL. Gently resuspend the cell pellet. Spread the entire cell suspension evenly onto LB agar plates containing the appropriate antibiotic. Use a sterile spreader for even distribution. Allow the liquid to absorb into the agar.
(4)Incubation: Incubate the plates upside down at 37°C overnight (12-16 h). Check for colony growth the following day.

**10.Colony PCR for Screening Recombinant Clones**
**Apparatus and materials:**
Pipettes and pipette tips, PCR tubes, Thermocycler, Recombinant clones from transformation plates, Forward primer (10 μM), Reverse primer (10 μM), 2× Rapid Taq Master Mix, ddH₂O.
**Procedure**
(1)Using pipette tip, gently touch a single colony from the transformation plate.
(2)Transfer the colony material to a fresh LB agar plate containing the appropriate antibiotic by gently streaking to create a backup colony.
(3)Dip the same pipette tip (with remaining bacterial material) into a PCR tube containing the prepared reaction mixture.
(4)Mix gently by pipetting up and down 2-3 times to release bacterial cells into the buffer.
(5)PCR Reaction Setup:

| Reagent | Volume (μL) |
| --- | --- |
| 2 × Rapid Taq Master Mix | 10 |
| Primer F (10 μM) | 2 |
| Primer R (10 μM) | 2 |
| ddH2O | 6 |
| Total | 20 |

(6)PCR Thermocycling Program:

| Step | Temperature | Time |
| --- | --- | --- |
| 1 | 94 ℃ | 5 min |
| 2 | 94 ℃ | 10 s |
| 3 | 50 ℃ | 20 s |
| 4 | 72 ℃ | 2-3 s/Kb |
| 5 | Go to step 2 | 30 Cycles |
| 6 | 72 ℃ | 4 min |
| 7 | 4 ℃ | infinite hold |

(7)Place the prepared PCR tubes into the thermocycler.
(8)Start the PCR program and wait for completion (approximately 2-3 h).
(9)Store completed reactions at 4°C if immediate analysis is not possible.

**11.Plasmid extraction and sequencing**
**Apparatus and materials:**
Column tube CP3, Collection tube, 1.5 mL centrifuge tube, 2 mL centrifuge tube, Centrifuge, Vortex mixer, Dry bath incubator, Buffer P1, Buffer P2, Buffer P3, Buffer PWT, Deionized distilled water, Inoculated bacterial culture (2 mL, containing target plasmid)
**Procedure**
(1)Harvest Bacterial Cells: Transfer 2 mL of the target plasmid-containing bacterial culture into a 2 mL centrifuge tube. Centrifuge at 8000 × g for 2 min. Discard the supernatant completely.
(2)Resuspension: Add 250 μL Buffer SP1 to the cell pellet. Resuspend thoroughly by vortexing or pipetting until the pellet is completely suspended.
(3)Lysis: Add 250 μL Buffer SP2. Immediately invert the tube gently 8 times to mix completely. Incubate at room temperature for 2-4 min (do not exceed 5 min).
(4)Neutralization: Add 350 μL Buffer SP3. Gently invert the tube up and down until a white flocculent precipitate forms and the solution below becomes clear.
(5)Supernatant Transfer: Centrifuge at 12000 × g for 3 min. Carefully transfer the clear supernatant to a CP3 column tube (adsorption column) placed in a collection tube.
(6)Binding: Centrifuge the column at 12000 × g for 1 min. Discard the flow-through from the collection tube.
(7)Wash: Add 500 μL Buffer PWT to the column. Centrifuge at 9000 × g for 1 min. Discard the flow-through. Repeat this wash step once more for a total of two washes.
(8)Drying the Column: Centrifuge the empty column at 9000 × g for 1 min to remove residual buffer.
(9)Elution: Place the CP3 column into a clean 1.5 mL centrifuge tube. Add 50-100 μL elution buffer or deionized distilled water to the center of the adsorption membrane. Incubate at room temperature for 1 min.
(10)Collection of Plasmid DNA: Centrifuge at 12000 × g for 1 min. The eluted plasmid DNA is now in the 1.5 mL tube. Store the plasmid DNA at -20 °C.
(11)Send the sample to the company for sequencing.

**12.Transformation of DNA into *E. coli* BL21(DE3)**
**Apparatus and Materials:**
Ice bucket, Water bath, Shaking incubator, centrifuge, Competent *E. coli* BL21(DE3) cells, Sequence-verified plasmids (pET28a-BSCBD, pET28a-gp17, pET28a-TFP), LB liquid medium, LB agar plates with antibiotic, 1.5 mL microcentrifuge tubes, Pipettes and sterile pipette tips, Sterile spreader.
**Procedure**
(1)Thawing and Sample Addition: Remove *E. coli* BL21(DE3) competent cells from the -80°C freezer. Thaw the competent cells on ice for 5 min. Add 10 μL of sequence-verified plasmids (pET28a-BSCBD, pET28a-gp17, pET28a-TFP) to the thawed competent cells. Mix gently by tapping the tube. Incubate the mixture on ice for 30 min to allow DNA uptake preparation.
(2)Heat Shock and Recovery: Quickly transfer the tubes to a 42°C water bath incubate for 90 s. Immediately transfer the tubes back to ice and incubate for 3-5 min. Add 700 μL of sterile LB liquid medium to the transformation mixture. Transfer to a shaking incubator set at 37°C, 200 rpm, incubate for 60 min.
(3)Centrifugation and Plating: Centrifuge the tubes at 5,000 rpm for 2 min to pellet the cells. Carefully discard the supernatant, leaving approximately 100 μL. Gently resuspend the cell pellet. Spread the entire cell suspension evenly onto LB agar plates containing the appropriate antibiotic. Use a sterile spreader for even distribution. Allow the liquid to absorb into the agar.
(4)Incubation: Incubate the plates upside down at 37°C overnight (12-16 h). Check for colony growth the following day.

