CONTRIBUTION

Our project aims to develop a rapid, ultra-sensitive, highly specific and user-friendly point-of-care testing (POCT) platform to revolutionize the field of food safety monitoring. The core of this platform is to construct a four-in-one nanoflower system, which integrates phage-derived binding protein (PBP, for specific recognition), horseradish peroxidase (HRP, for catalyzing signal generation), gold-platinum nanozymes (AuPt, for synergistic signal amplification), and calcium phosphate matrix (CaHPO₄, as a high surface area scaffold). This design enables the completion of the test within 30 minutes, with a detection limit as low as 10 CFU/mL, and can accurately distinguish target pathogens (such as E. coli), even in complex matrices like milk and tea, and can work reliably. The results can be quantified either by visual reading or by analyzing RGB values on a smartphone, significantly enhancing the application potential in resource-limited environments. Among them, PBP is crucial for the specific detection of bacteria. In this project, we provided a total of 4 new parts, and also added experimental data for the existing parts. The relevant parts are listed in Table 1.

Part number	Part name	Part type	Contribution type
BBa_25PDUACA	Tail Fiber Protein	Basic part	New part
BBa_25ZM5M81	pET28a-TFP	Composite part	New part
BBa_256Y31R2	BSCBD	Basic part	New part
BBa_25JHJB24	pET28a-BSCBD	Composite part	New part
BBa_K5532001	pET28a-gp17	Basic part	Add new data

1. BBa_25PDUACA (Tail Fiber Protein,TFP) and BBa_25ZM5M81(pET28a-TFP),
BBa_25PDUACA (Tail Fiber Protein,TFP)
Name: TFP
Base Pairs: 1659 bp
Amino acids: 553 aa
Origin: Escherichia phage T7
Properties:
Tail Fiber Protein (TFP) from Escherichia phage T7 is a structural protein consisting of 553 amino acids. It is encoded by the genome of Escherichia phage T7 (a member of the Teseptimavirus genus within the Autotranscriptaviridae family).
Usage and Biology:
Structural component of the short non-contractile tail. The tail comprises six fibers made of gp17 trimers, 2 nm in diameter and 32 nm in length. May attach to host lipopolysaccharides (LPS) to mediate primary attachment to the host cell [1].

BBa_25ZM5M81(pET28a-TFP)
Construction Design
We constructed a recombinant expression plasmid pET28a-TFP to enable soluble expression of TFP (Tail Fiber Protein from Escherichia phage T7) and support its application as a bacterial recognition module in nanoflower-based detection systems.
The TFP coding sequence was derived from the template plasmid pUC57-TFP (stored in E. coli DH5α), with the original sequence corresponding to NCBI Reference Sequence NP_042005.1 (553 amino acids). 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).

Engineering Principle
TFP is a structural protein from Escherichia phage T7 that mediates specific recognition and attachment to E. coli host cells by binding to surface receptors. In our detection system, TFP serves as a "biological recognition probe" to ensure selective capture of target bacteria from complex samples.

