Foodborne pathogens pose a significant public health concern, transmitted through contaminated food and water. Traditional detection methods such as polymerase chain reaction (PCR) are highly accurate but time consuming and labour intensive. Through our research, we found that gold nanoparticles (AuNPs) are of great interest unique in the development of rapid and visual sensors due to their simple synthesis, easy surface modification and good biocompatibility. Therefore, our team intends to develop a novel colorimetric biosensor based on AuNPs for direct detection and inactivation of food-borne pathogens. As a result, we provide the following new parts, which are designed, constructed and functionally validated.

Part Contributions
Part Number	Part Name	Contribution Type	Part Type
BBa_K5532001	gp17	New part	Basic part
BBa_K5532002	tailspike	New part	Basic part
BBa_K5532003	plyV12	New part	Basic part
BBa_K5532004	pET28a-gp17	New part	Composite part
BBa_K5532005	pET28a-tailspike	New part	Composite part
BBa_K5532006	pET28a-plyV12	New part	Composite part

Name: gp17
Base Pairs: 1689 bp
Origin: T3 phage tail fiber protein (TFP) gp17 (NCBI Reference Sequence: NP_523342.1)
The tail fiber protein (TFP) gp17 of T3 phage is a critical component involved in the phage's ability to recognize and attach to its host bacterium, typically Escherichia coli (E. coli). The protein forms a trimeric structure that plays a key role in binding to the bacterial surface receptors, facilitating the subsequent steps of infection [1]. The tail fibers are responsible for the initial contact and irreversible binding to the host cell, which is a crucial determinant of the phage's host range and specificity [2].
TFP gp17 can be utilized in various biotechnological applications, including biosensors for detecting specific bacterial strains. Its high specificity and binding affinity make it an excellent candidate for developing diagnostic tools [2].
To obtain the gp17 (TFP) protein, we transformed the successfully constructed pET28a-gp17 plasmid into E. coli BL21 (DE3) cells for protein expression. After inducing protein expression by adding 1mM IPTG, the SDS-PAGE gel results showed that the gp17 (TFP) protein was successfully expressed in the samples with 1mM IPTG. Following protein induction, we collected the cells and lysed them. The His-tagged TFP protein was purified using nickel-affinity chromatography. The purified TFP protein was then analyzed by SDS-PAGE, and the results demonstrated that the TFP protein was successfully purified (Fig. 1).

Fig. 1 SDS-PAGE analysis of the expression and purification of gp17 (TFP) protein After purifying the gp17 (TFP) protein, we conjugated the gp17 (TFP) protein with synthesized gold nanoparticles (AuNPs) via electrostatic physical adsorption. Using a microplate reader, we measured the absorption peaks of the AuNPs before and after conjugation across the full wavelength spectrum. The results indicated that after conjugation with the TFP protein.
To evaluate whether the conjugated AuNP@TFP could detect E. coli, we incubated the conjugated AuNP@TFP with varying concentrations of E. coli at room temperature for 30 min. After incubation, the supernatant was collected and analyzed using smartphone photography. The results showed that as the bacterial concentration increased, the color of the supernatant became progressively lighter. This change in color indicated that AuNP@TFP could be used to detect E. coli (Fig. 2).

Fig. 2 UV-Vis Absorption Spectra of AuNPs@TFP and bacteria detection colorimetric analysis of AuNP@ TFP Conjugates
Name: tailspike
Base Pairs: 2067 bp
Origin: Salmonella phage CKT1 tailspike protein (TSP) tailspike (GenBank: UJP30002.1)
The tailspike protein (TSP) of Salmonella phage CKT1 is a crucial component in the infection mechanism of this bacteriophage. TSPs are part of the phage's tail structure and are responsible for recognizing and binding to specific receptors on the surface of the host bacterium. They exhibit polysaccharide depolymerase activity, which allows them to cleave the O-antigen moiety of the lipopolysaccharide (LPS) on the bacterial surface. This enzymatic activity facilitates the penetration of the bacterial cell wall, enabling the phage to inject its genetic material and initiate infection [3][4].
TSPs can be used in agricultural and food safety applications to control pathogenic bacteria such as Salmonella, thereby reducing the incidence of foodborne illnesses [3].
Due to their high specificity for certain bacterial strains or serotypes, TSPs can be utilized in diagnostic assays to detect and monitor bacterial infections. For instance, TSPs can be incorporated into biosensors to identify the presence of Salmonella in clinical or environmental samples [4].
During the construction phase, we amplified the tailspike gene and the linearized pET28a vector backbone using PCR. After confirming the correct band sizes via gel electrophoresis, we recovered the amplified gene and vector fragments from the gel. These fragments were then ligated through a method of homologous recombination. The ligation product was subsequently transformed into DH5α competent cells. After culturing, we selected single colonies and verified them using colony PCR and sequencing. Once we identified the correct plasmid clones through PCR and sequencing, we extracted the plasmids and transformed them into the BL21(DE3) expression strain for subsequent protein expression and purification. Then we induced protein expression by adding IPTG. Following induction, we used a His-Tag protein purification kit to purify the protein. SDS-PAGE results confirmed the successful induction by IPTG and the successful purification of the protein (Fig. 3).