### Protein expression and purificatio

**13.Cultivation of *E. coli* BL21(DE3) with Plasmids**
**Apparatus and materials:**
Plasmid-carrying *E. coli* BL21(DE3): pET28a-BSCBD, pET28a-gp17, pET28a-TFP, Kanamycin, LB medium, Pipette and sterile pipettes.
**Preparation of Bacterial Cultures**
(1)Retrieve the prepared *E. coli* BL21(DE3) carrying the plasmids pET28a-BSCBD, pET28a-gp17, pET28a-TFP from the refrigerator.
(2)Ensure that these plates are stored at 4°C and are not older than a week for optimal bacterial viability.
(3)Take 8 sterile culture tubes and add 2 mL of LB medium to each container using a pipette.
(4)Using a separate pipette , add kanamycin to each container to achieve a final concentration of 0.2 μg/mL.
(5)Using a pipette, carefully pick a single colony from the plate of *E. coli* BL21(DE3) carrying the plasmid of interest (pET28a-BSCBD, pET28a-gp17, pET28a-TFP).
(6)Gently dip the pipette tip into the colony and transfer the bacterial sample into the appropriate 2 mL medium container.
(7)Repeat the process for each plasmid-containing strain, making sure to keep the strains separated and correctly labeled.
(8)Incubate overnight in a culture incubator at 37°C and 220 rpm.

**14.Scale-up Culture Protocol**
**Apparatus and Materials:**
Shaking incubator, Spectrophotometer, Sterile Erlenmeyer flasks, LB medium, IPTG (Isopropyl β-D-1-thiogalactopyranoside), Kanamycin, Pipette and sterile pipettes.
**Procedure**
(1)Inoculate the primary seed culture into 100 mL of fresh LB liquid medium in sterile Erlenmeyer flasks (1:50). Add kanamycin at 50 μg/mL for plasmid selection. Incubate in a culture incubator at 37°C and 220 rpm.
(2)Approximately 2 h or until log phase growth (OD600 until it reaches 0.4-0.6), add IPTG to a final concentration of 0.1 mM. Mix gently to ensure even distribution of IPTG.
(3)Incubate in a culture incubator at 25°C and 220 rpm.
(4)After 18-20 h post-induction, harvest the cells by centrifugation. Proceed with protein purification or store cell pellets at -80°C for future use.

**15.SDS-PAGE Gel Preparation**
**Apparatus and Materials:**
Gel casting apparatus with glass plates, Gel combs, Pipettes and pipette tips, Gel preparation cups, Lower-layer gel solution (2×), Lower-layer gel buffer (2×), Upper-layer gel solution (2×), Colored upper-layer gel buffer (2×), Modified coagulant, ddH2O, SDS-PAGE running buffer.
**Procedure**
(1)Gel Casting Assembly: Assemble the gel casting apparatus according to manufacturer's instructions.
(2)Ensure glass plates are clean and properly sealed to prevent leakage.
(3)Lower Layer Gel Preparation (for 1.00 mm thick mini gel): Take equal volumes (2.7 mL each) of Lower-layer gel solution (2×), Lower-layer gel buffer (2×). Mix the solutions thoroughly in a gel preparation cup.
(4)Lower Layer Gel Polymerization: Add 55 μL of modified coagulant to the mixed solution from step 2. Stir gently to mix well, avoiding the formation of air bubbles. Pour the mixed lower-layer gel solution into the gel casting mold, filling to a level 1.5 cm below the upper edge of the glass plate. Cover the gel surface with a thin layer of distilled water or isopropanol to ensure a flat surface and prevent air bubble formation.
(5)Lower Layer Gel Solidification: Allow the gel to stand at room temperature for 6-10 min. Check for solidification: A clear boundary between the gel and the overlay phase indicates complete polymerization.
(6)Upper Layer Gel Preparation (for 1.00 mm thick mini gel): Carefully pour off the water/alcohol overlay from the solidified lower gel. Take equal volumes (0.75 mL each) of Upper-layer gel solution (2×), Colored upper-layer gel buffer (2×). Mix thoroughly in a fresh gel preparation cup.
(7)Upper Layer Gel Polymerization: Add polymerization initiator: Add 15 μL of modified coagulant to the mixed solution from step 5. Mix carefully: Stir gently to ensure uniform mixing while avoiding air bubbles.
(8)Upper Layer Gel Casting: Add the upper-layer gel solution on top of the solidified lower gel until the solution reaches the top of the glass plate. Slowly insert the sample comb into the gel, ensuring no air bubbles are trapped beneath the comb teeth.
(9)Final Polymerization and Well Preparation: Allow the gel to stand for 10-15 min until the upper layer is completely solidified. Carefully remove the comb by pulling it straight up to avoid damaging the wells. Use a pipette tip or syringe filled with SDS-PAGE running buffer to rinse the sample wells and remove any unpolymerized gel material.
(10)Final Preparation: The gel is now ready for SDS-PAGE electrophoresis. Ensure all wells are clean and free of debris before sample loading.

**16.Bacterial Cell Sonication and His-tag Protein Purification**
**Apparatus and Materials:**
Centrifuge, Sonicator with probe, Ice bath, shaker, lysis Buffer, wash Buffer, elution Buffer, BeyoGold™ His-tag Purification Resin (50% slurry), Large centrifuge tubes, 2 mL microcentrifuge tubes, Pipettes and tips.
**Procedure**
(1)Cell Harvesting: Transfer bacterial cells from the shake flask to large centrifuge tubes. Centrifuge at 4,000 × g for 10 min at 8°C. Discard the supernatant completely and note the wet weight of the bacterial pellet. Add lysis buffer at 4 mL per gram of wet bacterial pellet and resuspend completely.
(2)Place the sample tube in an ice bath throughout the sonication process. Sonication parameters (Power: 200-300W, Pulse duration: 3 s on, Interval: 3 s off, Total times: 10 min). Keep the sample on ice continuously to prevent protein denaturation.
(3)Clarification of Lysate: Centrifuge the sonicated sample at 10,000 × g for 20-30 min at 4°C. Carefully collect the supernatant (soluble protein fraction) and place it in an ice-water bath. Reserve 20 μL of supernatant for subsequent protein analysis.
(4)Resin Preparation: Take 1 mL of well-mixed 50% BeyoGold™ His-tag Purification Resin. Centrifuge at 1,000 × g for 10 s at 4°C and discard the storage solution. Add 0.5 mL non-denaturing lysis buffer to the resin. Mix gently to equilibrate. Centrifuge at 1,000 × g for 10 s at 4°C. Discard the supernatant. Repeat equilibration 1-2 times.
(5)Protein binding: Add approximately 4 mL of bacterial lysate supernatant to the equilibrated resin. Incubate on a swing shaker or horizontal shaker at 4°C for 60 min.
(6)Column Loading: Load the mixture of lysate and BeyoGold™ His-tag Purification Resin into the empty column tube of the affinity chromatography column provided in the kit.
(7)Flow-through Collection: Open the cap at the bottom of the purification column. Allow the liquid to flow out under gravity. Collect approximately 20 μL of the flow-through for subsequent analysis.
(8)Column Washing: Wash the column 5 times with non-denaturing washing solution. Add 0.5-1 mL of washing solution each time. Collect approximately 20 μL of each wash fraction for subsequent analysis and detection.
(9)Protein Elution: Elute the target protein 2 times using elution buffer. Use 1 mL of elution buffer each time. Collect each elution fraction separately for analysis. Analyze elution fractions by SDS-PAGE to confirm protein purity and yield.

