iGEM 2024 — Team subcat-greatbay

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5532/wiki/desc-banner-1.png)

{{ self.title() }}

* Day1
* Day2
* Day3
* Day4
* Day5
* Day6
* Day7
* Day8
* Day9
* Day10-14
* Day15-17
* Day18-21
* Day22-38

Day1-20240726

Task
1.Safety training
2.LB Medium preparation and autoclaving
3.Incubate bacterical strains containing plasmids

Day2-20240727

Task
1.Plasmid extraction
2.Amplify target gene and vector by PCR
3.Agarose Gel Electrophoresis
Results

![](https://static.igem.wiki/teams/5532/wiki/240727.png)

Day3-20240728

Task
1.DNA gel extraction
2.Gibson assembly
3.Transform competent cells (DH5α)
4.Cell plating and incubate overnight at 37°C

Day4-20240729

Task
1.Selection of monoclonal colonies and sub-culturing
2.Colony PCR for identifying positive colonies
3.Selection of positive colony and inoculate in 10 mL liquid LB medium
Results

![](https://static.igem.wiki/teams/5532/wiki/240729.png)

![](https://static.igem.wiki/teams/5532/wiki/240729-2.png)

Day5-20240730

Task
1.Plasmid extraction
2.Transform competent cells (BL21)
3.Selection of BL21(DE3) positive colony and inoculate in 10 mL liquid LB medium

Day6-20240731

Task
1.Expansion of BL21 bacterial culture
2.Induction of protein expression with IPTG
3.Synthesis of AuNPs
4.Preparation of SDS-PAGE gel
Results

![](https://static.igem.wiki/teams/5532/wiki/240731.png)

Notes: Color Change Observed During the Synthesis of Gold Nanoparticles

Day7-20240801

Task
1.Protein extraction and purification
2.SDS-PAGE gel Electrophoresis
3.Buffer exchange of purified protein
Results

![](https://static.igem.wiki/teams/5532/wiki/240801.png)

Day8-20240802

Task
1.AuNPs@PBP preparation and detection
2.Cultivate bacteria for subsequent detection (Escherichia coli, Salmonella enterica, Staphylococcus aureus)
3.Gradient dilution of detection bacterial
Results

![](https://static.igem.wiki/teams/5532/wiki/240802.png)

Note: UV-Vis Absorption Spectra of AuNPs Conjugated with PBP Proteins

Day9-20240803

Task
1. Colorimetric detection of bacterial by AuNPs@PBP
Results

![](https://static.igem.wiki/teams/5532/wiki/240803.png)

Note: Colorimetric Analysis of Bacteria Detection Using AuNP@PBP Conjugates

Day10-14 20240804-20240808

Task
1.Summary and reporting of results of phased experiments
2.RGB Analytics and Data Analysis
Results

![](https://static.igem.wiki/teams/5532/wiki/20240804.png)

Note: Results of linear fitting between B/R values and bacterial concentrations

Day15-17 20240807-20240809

Task
1.Photothermal sterilisation test
2.LB solid medium preparation and flatbed coating operation
Results

![](https://static.igem.wiki/teams/5532/wiki/20240807.png)

Note: Photothermal sterilisation results of (A)AuNP@TFP and (B)AuNP@TSP

Day18-21 20240810-20240813

Task
1.Data aggregation and analysis
2.Check the national food safety standards and determine the testing standards
3.Design icon for app design
Results

![](https://static.igem.wiki/teams/5532/wiki/20240810.png)

Note: Icon displayed in the APP

Day22-38 20240814-20240830

Task
1.Discuss the features that need to be implemented in a smartphone app for bacteria detection
2.Participate in the design of the APP under the guidance of an external tutor
Results

![](https://static.igem.wiki/teams/5532/wiki/20240814.png)

Note: Smartphone APP designed for bacteria detection.

{% endblock %}

{% 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. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5532/wiki/desc-banner-1.png)

{{ self.title() }}

* Overview
* Round 1
* Round 2
* Round 3

Overview

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.

Round 1——pET28a-plyV12C（BBa\_K5532006）

Design

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).

![](https://static.igem.wiki/teams/5532/wiki/engineering/figure-1.png)

Figure 1 The plasmic structure of pET28a-plyV12

Build

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.

![](https://static.igem.wiki/teams/5532/wiki/engineering/figure-2.png)

Figure 2 Agarose gel electrophoresis of PCR amplified products for plyV12 gene and the linearized pET28a vector

![](https://static.igem.wiki/teams/5532/wiki/engineering/figure-3.png)

Figure 3 Colony PCR and sequencing results for the identification of pET28a-plyV12 plasmid

Test

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).

![](https://static.igem.wiki/teams/5532/wiki/engineering/figure-4.png)

Figure 4. SDS-PAGE analysis of the expression and purification of plyV12(CBD) protein

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.