Cultivation, Protein Expression and Validation
1. Plasmid Construction Validation
The recombinant plasmid was successfully constructed through a series of molecular procedures. Initially, PCR amplification of the TFP gene produced a 1674 bp amplicon, while the linearized pET28a vector showed a 5369 bp band, as confirmed by gel electrophoresis (Figure 2A), indicating successful amplification of both insert and backbone. Using Gibson Assembly, the purified TFP fragment and linearized vector were ligated with 2× CloneExpress Mix in an optimized system, followed by incubation at 50 °C for 30 minutes to facilitate homologous recombination. Subsequent colony PCR screening of transformants revealed a 1995 bp amplicon (Figure 2B), consistent with the expected size of the TFP insert plus partial vector sequence. Finally, Sanger sequencing demonstrated 100% identity between the cloned fragment and the target TFP sequence, confirming the accuracy of the plasmid assembly (Figure 2C). 2. Protein expression and purification
The verified pET28a-TFP plasmid was transformed into E. coli BL21(DE3) 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 both recombinant proteins (TFP: 65.4 kDa). The presence of strong induced bands confirms IPTG-induced expression in the precipitation (Figure 3). The purified TFP were then subjected to dialysis-assisted refolding to obtain soluble, functional proteins. Characterization
1.Catalytic performance
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-TFP-CaHPO₄), the four-in-one nanoflowers (HRP-TFP-CaHPO₄@AuPt) exhibited the highest catalytic performance (Figure 4). 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. 2.Optimization of reaction conditions
The catalytic activity of the synthesized TFP-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 the TFP-nanoflower constructs maintain optimal catalytic activity at physiological temperature (37°C) with reaction completion within 20-30 minutes (Figure 5). These optimized conditions will be applied in subsequent bacterial detection assays to ensure maximum sensitivity and reliability. 3.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 TFP. The detection results showed that the HRP-TFP-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 (Figure 6). We then 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), as shown in Figure 7. These standard curves can serve as a reference for us in the subsequent detection of unknown concentrations of bacteria. Additionally, we calculated the LOD and RSD values. The LOD value for HRP-TFP-CaHPO₄@AuPt was 19.4 CFU/mL. Within the detection range of 10¹ to 10⁴ CFU/mL, the RSD values were 3.07–5.23%, indicating satisfactory reproducibility and repeatability.

4.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 HRP-TFP-CaHPO₄@AuPt produced only weak signal values (B/R < 1) for B. subtilis, while exhibiting high signal values (B/R = 6~8) for E. coli (Figure 8). 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. 5.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 9). 6.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-TFP-CaHPO4@AuPt nanoflowers was significantly less in comparison to that of free HRP and HRP-TFP-CaHPO4 nanoflowers (Figure 10). These results suggested that the prepared HRP-TFP-CaHPO4@AuPt nanoflowers exhibited great resistance to high temperature and storage ability at room temperature because of the exceptional stability of AuPt.

BBa_256Y31R2 (BSCBD) Name: BSCBD
Base Pairs: 789 bp
Amino acids: 263 aa
Origin: Bacillus phage B103
Properties:
Endolysin from Bacillus phage B103 is a peptidoglycan-hydrolyzing enzyme consisting of 263 amino acids. It is encoded by the genome of Bacillus phage B103 (a member of the Beecentumtrevirus genus within the Picovirinae subfamily, Salasmaviridae family) [2].
Usage and Biology:
Endolysin with lysozyme activity that degrades host peptidoglycans and participates with the holin and spanin proteins in the sequential events which lead to the programmed host cell lysis releasing the mature viral particles. Once the holin has permeabilized the host cell membrane, the endolysin can reach the periplasm and break down the peptidoglycan layer.

BBa_25JHJB24(BSCBD) Construction Design
We constructed a recombinant expression plasmid pET28a-BSCBD to enable soluble 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 (stored in E. coli DH5α), 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).

Engineering Principle
BSCBD is a phage-derived binding protein (PBP) with specific recognition and high-affinity binding ability to target bacterial surface receptors. In our detection system, BSCBD serves as a "biological recognition probe" to selectively capture target bacteria from complex samples, ensuring the specificity and sensitivity of the nanoflower-based detection platform.

Cultivation, Protein Expression and Validation
1. Plasmid Construction Validation
The recombinant plasmid was successfully constructed through a series of molecular procedures. Initially, PCR amplification of the BSCBD gene produced a 804 bp amplicon, while the linearized pET28a vector showed a 5369 bp band, as confirmed by gel electrophoresis (Figure 12A), indicating successful amplification of both insert and backbone. Using Gibson Assembly, the purified BSCBD fragment and linearized vector were ligated with 2× CloneExpress Mix in an optimized system, followed by incubation at 50 °C for 30 minutes to facilitate homologous recombination. Subsequent colony PCR screening of transformants revealed a 1125 bp amplicon (Figure 12B), 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 12C). 2. Protein expression and purification
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).
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 [3].