**17.SDS-PAGE for Protein Analysis**
**Apparatus and Materials:**
Gel electrophoresis apparatus, Power supply, SDS-PAGE running buffer, Protein samples (treated with SDS loading buffer), Protein molecular weight marker, Prepared polyacrylamide gel, Pipettes and tips.
**Procedure**
(1)Gel Set up and Sample Loading: Load the prepared polyacrylamide gel into the electrophoresis tank . Add SDS-PAGE running buffer to the tank, ensuring both upper and lower chambers are properly filled. Using a micropipette, carefully load 5 μL of treated protein sample into each well of the gel.
(2)Electrophoresis: Connect the electrophoresis apparatus to the power supply, ensuring correct polarity (proteins migrate toward the positive electrode). Initial voltage: 80-100V for sample stacking. Running voltage: 120-150V for separation.
(3)Electrophoresis Completion: Monitor the progress by observing the dye front (tracking dye) movement. When the indicator dye reaches the bottom of the gel, turn off the power supply.
(4)Gel Removal: Carefully remove the gel cassette from the electrophoresis tank. Use appropriate tools to pry open the glass plates. Carefully peel off the polyacrylamide gel completely. Handle gently to avoid tearing or damaging the gel.
(5)Protein Staining: Place the gel into a clean plastic container or petri dish. Pour blue nucleic acid dye solution over the gel, ensuring the solution completely submerges the entire gel. Gently shake the container to ensure the dye solution makes full contact with the gel surface.
(6)Imaging: Turn off room lights and activate the lamp. Stained protein bands will emit bright and can be directly observed and photographed for documentation.

**18.Bradford method for protein concentration determination**
**Apparatus and Materials:**
Microplate reader, 96-well microplate, Micropipettes and tips, 1.5 mL microcentrifuge tubes, BSA (Bovine Serum Albumin) standard solution (0.2 mg/mL), 1× PBS (Phosphate Buffered Saline), Bradford working solution.
**Procedure**
(1)BSA Standard Preparation: Prepare BSA stock solution at 0.2 mg/mL concentration in 1× PBS.
(2)Standard Curve Preparation: Take 8 centrifuge tubes and label them 0-7. Prepare standard dilutions according to the following table. Vortex each tube to ensure complete mixing of the standard solutions.

| Tube No. | 1× PBS (μL) | 0.2 mg/mL BSA (μL) | Final BSA Concentration (μg/mL) |
| --- | --- | --- | --- |
| 0 (Blank) | 300 | 0 | 0 |
| 1 | 285 | 15 | 10 |
| 2 | 270 | 30 | 20 |
| 3 | 240 | 60 | 40 |
| 4 | 210 | 90 | 60 |
| 5 | 180 | 120 | 80 |
| 6 | 150 | 150 | 100 |
| 7 | 75 | 225 | 150 |

(3)Microplate Assay Setup: Add 20 μL of each BSA standard solution to designated wells in the microplate. Add 20 μL of appropriately diluted protein samples to separate wells. Add 200 μL of Bradford working solution to each well containing standards and samples.
(4)Incubation and Measurement: Incubate the microplate at room temperature for 5 min. Using a microplate reader, measure the A595 value of each well. Use tube 0 (blank control) to zero the instrument before measurements.
(5)Data Analysis: Plot a standard curve. Fit a linear trendline to determine the equation of the standard curve.

### Preparation and test of Nanoparticle

**19.AuPt Nanoparticle Solution Preparation**
**Apparatus and Materials:**
Magnetic stirrer with heating capability, Analytical balance, ddH2O, Chloroplatinic acid (H₂PtCl₆), Chloroauric acid (HAuCl₄), Trisodium citrate (C6H5Na3O7), Calcium chloride (CaCl₂), Potassium Carbonate (K₂CO₃), 10 × PBS.
**Procedure**
(1)AuPt Nanoparticle Synthesis: In a 100 mL flask, combine: ddH2O: 45 mL, Chloroplatinic acid: 0.38 mL (10 mg/mL), Chloroauric acid: 0.3 mL (10 mg/mL).
(2)Heating and mixing: Stir the mixture continuously using a magnetic stirrer. Heat to boiling and maintain for 10 min.
(3)Reduction step: Add 0.2 mL of trisodium citrate solution (114.1 mg/mL) to the boiling mixture. Allow the reaction to proceed under condensation reflux for 30 min.
(4)Cooling step: Gradually cool the resulting AuPt nanoparticle solution to ambient temperature. Store at 4°C for future use.
(5)Supporting Reagent Preparation: Prepare the following reagents according to the specified concentrations and volumes.

| Reagent | Concentration | Volume (mL) | Preparation Notes |
| --- | --- | --- | --- |
| K₂CO₃ | 0.1 M | 100 | Dissolve 1.38 g in ddH2O |
| Trisodium Citrate | 114.1 mg/mL | 100 | Dissolve 11.41 g in ddH2O |
| CaCl₂ | 200 mM | 100 | Dissolve 2.94 g in ddH2O |
| 0.5 × PBS | 0.5 × | 500 | Dilute 10× PBS stock 1:20 with ddH2O |
| 0.5 × PBS (0.05% Tween-20) | contain 0.05% Tween-20 | 200 | Add 100 μL Tween-20 to 200 mL 0.5× PBS |
| Chloroauric acid | 10 mg/mL | 3.8 | Dissolve 38 mg in ddH2O |
| Chloroplatinic acid | 10 mg/mL | 3 | Dissolve 30 mg in ddH2O |

(6)Storage: AuPt nanoflowers store at 4°C, protected from light. Buffer solutions store at 4°C, check for precipitation before use. Metal salt solutions store at room temperature.