![](https://static.igem.wiki/teams/5532/wiki/engineering/figure-5.png)

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

Learn

| 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. |

Round 2——pET28a-gp17（BBa\_K5532004）

Design

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).

![](https://static.igem.wiki/teams/5532/wiki/engineering/figure-6.png)

Figure 6 The plasmic structure of pET28a-gp17

Build

To construct the pET28a-gp17 plasmid, we amplified the gp17 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.

![](https://static.igem.wiki/teams/5532/wiki/engineering/figure-7.png)

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

![](https://static.igem.wiki/teams/5532/wiki/engineering/figure-8.png)

Figure 8 Colony PCR and sequencing results for the identification of pET28a-gp17 plasmid

Test

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).

![](https://static.igem.wiki/teams/5532/wiki/engineering/figure-9.png)

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.

![](https://static.igem.wiki/teams/5532/wiki/engineering/figure-10.png)

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

Learn

| 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. |

Round 3——pET28a-Tailspike（BBa\_K5532005）

Design

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 11).

![](https://static.igem.wiki/teams/5532/wiki/engineering/figure-11.png)

Figure 11 The plasmic structure of pET28a-tailspike

Build

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.

![](https://static.igem.wiki/teams/5532/wiki/engineering/figure-12.png)

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

![](https://static.igem.wiki/teams/5532/wiki/engineering/figure-13.png)

Figure 13 Colony PCR (red box) and sequencing results for of pET28a-tailspike plasmid

Test

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).

![](https://static.igem.wiki/teams/5532/wiki/engineering/figure-14.png)

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.

![](https://static.igem.wiki/teams/5532/wiki/engineering/figure-15.png)

Figure 15 UV-Vis absorption spectra of AuNPs@TSP and bacteria detection colorimetric analysis of AuNP@TSP conjugates

Learn

| 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. |

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5532/wiki/desc-banner-1.png)

{{ self.title() }}

* Plasmid extraction
* PCR
* Agarose Gel Electrophoresis
* DNA Gel Extraction
* Gibson assembly
* Transformation of Competent Cells
* Colony PCR
* Expression of His-Tagged Fusion PBP Proteins
* Purification of His-Tagged Fusion PBP Proteins
* SDS-PAGE Gel Electrophoresis
* Coomassie Brilliant Blue Staining
* Synthesis of AuNPs and Preparation of AuNPs@PBP Conjugation
* TEM for Detection of Gold Nanoparticles (AuNPs)
* Bacterial Detection Using AuNPs @ PBP and Smartphone-Based Colorimetric Assay
* Photothermal sterilization of AuNP@PBP

Plasmid extraction (DiaSpin Plasmid Mini-Preps Kit, B110091-0050)

1.Material and Apparatus

|  |
| --- |
| Buffer S |
| Buffer SP1 |
| Buffer SP2 |
| Buffer SP3 |
| Buffer DW1 |
| Wash solution |
| Elution buffer |
| Absorption column |
| Collection tube |
| Centrifuge |
| EP tube |

2.Procedure

1) Preparation

a.Check that RNase A has been added to Buffer SP1.

b.Check whether 100% ethanol has been added to the Wash Solution.

c.Check whether Buffer SP2 and SP3 are precipitated.

2) Equilibrate column.

a.Place a Miniprep column into a collection tube.

b.Add 500 μL Buffer S to the adsorption column, centrifuge at 12,000 ×g for 1 min, discard the liquid in the collection tube, and put the column back into the collection tube.

3) Take 5 mL of overnight cultured bacterial, centrifuge at 8,000 ×g for 2 min to collect bacterial, discard the supernatant.
4) Resuspend the bacterial were completely suspended by adding 250 μL Buffer SP1, make the pellet complete resuspended.
5) Add 250 µL Buffer SP2 and gently invert the tube for 10 times. And incubate at room temperature for 4 min.
6) Add 350 µL Buffer SP3 and gently invert the centrifuge tube 10 times for mixing.
7) Centrifuge at 12,000 ×g for 10 min. Transfer the supernatant into the adsorption column, centrifuge 8,000 ×g for 30 sec, discard the liquid in the collection tube.
8) Add 500 μL Buffer DW1, centrifuge at 9,000 ×g for 30 sec, and discard the liquid in the collection tube.
9) Add 500 μL Wash Solution, centrifuge at 9,000 ×g for 30 sec, and discard the liquid in the collection tube.
10) Repeat Step 9.
11) Place the column back into the collection tube and centrifuged at 9,000 ×g for 1 min.
12) Put the adsorption column into a clean 1.5 mL centrifuge tube, add 75 μL Elution Buffer in the center of the adsorption membrane, incubate at room temperature for 1 min, and centrifuge at 9,000 ×g for 1 min. Preserve the DNA solution in the tube.