Existing part: BBa_K5532004 (pET28a-gp17) Summary
In this project, we utilized the existing part BBa_K5532004 (pET28a-gp17) as the recognition module for E. coli, and applied it to the preparation of the four-component nanoflower(HRP-gp17-CaHPO4@AuPt) and the detection of bacteria. We investigated the optimal reaction conditions, bacterial detection capabilities, specificity and storage stability of HRP-gp17-CaHPO4@AuPt. These experimental results further demonstrated the potential of gp17 as a bacterial recognition module, and supplemented the existing components (gp17) with data on bacterial detection performance.

Documentation:
1.Usage and Biology
gp17 is a structural protein from Escherichia phage T3 that mediates specific recognition and attachment to E. coli host cells by binding to surface receptors. In our detection system, gp17 serves as a "biological recognition probe" to ensure selective capture of target bacteria from complex samples.
2.Measurement
Construction Design
We constructed a recombinant expression plasmid pET28a-gp17 to enable soluble expression of gp17 (Tail Fiber Protein from Escherichia phage T3) and support its application as a bacterial recognition module in nanoflower-based detection systems. The gp17 coding sequence was derived from the template plasmid pUC57-gp17 (stored in E. coli DH5α), with the original sequence corresponding to NCBI Reference Sequence 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).

Protein expression and purification
The verified pET28a-gp17 plasmid was transformed into E. coli BL21(DE3) 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 both recombinant proteins (gp17:65.8 kDa). The presence of strong induced bands confirms IPTG-induced expression in the precipitation (Figure 15). The purified gp17 were then subjected to dialysis-assisted refolding to obtain soluble, functional proteins. Catalytic performance
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-gp17-CaHPO₄), the four-in-one nanoflowers (HRP-gp17-CaHPO₄@AuPt) exhibited the highest catalytic performance (Figure 16). 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. Optimization of reaction conditions
The catalytic activity of the synthesized 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 the gp17-nanoflower constructs maintain optimal catalytic activity at physiological temperature (37°C) with reaction completion within 20-30 minutes (Figure 17). These optimized conditions will be applied in subsequent bacterial detection assays to ensure maximum sensitivity and reliability. 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 gp17. The detection results showed that the HRP-gp17-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 (Figure 18). We then 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 0.9286), as shown in Figure 19. These standard curves can serve as a reference for us in the subsequent detection of unknown concentrations of bacteria. Additionally, we calculated the LOD and RSD values. The LOD value for HRP-gp17-CaHPO₄@AuPt was 10.1 CFU/mL. Within the detection range of 10¹ to 10⁴ CFU/mL, the RSD values were 3.54–6.51%, indicating satisfactory reproducibility and repeatability.

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 HRP-gp17-CaHPO₄@AuPt produced only weak signal values (B/R < 1) for B. subtilis, while exhibiting high signal values (B/R = 3~4) for E. coli (Figure 20). 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. 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-gp17-CaHPO4@AuPt nanoflowers was significantly less in comparison to that of free HRP and HRP-gp17-CaHPO4 nanoflowers (Figure 21). These results suggested that the prepared HRP-gp17-CaHPO4@AuPt nanoflowers exhibited great resistance to high temperature and storage ability at room temperature because of the exceptional stability of AuPt. Reference
[1] Cuervo, A., Pulido-Cid, M., Chagoyen, M., Arranz, R., González-García, V. A., Garcia-Doval, C., Castón, J. R., Valpuesta, J. M., van Raaij, M. J., Martín-Benito, J., & Carrascosa, J. L. (2013). Structural characterization of the bacteriophage T7 tail machinery. The Journal of biological chemistry, 288(36), 26290–26299.
[2] Pecenková T, Benes V, Paces J, Vlcek C, Paces V. Bacteriophage B103: complete DNA sequence of its genome and relationship to other Bacillus phages. Gene. 1997 Oct 15;199(1-2):157-63.
[3] 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.