**20.Synthesis of HRP-PBP-CaHPO₄@AuPt Nanoflowers**
**Apparatus and Materials:**
2 mL microcentrifuge tubes, Centrifuge, Vortex mixer, pH meter, HRP (Horseradish Peroxidase), PBP (Phage-derived Binding Proteins, include gp17, TFP), 0.5 × PBS, CaCl₂ solution (200 mM), AuPt nanoparticle solution (prepared as per previous protocol), K₂CO₃ solution (0.1 M), Pipettes and sterile tips
**Procedure**
(1)HRP-PBP-CaHPO₄ Nanoflower Synthesis: In a 2 mL microcentrifuge tube, combine HRP solution 250 μL (2 mg/mL), PBP solution 100 μL (1 mg/mL), 0.5 × PBS 1 mL (5 mM, pH 7.4). Add 20 μL of CaCl₂ solution (200 mM) to the mixture. Mix gently by pipetting to ensure uniform distribution. Incubate at room temperature for 18 h to allow biomineralization and nanoflower formation.
(2)Purification of nanoflowers: Centrifuge the reaction mixture to pellet the nanoflowers. Wash five times with PBS to remove unreacted components. After each wash, gently vortex to resuspend the pellet. Resuspend the purified HRP-PBP-CaHPO₄ nanoflowers in 200 μL PBS.
(3)AuPt Nanoparticle Coupling: pH adjustment for electrostatic coupling, take 500 μL of AuPt nanoparticle solution (from previous synthesis). Adjust pH to 8.0 using K₂CO₃ solution to optimize electrostatic interactions. Add 200 μL of HRP-PBP-CaHPO₄ nanoflowers (1 mg/mL) to the pH-adjusted AuPt solution
(4)Incubate at room temperature for 30 min under static conditions to allow electrostatic adsorption
(5)Purification of hybrid nanoflowers: Centrifuge at 12,000 rpm for 2 min at 4°C to pellet the hybrid nanoflowers. Wash the pellet with PBS to remove unbound AuPt nanoparticles. Resuspend the HRP-PBP-CaHPO₄@AuPt nanoflowers in 200 μL PBS.
(6)Storage: Store the final hybrid nanoflowers at 4°C for future use. Label with preparation date and concentration.

**21.Transmission Electron Microscopy**
**Apparatus and Materials:**
Nanoflower sample, Deionized (DI) water, TEM Grids, Transmission Electron Microscope: Talos F200x TEM (Thermo Fisher Scientific, USA) or an equivalent analytical TEM, Sonicator, Fine-tipped tweezers for handling TEM grids.
**Procedure**
•**Sample preparation**
(1)Sample Dilution: Dilute the raw nanoflower solution with deionized (DI) water at a 1:2 ratio.
(2)Dispersion: Briefly sonicate the diluted sample for 30-60 seconds in a bath sonicator to ensure a homogeneous suspension and break up large aggregates before deposition.
(3)Sample Deposition: Using fine tweezers, hold a TEM grid (carbon film facing up). Pipette 5 µL of the diluted and sonicated nanoflower solution and carefully drop it onto the center of the carbon film.
(4)Drying: Place the grid inside a covered petri dish to protect it from dust and allow it to dry completely at room temperature for at least 1-2 hours or until all solvent has evaporated.
•**TEM Imaging:**
(1)Grid Loading: Once completely dry, carefully load the prepared TEM grid into a suitable TEM holder according to the instrument’s specific operating manual.
(2)Insertion: Insert the holder into the TEM column and allow the system to achieve acceptable vacuum.
(3)Microscope Setup: Align the microscope according to standard operating procedures. Begin observation at a low magnification to locate a suitable area with well-dispersed nanoparticles.
(4)Image Acquisition: Increase magnification to desired levels for detailed morphological analysis.

**22.Catalytic Performance Determination**
**Apparatus and Materials:**
Microplate reader, 96-well microplates, Incubators, Timer, AuPt nanoparticles, HRP-PBP-CaHPO₄ nanoflowers, HRP-PBP-CaHPO₄@AuPt nanoflowers, TMB (3,3',5,5'-tetramethylbenzidine) solution, H₂O₂ (hydrogen peroxide) solution, pipettes and tips, Microcentrifuge tubes.
**Procedure**
(1)Comparative Catalytic Activity Assessment
Replicates: All experiments performed in triplicate
Test samples: Three different catalyst systems
a.AuPt nanoparticles alone
b.HRP-PBP-CaHPO₄ nanoflowers
c.HRP-PBP-CaHPO₄@AuPt hybrid nanoflowers
Catalytic Activity Assay: Reaction mixture preparation: For each well, add the following components in order 10 μL of respective nanoflower suspension. 50 μL TMB solution. 150 μL H₂O₂ solution.
Reaction conditions: Room temperature (25°C). Reaction 30 min. Gently mix by pipetting or plate shaking.
Detection and documentation: Measure absorbance at 650 nm using a microplate reader. Take photos with smartphone to document color development. Record all measurements in triplicate.
(2)Reaction Condition Optimization
a.Temperature Optimization: Test catalytic activity at 4°C, 25°C, 30°C, 37°C, 50°C
Use the same reaction system as Part 1. Reaction time: 30 min at each temperature. Place reaction plates in appropriate temperature-controlled environments.
b.Time Course Optimization: Monitor catalytic activity at 5, 10, 15, 20, 25, 30 min.
Use the same reaction system with optimal temperature from previous experiment. Take absorbance readings at each time point. Document color development progression.