PCR (Polymerase Chain Reaction)

1.Material and Apparatus

|  |
| --- |
| PrimeStar Max Premix (2×) |
| Primers |
| Template DNA |
| ddH2O |
| PCR Thermal Cycler |
| Pipettes and Tips |
| PCR tube |

2.Procedure

1) Reaction Setup

a.Thaw all reagents on ice and keep them on ice throughout the setup.

b.Prepare a master mix for the number of reactions plus one extra to account for pipetting errors. The master mix should include the following per 50 μL reaction:

| Reagent | Volume (µL) |
| --- | --- |
| PrimeStar Max Premix (2×) | 25 |
| Primer F | 1 |
| Primer R | 1 |
| DNA template | 1 (~10 ng) |
| ddH2O | 22 |
| Total | 50 |

| Primer name | Sequence |
| --- | --- |
| plyV12C-R | agtggtggtggtggtggtgctcgagttatttgaaagtaccccacgcctc |
| plyV12C-F | gggtcgcggatccgaattcatgctaaatgggggatcaacaccccc |
| plyV12C-pET28a-F | gcgtggggtactttcaaataactcgagcaccaccaccaccaccactgaga |
| plyV12C-pET28-R | tgatcccccatttagcatgaattcggatccgcgacccatttgctgtccac |
| tailspike-F | gtggtggtggtgctcgagttaggctaacgtcagacgagtatagc |
| tailspike-R | tcgcggatccgaattcatgagttcaggatgtggggatgtactatctc |
| tailspike- pET28a-F | ctgacgttagcctaactcgagcaccaccaccaccaccactgagatccgg |
| tailspike- pET28a-R | ccccacatcctgaactcatgaattcggatccgcgacccatttgctgtcca |
| gp17-R | ggtggtggtgctcgagttaaacatcttcaaccgcaat |
| gp17-F | tcgcggatccgaattcatggctaatgttataaaaactgtactaacata |
| gp17- pET28a-F | attgcggttgaagatgtttaactcgagcaccaccaccaccaccactg |
| gp17- pET28a-R | ttttataacattagccatgaattcggatccgcgacccatttgctgtc |

2) Mix gently and briefly centrifuge to ensure all components are at the bottom of the tube.
3) Place the PCR tubes in the PCR thermal cycler and set the following parameters:

| Process | Temperature | Time | Cycle |
| --- | --- | --- | --- |
| Pre-Denaturation | 98℃ | 2 min | 1 |
| Denaturation | 98℃ | 10 sec |  |
| Annealing | 70℃ | 30 sec | 30 |
| Extension | 72℃ | 2.5 min |  |
| Final Extension | 72℃ | 10 min | 1 |
| Hold | 4℃ | until samples are removed | 1 |

Agarose Gel Electrophoresis

1.Material and Apparatus

|  |
| --- |
| Agarose Powder |
| TAE or TBE Buffer |
| Loading Dye |
| GelRed (Beyotime- D0139) |
| Trans15K DNA Marker |
| Microwave oven |
| Gel Casting Tray and Comb |
| UV Transilluminator |
| Electrophoresis Apparatus |

2.Procedure

1) Gel Preparation

a.Dissolve Agarose: For a 1% gel, weigh out 1 gram of agarose in 100 mL of 1X TAE buffer. And heat the mixture in a microwave until the agarose is completely dissolved. Be cautious of boiling over.

b.Cool the Agarose: Allow the molten agarose to cool to about 50-60°C before pouring. Add 10 µL GelRed and mix thoroughly.

2) Casting the Gel

a.Set Up the Gel Tray: Place the gel tray in the casting stand and insert the comb to form wells.

c.Pour the Agarose Solution: Pour the cooled agarose solution into the gel tray, ensuring no bubbles form. Allow the gel to solidify at room temperature for 20-30 min.

d.Remove the Comb: Carefully remove the comb once the gel is set.

3) Add Loading Dye: Mix DNA samples with loading dye to a final concentration of 1X.
4) Setting Up Electrophoresis

a.Place the Gel in the Electrophoresis Tank: Position the gel in the electrophoresis apparatus and cover it with 1× TAE buffer until the gel is fully submerged.

b.Load the Samples: Carefully pipette the DNA samples and the DNA ladder into the wells of the gel.

c.Run the gel at 150 V for 25 min

5) Visualizing the DNA: Place the gel on a UV transilluminator to visualize the DNA bands. And capture images of the DNA bands Documenting Results
6) Analyze the Results: Compare the bands of your samples to the DNA ladder to determine the sizes of the DNA fragments.

