Foodborne bacteria significantly impact daily life. According to WHO and FAO, food-borne diseases in the U.S. cause 3.3 to 12.3 million illnesses and 3,900 deaths annually, resulting in economic losses of $6.5 to $34.9 billion. Effective bacterial detection and sterilization are thus crucial. Current methods like microbial analysis, PCR, fluorescent labeling, and ELISA are costly, time-consuming, and require professional equipment. To address these issues, we propose using AuNP@PBP. By coupling AuNPs with proteins plyV12, gp17, and tailspike, the nanoparticles change color based on bacterial concentration, which can be analyzed via smartphone RGB values. Under near-infrared light, AuNPs generate heat to kill bacteria. This method is quick (30 min), cost-effective, easy to use, and scalable, offering a viable solution to the global food security crisis.

We selected the pET28a vector for constructing the pET28a-plyV12 plasmid due to its T7 promoter, which facilitates protein expression, and its His tag, which aids in protein purification. This choice was made to efficiently express and purify the plyV12 protein (CBD) (Figure 1).

Figure 1 The plasmic structure of pET28a-plyV12 We amplified the plyV12 gene and linearized the pET28a vector using PCR. The PCR products were then analyzed by agarose gel electrophoresis to verify the amplification and size of the DNA fragments. The agarose gel electrophoresis results demonstrated successful amplification of the plyV12 genes and the linearized pET28a vector fragments (Figure 2). Then we recovered the plyV12 gene and the linearized pET28a vector fragments. These were then ligated using homologous recombination. The recombinant plasmid was transformed into DH5α competent cells. Post-cultivation, we selected single colonies for colony PCR, then colony PCR products were analyzed via agarose gel electrophoresis. The gel electrophoresis results revealed the bands corresponding to the expected sizes of plyV12 gene (Figure 3). These positive colonies were further verified by sequencing and the results confirmed that the gene sequences in the plasmids were correct. Then the constructed pET28a-plyV12 plasmid was extracted from DH5a and transferred into the BL21(DE3) expression strain for subsequent protein expression and purification.

Figure 2 Agarose gel electrophoresis of PCR amplified products for plyV12 gene and the linearized pET28a vector
Figure 3 Colony PCR and sequencing results for the identification of pET28a-plyV12 plasmid After the successful construction of the pET28a-plyV12 plasmid, we transformed it into E. coli BL21 (DE3) cells for protein expression. Protein expression was induced by adding 1 mM IPTG to the culture medium. The SDS-PAGE gel showed distinct bands at the expected molecular weight for the plyV12 (CBD) protein in the induced samples, indicating that the plyV12 (CBD) target protein was successfully expressed upon IPTG induction (Figure 4A). Post-induction, the cells were harvested and lysed, and the His-tagged CBD protein was purified using nickel-affinity chromatography. The purified CBD protein was analyzed by SDS-PAGE, and the results demonstrated that the CBD protein was successfully purified (Figure 4B).

After the purification of the plyV12 (CBD) protein, we conjugated the plyV12 (CBD) protein with synthesized gold nanoparticles (AuNPs) via electrostatic physical adsorption. Upon successful conjugation, the maximum absorption peak was expected to shift from 530 nm to 533 nm. During the conjugation process, we added 100 μg of CBD protein to 1 mL of AuNPs and observed the solution change from purple-red to colorless. Furthermore, no significant absorption peaks at 530 nm or 533 nm were observed in the spectrum (Figure 5A). These observations indicate that the conjugation of the CBD protein to AuNPs failed.
To evaluate the ability of the conjugated AuNP@CBD to detect Staphylococcus aureus (S. aureus), we incubated the conjugated AuNP@CBD with S. aureus at room temperature for 30 min. After incubation, the supernatant was collected and analyzed using smartphone photography. Since the preparation of AuNP@CBD was unsuccessful, no change in the color of the supernatant was observed after incubation with S. aureus, remaining almost unchanged (Figure 5B). This further indicates that the conjugation of CBD protein to AuNPs failed.

Figure 5. UV-Vis absorption spectra of AuNPs@CBD and bacteria detection colorimetric analysis of AuNP@ CBD conjugates

Issue	Analysis	Solution
The conjugation of gold nanoparticles (AuNPs) with the CBD protein failed. After add CBD protein into AuNP solution, the solution change from purple-red to colorless. No significant absorption peaks at 533 nm were observed in the spectrum	The failure in conjugation may be attributed to the electrostatic physical adsorption method, which might not be suitable for the CBD protein. Electrostatic adsorption relies on the charge interactions between the protein and the AuNPs, which may not be strong or specific enough to ensure stable conjugation for the CBD protein. As a result, the CBD protein may not have been effectively attached to the AuNPs, leading to the failure in bacterial detection.	To address this issue, we propose to explore alternative conjugation methods. One potential approach is to activate the surface of the AuNPs and then facilitate covalent bonding with specific functional groups on the CBD protein, such as amine groups. Covalent conjugation generally provides stronger and more stable binding compared to electrostatic adsorption, which could improve the effectiveness of the conjugation and, consequently, the bacterial detection capability of the AuNP@CBD conjugates.

Since the pET28a vector backbone contains a T7 promoter for protein expression and a His-tag to facilitate protein purification, we chose the pET28a vector backbone to construct the pET28a- tailspike plasmid for the expression and purification of the gp17 protein (TSP) (Figure 6).

Figure 6 The plasmic structure of pET28a-gp17 To construct the pET28a-gp17 plasmid, we amplified the plyV12 gene and the linearized pET28a vector backbone using PCR firstly. After confirming the correct band sizes via gel electrophoresis (Figure 7), we extracted the amplified gene and vector fragments through gel extraction. Subsequently, we ligated these fragments using homologous recombination.
The ligation products were then transformed into DH5α competent cells. Following transformation, single colonies were selected and subjected to colony PCR and sequencing for verification. Correct plasmid clones were identified through colony PCR and sequencing analysis (Figure 8).
After confirming the correct plasmid constructs, the plasmids were extracted and transformed into BL21(DE3) expression strains for subsequent protein expression and purification.