**23.Bacterial Detection**
**Apparatus and Materials:**
96-well microplates, Microplate reader, Incubator, PBP solution (10 μg/mL in PBS), HRP-PBP-CaHPO₄@AuPt nanoflowers (1 mg/mL), 0.5 × PBS (0.05% Tween-20), Blocking solution (5% BSA in PBS), TMB solution, H₂O₂ solution, Bacterial samples
**Procedure**
(1)PBP Immobilization in 96-Well Plates
a.Protein coating: Add 100 μL of PBP solution (10 μg/mL in PBS) to each well of the 96-well plate.
b.Incubation: Cover the plate and incubate overnight at 4°C to allow protein adsorption to the plate surface.
c.Washing: Remove unbound PBP by washing with 200 μL washing solution. Perform 3-5 wash cycles. After each wash, gently tap the plate and invert on absorbent paper to remove residual liquid.
d.Blocking: Add 200 μL blocking solution (5% BSA in PBS) to each well. Incubate at room temperature for 1 h to ensure effective blocking. Store at 4°C if not used immediately (avoid freeze-thaw cycles).
(2)Bacterial Detection Assay
a.Sample Preparation: Prepare bacterial samples at concentrations of 10¹-10⁷ CFU/mL in PBS. Use PBS as negative control. Perform 5 independent experiments, each with triplicate wells.
b.Detection Protocol: Add 200 μL of diluted bacterial sample to each test well. Add 10 μL of HRP-PBP-CaHPO₄@AuPt nanoflowers (1 mg/mL),
c.Incubation: Place in 37°C incubator for 20 min to allow bacterial binding and nanoflower attachment.
d.Washing and Signal Development: Remove liquid and tap dry on absorbent paper. Add 200 μL washing solution to each well. Let stand for 1 minute, then discard washing solution. Tap dry on absorbent paper. Repeat washing 5 times,
e.Colorimetric detection: Add 50 μL TMB substrate and 50 μL H₂O₂ solution to each well. For enhanced color reaction, use H₂O₂ at 0.01%-1% concentration. Incubate at 37°C in the dark for 10 min.
f.Measurement and documentation: Measure optical density at 650 nm using microplate reader. Photograph results with smartphone for visual documentation.
(3)Specificity Test
Test nanoflowers functionalized with TFP/gp17 against:
•*E. coli*
•B. subtilis
•Mixed culture of *E. coli* and B. subtilis
- Compare RGB values with controls.
(4)Storage Stability
Store HRP, HRP-TFP-CaHPO₄, and HRP-TFP-CaHPO₄-AuPt NPs at room temperature (25°C) for 4 weeks, measuring their catalytic activity weekly using the same detection method as in point 21.
(5)Real Sample Detection
- Dilute milk/tea with PBS (1:10, w/v); mix thoroughly.
- Spike with bacteria (100, 102, 104 CFU/mL);
- Detect as above.
(6)Data Analysis
a.RGB Analysis Protocol: Use an online tool Image Color Picker to analyze smartphone photographs. Extract Red (R), Green (G), and Blue (B) values from each well. Calculate B/R ratios for enhanced discrimination.
b.Statistical Analysis: Plot bacterial concentration vs. RGB values and B/R ratios. Establish linear relationship equations.
Calculate Limit of Detection (LOD) using the formula:
                                                            LOD = 3 × (SD/K)
Calculate Relative Standard Deviation (RSD) using the formula
                                                            RSD = (SD/mean) × 100%
(Where LOD = Limit of Detection, the lowest concentration or amount that the method can reliably detect. 3 = Confidence level constant representing 99% confidence. SD = standard deviation of blank response. K = slope of calibration curve. Mean = Arithmetic mean value of the dataset).

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{% 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 %}
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# RESULTS

![vertical line](https://static.igem.wiki/teams/5736/img/desc/p1-img1.webp)
![卡通图片](https://static.igem.wiki/teams/5736/img/desc/p1-img2.webp)

![shrimp](https://static.igem.wiki/teams/5736/img/desc/p2-nav-image.webp)

[Overview](#content1)
[PBP preparation](#pbp_preparation)
[Protein expression and purification](#pbp_expression)
[Synthesis and performance test of four-in-one nanoflowers](#content2)
[Nanoflowers preparation](#nanoflowers_prep)
[Nanoflowers Characterization](#nanoflowers_char)
[Catalytic Performance Testing](#catalytic_test)
[Optimization of reaction conditions](#optimization)
[Bacterial detection performance test](#bacterial_detection)
[Specificity Test](#specificity)
[Detection of real samples](#real_samples)
[Storage stability test](#stability)
[Discussion](#content3)
[Future plan](#content4)
[Reference](#content5)

![shrimp](https://static.igem.wiki/teams/5736/img/desc/p2-middle-image.webp)

### Overview

Our team designed an ​​integrated four-in-one nanoflower system​​ for ultra-sensitive, rapid, and low-cost bacterial detection. This platform combines ​​specific recognition, catalytic signal generation, and dual signal amplification​​ within a single nanostructure, enabling highly efficient pathogen identification. Our experimental part can mainly be divided into two parts:
•The first part is the preparation of Phage-derived Binding Proteins (PBP) through molecular biology.
•The second part is the preparation and performance testing of the four-in-one nano flowers.

**PBP preparation**
We obtained the amino acid sequences of TFP, gp17 and BSCBS from GenBank, detailed information is shown in Table 1.

**Table 1** Information of three PBP.

| Name | Source | Genbank |
| --- | --- | --- |
| TFP | tail fiber protein [Escherichia phage T7] | NP\_042005.1 |
| gp17 | tail fiber protein [Enterobacteria phage T3] | NP\_523342.1 |
| BSCBD | Bacillus phage B103 | NP\_690649.1 |

After sending the genes to GenScript Company for synthesis, we obtained a strain containing the target gene. In order to obtain a large amount of target proteins, we cloned the target gene into the plasmid vector of pET28a, which is a prokaryotic expression vector featuring a T7 promoter (for IPTG-inducible strong expression) and a C-terminal His-tag (for affinity purification). Thus, we constructed three recombinant plasmids (Figure 1).

![Figure 1](https://static.igem.wiki/teams/5736/img/results/figure-1.webp)

**Figure 1**
Structure diagram of plasmids of (A)pET28a-TFP, (B) pET28a-gp17 and (C) pET28a-BSCBD.

**Plasmid Construction**
The insert fragments (TFP, gp17 and BSCBD) were amplified by PCR from their respective plasmids using gene-specific primers. All samples were separated on a 1% agarose gel stained with YeaGreen nucleic acid dye. Electrophoresis was performed at 180 V for 15 minutes. The gel confirms the successful amplification of target genes. The PCR products for TFP, gp17 and BSCBD are single, sharp bands at the correct sizes, indicating high purity and specificity of amplification (Figure 2).