Fig. 3 SDS-PAGE analysis of the expression and purification of plyV12(CBD) protein After purifying the tailspike (TSP) protein, we conjugated the TSP protein with synthesized gold nanoparticles (AuNPs) via electrostatic physical adsorption. The expected shift in the maximum absorption peak from 530 nm to 533 nm was confirmed using a microplate reader, indicating successful conjugation. Then we incubated varying concentrations of Salmonella with AuNP@TSP at room temperature for 30 min. Analysis of the supernatant using smartphone photography showed that higher bacterial concentrations resulted in a progressively lighter color of the supernatant, demonstrating that AuNP@TSP can be used to detect Salmonella (Fig. 4).

Name: plyV12
Base Pairs: 525 bp
Origin: Staphylococcus aureus phage plyV12 endolysin cell wall binding domain (CBD) (GenBank accession No. AAT01859.1)
Function: The cell wall binding domain (CBD) of the endolysin plyV12 is a critical component of the bacteriophage's lytic system. It specifically binds to the cell wall of Staphylococcus aureus (S. aureus), facilitating the enzymatic activity of the endolysin to degrade the bacterial cell wall peptidoglycan. This binding is highly specific and crucial for the targeted lysis of S. aureus cells, which enables the release of progeny phages [5].
The specificity of plyV12 for S. aureus can be utilized in diagnostic assays to detect the presence of this pathogen in clinical and environmental samples. This can aid in the rapid identification and monitoring of S. aureus infections [5]. Biocontrol in Food Safety: plyV12 can be applied in the food industry to control S. aureus contamination. Its use as a biocontrol agent can help reduce the incidence of foodborne illnesses caused by this pathogen [6].

Fig. 5 SDS-PAGE Analysis of the expression and purification of plyV12(CBD) protein

The composite part are genetically engineered plasmids, mainly consisting of T7 Promoter, His-Tag sequence, Target gene (gp17, tailspike, plyV12) and T7 Terminator T7 Promoter is a strong promoter derived from the T7 bacteriophage. It can drive high-level transcription of the downstream gene when T7 RNA polymerase is present. It is specifically recognized by the T7 RNA polymerase, ensuring targeted and efficient initiation of transcription.
T7 Terminator is a transcriptional terminator derived from the T7 bacteriophage. It ensures efficient termination of transcription by T7 RNA polymerase. This prevents read-through transcription into downstream sequences, enhancing the stability and yield of the target mRNA.
His-Tag is a sequence of six histidine residues added to the N-terminus of the gp17, tailspike, and plyV12. His-Tag can facilitate the purification of the expressed protein via immobilized metal affinity chromatography (IMAC). The His-tag binds to metal ions such as nickel or cobalt, allowing for selective isolation of the gp17, tailspike, and plyV12 protein from the cell lysate. These parts will be integrated into the recombinant protein expression vector pET28a, which enables efficient expression of the target protein.
To construct the pET28a-gp17, pET28a-tailspike and pET28a-plyV12 plasmid, we amplified the gp17, tailspike and plyV12 genes and the linearized pET28a vector backbone using PCR. After confirming the correct band size by gel electrophoresis (Fig. 6), we recovered the amplified gene and vector fragments from the gel. These were then ligated using homologous recombination. The ligation product was transformed into DH5α competent cells. Following transformation, single colonies were picked and screened by colony PCR and sequencing for validation (Fig. 7). After identifying the correct plasmid clone through PCR and sequencing, the plasmid was extracted and transformed into the BL21(DE3) expression strain for subsequent protein expression and purification. We induced protein expression by adding IPTG. Subsequently, we purified the protein using a His-Tag protein purification kit. The results from SDS-PAGE showed successful induction by IPTG and successful protein purification (Fig. 1, 3, 5).