Figure7 Agarose gel electrophoresis of PCR amplified products for gp17 gene and the linearized pET28a vector

Figure 8 Colony PCR and sequencing results for the identification of pET28a-gp17 plasmid 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 1 mM IPTG, the SDS-PAGE gel results showed that the gp17 (TFP) protein was successfully expressed in the samples with 1 mM IPTG (Figure 9A). 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 (Figure 9B).

Figure 9 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. Upon successful conjugation, the maximum absorption peak was expected to shift from 530 nm to 533 nm. We added 100 μg of TFP protein to 1 mL of AuNPs and incubated at room temperature for 30 min. Following this, we added 1% BSA and incubated at room temperature for another 30 min to block the unconjugated sites of AuNPs. 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, the maximum absorption peak of the AuNPs shifted from 530 nm to 533 nm (Figure 10A). This observation confirmed the successful conjugation of the TFP protein with the AuNPs.
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 (Figure 10B). This change in color indicated that AuNP@TFP could be used to detect E. coli.


Figure 10 UV-Vis absorption spectra of AuNPs@TFP and bacteria detection colorimetric analysis of AuNP@TFP conjugates

Issue	Analysis	Solution
After the PCR amplification of the gp17 target gene, we observed bright, short non-specific bands in the DNA gel electrophoresis results.	Primer Dimerization: The primers may be forming dimers due to non-specific binding with each other, resulting in short, non-specific amplification products. Suboptimal Primer Design: The primers may not be sufficiently specific to the target sequence. Excessive Primer Concentration: High primer concentration can increase the likelihood of non-specific binding and amplification.	Optimize Primer Design: Use software tools to design primers with high specificity for the gp17 gene, ensuring minimal cross-reactivity with other sequences. Reduce Primer Concentration: Lower the concentration of primers in the PCR reaction to reduce the likelihood of non-specific amplification.

Since the pET28a vector backbone contains a T7 promoter for protein expression and a His-tag to facilitate protein purification, we chose the pET28a vector backbone to construct the pET28a- tailspike plasmid for the expression and purification of the gp17 protein (TSP) (Figure 6).

Figure 11 The plasmic structure of pET28a-tailspike
To construct the pET28a-tailspike plasmid, we first amplified the tailspike gene and linearized the pET28a vector backbone using PCR. After confirming the correct sizes of the target gene and vector bands via gel electrophoresis (Figure 12), we extracted the amplified gene and vector fragments through gel extraction. These fragments were then ligated using homologous recombination. The ligation products were transformed into DH5α competent cells, and single colonies were selected for colony PCR. Upon identifying a potential positive clone through colony PCR (Figure 13-red box), one or two positive clones were chosen for sequencing verification. After confirming the correct plasmid clones through colony PCR and sequencing, the plasmids were extracted and transformed into BL21(DE3) expression strains for subsequent protein expression and purification.

Figure 12 Agarose gel electrophoresis of PCR amplified products for tailspike gene and the linearized pET28a vector

Figure 13 Colony PCR (red box) and sequencing results for of pET28a-tailspike plasmid To express and purify the tailspike protein (TSP), we transformed the successfully constructed pET28a-tailspike plasmid into E. coli BL21 (DE3) cells. The BL21(DE3) strain containing the pET28a-tailspike plasmid was then cultured, and protein expression was induced by adding 1 mM IPTG to the culture medium. The SDS-PAGE gel showed that the tailspike protein was successfully expressed in the samples with IPTG (Figure 14A). After induction, the cells were harvested and lysed, and the His-tagged TSP protein was purified using nickel-affinity chromatography. The purified TSP protein was analyzed by SDS-PAGE, and the results demonstrated that the TSP protein was successfully purified (Figure 14B).

Figure 14 SDS-PAGE analysis of the expression and purification of tailspike (TSP) protein
After purifying the tailspike (TSP) protein, we conjugated the TSP protein with synthesized gold nanoparticles (AuNPs) via electrostatic physical adsorption. Upon successful conjugation, the maximum absorption peak was expected to shift from 530 nm to 533 nm. We added 100 μg of TSP protein to 1 mL of AuNPs and incubated at room temperature for 30 min. Following this, we added 1% BSA and incubated at room temperature for another 30 min to block the solution. The expected shift in the maximum absorption peak from 530 nm to 533 nm was confirmed using a microplate reader, indicating successful conjugation (Figure 15A).
To evaluate the detection capability of the conjugated AuNP@TSP for Salmonella, 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 (Figure 15B), demonstrating that AuNP@TSP can be used to detect Salmonella.

Issue	Analysis	Solution
When use single clones performing colony PCR to select positive clones, we found that the tailspike gene was either difficult to amplify or produced very faint bands.	The difficulty in amplifying the tailspike gene or the faintness of the bands may be attributed to the use of a standard PCR mix instead of a high-fidelity enzyme. Standard PCR mixes may lack the accuracy and efficiency required to amplify specific and potentially complex sequences like the tailspike gene. High-fidelity enzymes, on the other hand, offer greater specificity and accuracy, which are crucial for amplifying target genes effectively.	To address this issue, we recommend switching to a high-fidelity enzyme for colony PCR. High-fidelity enzymes provide improved accuracy and efficiency in amplifying specific sequences, which should enhance the amplification of the tailspike gene and produce clearer, more distinct bands. By using a high-fidelity enzyme, we can increase the likelihood of successfully identifying and verifying the correct clones for subsequent experiments.