![Figure 2](https://static.igem.wiki/teams/5736/img/results/figure-2.webp)

**Figure 2**
Agarose gel electrophoresis of TFP, gp17, BSCBD and plasmid vector.

Using Gibson Assembly, the purified target genes and linearized vector were ligated with 2× CloneExpress Mix in a stoichiometrically optimized system to facilitate homologous recombination. Subsequent colony PCR tests were conducted on the transformed organisms, as shown in Figure 3, which revealed the presence of expected amplification products (TFP: 1995 bp; gp17: 2010 bp; BSCBD: 1125 bp). Finally, Sanger sequencing demonstrated that the cloned fragments were 100% consistent with the target sequence, thereby confirming the accuracy of the plasmid assembly (Figure 3C).

![Figure 3](https://static.igem.wiki/teams/5736/img/results/figure-3.webp)

**Figure 3**
Transformation of plasmids pET28a-TFP, pET28a-gp17 and pET28a-BSCBD.
(A)Transformation result; (B) Colony PCR verification; (C) Sequencing result.

**Protein expression and purification**
The sequence-verified recombinant plasmids pET28a-BSCBD was then transformed into chemically competent *E. coli* BL21(DE3) cells via heat shock method. After colony PCR verification, positive transformants were cultured for protein expression. The protein expression was induced with 0.1 mM IPTG at 25°C overnight. Cells were harvested by centrifugation, lysed by sonication, and the soluble and insoluble fractions were separated. His-tagged proteins were purified using Ni-NTA affinity chromatography. Samples were analyzed by 12% SDS-PAGE followed by Coomassie Blue staining. The results demonstrate successful expression of TFP (Theoretical molecular weight: 65.4 kDa), and gp17 (Theoretical molecular weight: 65.8 kDa). The presence of strong induced bands confirms IPTG-induced expression in the precipitation (Figure 4). The purified recombinant proteins (TFP/gp17) were then subjected to dialysis-assisted refolding to obtain soluble, functional proteins. However, the SDS-PAGE result showed that no target band of BSCBD was observed (Theoretical molecular weight: 33.1 kDa).

The failure of BSCBD expression in *E. coli* is likely attributable to **lysin-induced host cell toxicity** and **insufficient control of premature expression** during the pre-induction phase. Lysins, such as BSCBD, are designed to hydrolyze bacterial cell walls. When produced intracellularly in *E. coli*, even low levels of leakage expression can compromise cell wall integrity, leading to reduced viability, poor growth, and ultimately failed protein production [1].

![Figure 4](https://static.igem.wiki/teams/5736/img/results/figure-4.webp)

**Figure 4**
SDS-PAGE Analysis of Recombinant Protein Expression and Purification.
M: Marker, S: Supernatant, P: Precipitate, FL: Flow liquid, W: Wash, E: Elution

### Synthesis and performance test of four-in-one nanoflowers

**Nanoflowers preparation**
After successfully obtaining the TFP and gp17 proteins targeting *E. coli*, we will prepare four-in-one nano-flowers for subsequent bacterial detection. This four-in-one nano flower is mainly composed of the following components:
Phage-derived Binding Proteins (PBPs): Engineered TFP and gp17 proteins provide species-specific bacterial recognition.
Horseradish Peroxidase (HRP): Catalyzes 3,3',5,5'-Tetramethylbenzidine (TMB) oxidation for visible signal generation.
Gold-Platinum Nanozyme (AuPt): Enhances catalytic activity with peroxidase-like properties.
Calcium Phosphate Matrix (CaHPO4): Forms 3D nanoflowers structure providing high surface area.
The preparation of the four-in-one nanoflower mainly consists of three steps. The first step is to synthesize AuPt nanoparticles. The second step is to prepare a calcium-based nano-flower scaffold (i.e., a three-in-one nanoflower). The third step is to adsorb AuPt nanoparticles onto the nanoized scaffold through electrostatic adsorption, thereby obtaining a four-in-one nano-flower (Figure 5). For the detailed preparation method, please refer to the [Experiment](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page.

![Figure 5](https://static.igem.wiki/teams/5736/img/results/figure-5.webp)

**Figure 5**
The preparation process and detection principle of the four-in-one nano flower [2].

**Nanoflowers Characterization**
The synthesized ​​four-in-one nanoflowers​​ were subjected to detailed microstructural and compositional characterization using ​​transmission electron microscopy (TEM)​​. TEM analysis confirmed the successful formation and uniform dispersion of ​​gold-platinum (AuPt) nanozyme particles across the nanoflower scaffold. These metallic nanoparticles were consistently anchored across the calcium phosphate-based matrix via ​​electrostatic adsorption and surface complexation. This homogeneous distribution is critical for maximizing catalytic accessibility and enhancing signal amplification efficiency in subsequent biosensing applications.

![Figure 6](https://static.igem.wiki/teams/5736/img/results/figure-6.webp)

**Figure 6**
Transmission Electron Microscopy Characterization of HRP-PBP-CaHPO₄@AuPt

**Catalytic Performance Testing**
After successfully obtaining the four-in-one nanoflowers, we measured and compared their catalytic performance. The experimental results indicated that, compared to individual AuPt nanoparticles and the three-in-one nanoflowers (HRP-PBP-CaHPO₄), the four-in-one nanoflowers (HRP-PBP-CaHPO₄@AuPt) exhibited the highest catalytic performance (Figure 7). This is attributed to the surface of the four-in-one nanoflowers being able to adsorb a large number of AuPt nanoparticles, endowing them with a dual signal amplification function. This also suggests that we can potentially use trace amounts of the four-in-one nanoflowers to achieve significant detection results.

![Figure 7](https://static.igem.wiki/teams/5736/img/results/figure-7.webp)

**Figure 7**
Catalytic performance of AuPt NPs, Three-in-one NFs and Four-in-one NFs of (B) TFP and (C) gp17.

**Optimization of reaction conditions**
The catalytic activity of the synthesized TFP-nanoflowers and gp17-nanoflowers was then assessed under varying conditions to determine optimal performance. For time optimization, reactions were conducted at fixed temperature (25°C) with TMB/H₂O₂ substrate, and absorbance at 650 nm was measured at different time points (5, 10, 15, 20, 25, 30 min). For temperature optimization, reactions were carried out for a fixed duration (30min) at different temperatures (4°C, 25°C, 30°C, 37°C, 50°C). All measurements were performed in triplicate. The results demonstrate that both nanoflower constructs maintain optimal catalytic activity at physiological temperature (37°C) with reaction completion within 20-30 minutes (Figure 8). These optimized conditions will be applied in subsequent bacterial detection assays to ensure maximum sensitivity and reliability.