Fig.6 Agarose gel electrophoresis of PCR amplified products for plasmid construction
Fig.7 Colony PCR and sequencing results for the identification of positive clones To evaluate the ability of AuNPs conjugated with PBP proteins (AuNP@PBP) to detect specific bacteria, we incubated the AuNP@PBP with their respective target bacteria at room temperature for 30 min. The specific conjugates and corresponding bacteria were as follows: AuNP@TFP for E. coli detection, AuNP@TSP for Salmonella detection, and AuNP@CBD for S. aureus detection. After incubation, the supernatant was collected and analyzed using smartphone photography.
When AuNP@pET28a (empty vector control) was incubated with all three types of bacteria, the supernatant remained purple-red, indicating that AuNP@pET28a did not bind to the bacteria (Fig. 8A). For AuNP@TFP and AuNP@TSP, incubation with varying concentrations of E. coli and Salmonella, respectively, as the bacterial concentration increased from (10^3) to (10^9) CFU/mL, the supernatant color changed from purple-red to colorless (Fig. 8B, C). This indicates that AuNP@TFP and AuNP@TSP successfully bound to their respective target bacteria. Since the preparation of AuNP@CBD was unsuccessful, incubation with S. aureus did not result in a significant color change in the supernatant, which remained almost unchanged (Fig. 8D).

Fig.8 Colorimetric analysis of bacteria detection using AuNP@PBP conjugates
We performed photothermal sterilisation tests on the prepared AuNP@TFP and AuNP@TSP. The experimental results showed that the temperatures of AuNPs@TFP and AuNPs@TSP gradually increased (28℃-45℃) with the increase of irradiation time (0-10 min). The treated samples were subjected to plate-coating operation and after incubation overnight, we found that the number of bacteria decreased, indicating the effectiveness of photothermal sterilization (Fig. 9).

Fig. 9 Photothermal sterilisation results of (A)AuNP@TFP and (B)AuNP@TSP

1.Kato H, Fujisawa H, Minagawa T. Subunit arrangement of the tail fiber of bacteriophage T3. Virology. 1986 Aug;153(1):80-6. doi: 10.1016/0042-6822(86)90009-7. PMID: 2943077.
2.Taslem Mourosi J, Awe A, Guo W, Batra H, Ganesh H, Wu X, Zhu J. Understanding Bacteriophage Tail Fiber Interaction with Host Surface Receptor: The Key "Blueprint" for Reprogramming Phage Host Range. Int J Mol Sci. 2022 Oct 12;23(20):12146. doi: 10.3390/ijms232012146. PMID: 36292999; PMCID: PMC9603124.
3.Opeyemi U. Lawal1，Lawrence Goodridge. TSPDB: A curated resource of tailspike proteins with 2 potential applications in phage research. bioRxiv preprint doi: https://doi.org/10.1101/2024.05.23.595625;
4.Schmidt, A., Rabsch, W., Broeker, N.K. et al. Bacteriophage tailspike protein based assay to monitor phase variable glucosylations in Salmonella O-antigens. BMC Microbiol 16, 207 (2016). https://doi.org/10.1186/s12866-016-0826-0
5.Liu J, Zhang X, Yang H, Yuan J, Wei H, Yu J, Fang X. Study of the interactions between endolysin and bacterial peptidoglycan on S. aureus by dynamic force spectroscopy. Nanoscale. 2015 Oct 7;7(37):15245-50. doi: 10.1039/c5nr03525b. PMID: 26324763.
6. Rahman MU, Wang W, Sun Q, Shah JA, Li C, Sun Y, Li Y, Zhang B, Chen W, Wang S. Endolysin, a Promising Solution against Antimicrobial Resistance. Antibiotics (Basel). 2021 Oct 20;10(11):1277. doi: 10.3390/antibiotics10111277. PMID: 34827215; PMCID: PMC8614784.