ENGINEERING

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.

  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).

  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).


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.

  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.

  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.

  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).

  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).


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.

  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).

  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.

  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.

  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 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.

  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.


•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.
•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 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.


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.

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).

	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.
•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).
•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.

  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.

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).