![Figure 8](https://static.igem.wiki/teams/5736/img/results/figure-8.webp)

**Figure 8**
Optimization of reaction conditions of (A, B) HRP-TFP-CaHPO4@AuPt, and (C, D) HRP-gp17-CaHPO4@AuPt

**Bacterial detection performance test**
Subsequently, serial dilutions of *E. coli* (ranging from 10¹ to 104CFU/mL) were prepared and detected using four different detection systems for each protein construct (TFP and gp17). The detection results showed that the four-component nanoflower systems (HRP-PBP-CaHPO₄-AuPt NPs) demonstrate significantly enhanced detection sensitivity compared to three-component and control systems. The incorporation of AuPt nanoparticles provides synergistic catalytic amplification, greatly improving the detection limit and dynamic range. The performance trends of TFP and gp17 are similar, indicating that this nano-flower-like structure has extremely high sensitivity in bacterial detection (Figure 9). Compared with the four-component nano-flower of gp17, TFP has a higher detection signal value. We have verified this on the [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) page. The results show that TFP has a higher affinity than gp17, enabling it to bind to more *E. coli* during the detection process, thereby achieving a better detection outcome.

![Figure 9](https://static.igem.wiki/teams/5736/img/results/figure-9-new.webp)

**Figure 9**
Bacterial Test Results of (A) HRP-TFP-CaHPO4@AuPt, and (B) HRP-gp17-CaHPO4@AuPt

We performed linear regression analysis on bacterial concentration versus B/R ratio and found a good linear relationship for both quadruple-combination nanoflowers within the detection range of 10¹–10⁴ CFU/mL (R² values of 0.9434 and 0.9286, respectively), as shown in Figure 10. These standard curves can serve as a reference for us in the subsequent detection of unknown concentrations of bacteria.

![Figure 10](https://static.igem.wiki/teams/5736/img/results/figure-10.webp)

**Figure 10**
The linear curve between B/R ratio and *E. coli* concentration of (A) HRP-TFP-CaHPO4@AuPt, and (B) HRP-gp17-CaHPO4@AuPt

Additionally, we calculated the LOD and RSD values, with results shown in the table below. The LOD values for both quadruple-combination nanoflowers ranged from 10.1 to 19.4 CFU/mL. Within the detection range of 10¹ to 10⁴ CFU/mL, the RSD values were 3.07–5.23% and 3.54–6.51%, respectively, indicating satisfactory reproducibility and repeatability (Table 2).

**Table 2** The calculation results of LOD and RSD.

|  | LOD (CFU/mL) | RSD (%) |
| --- | --- | --- |
| HRP-TFP-CaHPO₄@AuPt | 19.4 | 3.07-5.23 |
| HRP-gp17-CaHPO₄@AuPt | 10.1 | 3.54-6.51 |

**Specificity Test**
To test the specificity of the four-in-one nano-flower, we introduced different types of strains (B. subtilis and *E. coli*). Test results indicated that both types of quadruple-combination nanoflowers produce only weak signal values (B/R < 1) for B. subtilis, while exhibiting high signal values (B/R = 3~4) for *E. coli* (Figure 11). Furthermore, in a mixed culture of B. subtilis and *E. coli*, the nanoflowers maintain high signal values, demonstrating that their detection performance remains unaffected in the presence of different bacterial species.

![Figure 11](https://static.igem.wiki/teams/5736/img/results/figure-11.webp)

**Figure 11**
Detection specificity of (A) HRP-TFP-CaHPO4@AuPt and (B) HRP-gp17-CaHPO4@AuPt.
BS: B. subtilis; EC: *E. coli*; EC+BS: *E. coli* and B. subtilis mixture

**Detection of real samples**
In order to test whether the four-in-one nano flowers can perform detection in complex environments, we conducted tests on real samples. We employed HRP-TFP-CaHPO₄@AuPt to detect *E. coli* in real samples. The results demonstrated successful detection of bacteria in milk (milky white) and tea (light brown). Signal intensity increased proportionally with bacterial concentration, indicating that our detection method reliably avoids interference from sample coloration and exhibits both reliability and universality (Figure 12).

![Figure 12](https://static.igem.wiki/teams/5736/img/results/figure-12.webp)

**Figure 12**
Real sample testing of HRP-TFP-CaHPO4@AuPt.(A) milk, (B) tea.

**Storage stability test**
Finally, we conducted an investigation into the storage stability of the four-in-one nano flowers in room temperature (25 ℃）). The findings indicated that the reduction in enzyme activity of the HRP-PBP-CaHPO4@AuPt nanoflowers was significantly less in comparison to that of free HRP and HRP-PBP-CaHPO4 nanoflowers (Figure 13). These results suggested that the prepared HRP-PBP-CaHPO4@AuPt nanoflowers exhibited great resistance to high temperature and storage ability at room temperature because of the exceptional stability of AuPt.

![Figure 13](https://static.igem.wiki/teams/5736/img/results/figure-13.webp)

**Figure 13**
Storage stability in PBS at room temperature of HRP-PBP-CaHPO4@AuPt nanoflowers.

### Discussion

In this project, we successfully obtained four-in-one nanoflowers and conducted a detailed study on its detection performance. Compared with the traditional colorimetric method, this method has the following advantages (Table 3):
✧Short detection time: The entire detection process (bacterial binding + display) takes no more than 30 minutes.
✧High detection sensitivity: Even at low concentrations (as low as 10 CFU/mL), it can detect the signal value.
✧High detection specificity: It can also identify the target bacteria in complex matrices, avoiding false positives caused by environmental factors.
✧User-friendly: This detection method can be combined with a mini program in the future. Just by taking a photo with a mobile phone, it can intelligently analyze the RGB values and output the bacterial concentration.

**Table 3** Different type of colorimetric methods for *E. coli* detection.

| Methods | Device Structure | LOD (CFU/mL) | Time | Linear range | Ref |
| --- | --- | --- | --- | --- | --- |
| Phage-activated DNAzyme hydrogel sensor | Naked-eye detection, Smartphone | 10 CFU/mL | Not mentioned | 10¹–10⁷ CFU/mL | [4] |
| Phage lysis of β-gal/pH-CuO₂ nanoenzyme cascade | Smartphone, Microplate reader | 15 CFU/mL | >30 min | 10¹–10⁷ CFU/mL | [5] |
| Click-chemistry-mediated nanozyme colorimetric method | Microplate reader | 5 CFU/mL | 20 min | 5–10⁶ CFU/mL | [6] |
| Loop-mediated isothermal amplification colorimetric dual DNAzyme reaction | Naked-eye detection, Smartphone, LAMP | 100 CFU/mL | 1.5 h | 10¹–10⁹ CFU/mL | [7] |
| LAMP colorimetric method based on FTA card | Naked-eye detection, LAMP, FTA Card | 25 CFU/mL | 35 min | Not mentioned | [8] |
| Four-in-one Dual-Signal Amplifying Nanoflowers | Naked-eye detection, Smartphone | 10-20 CFU/mL | 30 min | 10¹–104 CFU/mL | Our project |
| LAMP: A colorimetric loop-mediated isothermal amplification | | | | | |

### Future plan

**1.Increase the soluble expression of TFP and gp17**
Although we successfully obtained two peptide-binding proteins that can bind to Escherichia coli, namely TFP and gp17, both of these proteins are expressed in an insoluble aggregate form. Therefore, additional denaturation and refolding steps are required to restore them to a soluble and functionally active form. This process involves dissolving the aggregate proteins using a strong denaturant (8M urea), and then gradually removing the denaturant under controlled buffer conditions to facilitate their proper folding. To improve the solubility and yield of functional folded proteins in future cycles, we propose the following evidence-based strategies:
✧**Introduce Fusion Tags to Prevent Aggregation**
Fuse the target proteins with ​​solubility-enhancing partners​​ such as ​​MBP (Maltose-Binding Protein)​​ or ​​SUMO (Small Ubiquitin-like Modifier)​​. These tags serve as ​​chaperone-like carriers​​ that improve proper folding, enhance stability, and maintain solubility during expression. MBP is particularly effective due to its high solubility and ability to promote correct folding in fusion constructs, as demonstrated in numerous recombinant protein production systems [3].
✧**Optimize Induction Conditions for Proper Folding**
Lower induction temperature​​ and ​​reduce IPTG concentration​​ to slow down protein synthesis rates. This minimizes misfolding and aggregation by allowing the cellular chaperone machinery more time to assist in proper folding.
These strategies are widely adopted in recombinant protein production to shift the equilibrium from inclusion body formation toward soluble expression, thereby reducing dependency on costly and inefficient refolding procedures.

**2.Improve the expression strategy of BSCBD**
The failure of BSCBD expression in *E. coli* is likely attributable to ​​lysin-induced host cell toxicity​​ and ​​insufficient control of premature expression​​ during the pre-induction phase. Lysins, such as BSCBD, are designed to hydrolyze bacterial cell walls. When produced intracellularly in *E. coli*, even low levels of leakage expression can compromise cell wall integrity, leading to reduced viability, poor growth, and ultimately failed protein production. This aligns with the observation that phage lysins often accumulate and damage *E. coli*'s own cell walls when expressed recombinantly, resulting in bacterial death and low yields [3].
To address this, we propose employing ​​specialized *E. coli* strains equipped with tighter transcriptional control mechanisms​​, such as ​​BL21(DE3) pLysS or BL21(DE3) pLysE​​. These strains carry the ​​pLys plasmid​​ encoding T7 lysozyme, which inhibits basal expression by suppressing T7 RNA polymerase activity prior to induction. This minimizes premature lysin production, thereby reducing host toxicity and improving cell survival until induction. Additionally, optimizing ​​induction conditions​​—such as lowering induction temperature, reducing inducer (IPTG) concentration, and delaying induction until high cell density is achieved—can further mitigate metabolic burden and enhance functional protein folding.

### Reference

[1] Love, M. J., Abeysekera, G. S., Muscroft-Taylor, A. C., Billington, C., & Dobson, R. C. J. (2020). On the catalytic mechanism of bacteriophage endolysins: Opportunities for engineering. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1868(1), 140302.
[2] Hong, B., Qin, T., Wang, W., Luo, L., Li, Y., Ma, Y., & Wang, J. (2025). Rapid and ultrasensitive detection of Salmonella typhimurium based on dual signal amplification of four-in-one AuPt nanozyme coated enzyme-antibiotic-inorganic nanoflowers. Sensors and Actuators B: Chemical, 426, 137097.
[3] Raran-Kurussi S, Waugh DS. The ability to enhance the solubility of its fusion partners is an intrinsic property of maltose-binding protein but their folding is either spontaneous or chaperone-mediated. PLoS One. 2012;7(11): e49589.
[4] H. Mann, S. Khan, A. Prasad, F. Bayat, J. Gu, K. Jackson, Y. Li, Z. Hosseinidoust, T. F. Didar, C. D. M. Filipe, Bacteriophage-Activated DNAzyme Hydrogels Combined with Machine Learning Enable Point-of-Use Colorimetric Detection of Escherichia coli. Adv. Mater. 2024, 37, 2411173.
[5] Zeng, Q., Deng, T., Yang, Y., Wu, W., Jiang, Z., Wu, H., Deng, C. (2025). pH-Adaptable CuO2 photo-responsive oxidase with phage-lysed β-galactosidase based cascade reaction for colorimetric detection of Escherichia coli in drinking water with high specificity and sensitivity. Journal of Hazardous Materials, 492, 138295.
[6] Alzahrani A. A novel strategy for Escherichia coli detection in raw beef in combination with click chemistry. NPJ Sci Food. 2025 Apr 24;9(1):59.
[7] Sewid AH, Ramos JH, Dylewski HC, Castro GI, D'Souza DH, et al. (2025) Colorimetric dual DNAzyme reaction triggered by loop-mediated isothermal amplification for the visual detection of Shiga toxin-producing Escherichia coli in food matrices. PLOS ONE 20(4): e0320393.
[8] Fumin Chen, Junyu Wang and Weiguang Li et al. Visual and Rapid Detection of Escherichia coli O157:H7 in Stool Samples by FTA Card-based Loop-mediated Isothermal Amplification. Zoonoses. 2023. Vol. 3(1).

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