DNA Gel Extraction (DiaSpin DNA Gel Extraction Kit, B110092-0050)

1.Material and Apparatus

|  |
| --- |
| Wash Solution |
| Buffer B2 |
| Elution Buffer |
| Column with collection tube |
| Centrifuge |
| Razor Blade |
| Microcentrifuge Tubes |
| Pipettes and Tips |

2.Procedure

1) Excising the DNA Fragment

a.Visualize the DNA Bands: Place the agarose gel on a UV transilluminator and identify the DNA band of interest.

b.Excise the DNA Band: Using a clean razor blade, carefully cut out the DNA band from the gel. Minimize the amount of excess agarose around the band. Place the excised gel slice into a clean microcentrifuge tube.

2) Add 3 volumes of Buffer B2 to 1 volume of the gel slice (e.g., if the gel slice is 100 mg, add 300 μL of binding buffer).
3) Incubate the tube at 50°C water bath for 5-10 min or until the gel is completely dissolved. Vortex the tube briefly every 2-3 min to facilitate dissolution.
4) Place a column into a collection tube. And transfer the dissolved gel mixture to the column.
5) Centrifuge at 8000 ×g for 30 sec. Discard the flow-through and place the column back into the collection tube.
6) Add 500 μL wash buffer to the column. And centrifuge at 9000 ×g for 30 sec. Discard the flow-through and place the column back into the collection tube.
7) Repeat step 6
8) Spin the column at 9000 ×g for an additional 1 min to remove any residual wash buffer.
9) Place the column into a clean microcentrifuge tube. Add 35 μL elution buffer directly onto the center of the column. Let it sit at room temperature for 1 min to increase yield.
10) Spin the column at 10,000 - 16,000 ×g for 1 min to elute the DNA.

Gibson assembly (ClonExpress Ultra One Step Cloning Kit, C115-01)

1.Material and Apparatus

|  |
| --- |
| 2× ClonExpress Mix |
| Linearized Vector |
| Gene fragment |
| PCR Tube |
| Pipettes and Tips |
| PCR Thermal Cycler |

2.Procedure

1) Thaw the 2× ClonExpress Mix on ice.
2) Prepare the Cloning Reaction in a PCR tube, set up the reaction as follows:

| Reagent | Volume (µl) |
| --- | --- |
| Linearized Vector | [0.02 × length bp] ng |
| Gene fragment | [0.04 × length bp] ng |
| 2× ClonExpress Mix | 5 |
| ddH2O | - |
| Total | 10 |

3) Gently mix the reaction by pipetting up and down. Incubate at 50°C for 5 min.

Transformation of Competent Cells

1.Material and Apparatus

|  |
| --- |
| Competent Cells |
| SOC Medium |
| LB Agar Plates with Antibiotics |
| Water Bath |
| Incubator |
| Pipettes and Tips |

2.Procedure

1) Thaw competent cells on ice.
2) Add 10 μL of cloning reaction to 50 μL of competent cells. Gently mix by flicking the tube.
3) Incubate the mixture on ice for 30 min.
4) Heat-shock the cells at 42°C for 45 sec.
5) Recover Cells: Immediately transfer the cells back to ice for 2 min.
6) Add 700 μL of pre-warmed SOC medium to the cells.
7) Incubate the cells at 37°C with shaking (200-250 rpm) for 1 hour.
8) Spread 200 μL of the transformed cells onto the LB agar plate with kanamycin.
9) Incubate the plates at 37°C overnight.

Colony PCR

1.Material and Apparatus

|  |
| --- |
| PCR Master Mix |
| Primers |
| PCR Tube |
| Sterile Pipette Tips |
| PCR Thermal Cycler |

2.Procedure

1) Using a sterile toothpick or pipette tip, gently pick a single bacterial colony from the LB agar plate.
2) Suspend the picked colony in 100 μL LB medium with kanamycin in a microcentrifuge tube. Incubate at 37°C for 1 hour.
3) Use 1-2 μL of this bacterial suspension as the template for the PCR reaction.
4) PCR reaction setup as follows

| Reagent | Volume (µl) |
| --- | --- |
| PCR Master Mix (2×) | 5 |
| Primer F | 1 |
| Primer R | 1 |
| DNA template | 1 |
| ddH2O | 2 |
| Total | 10 |

5) Mix gently and briefly centrifuge to ensure all components are at the bottom of the tube.
6) Place the PCR tubes in the PCR thermal cycler and set the following parameters:

| Process | Temperature | Time | Cycle |
| --- | --- | --- | --- |
| Pre-Denaturation | 95℃ | 2 min | 1 |
| Denaturation | 95℃ | 10 sec | 30 |
| Annealing | 70℃ | 30 sec |
| Extension | 72℃ | 2.5 min |
| Final Extension | 72℃ | 10 min | 1 |
| Hold | 4℃ | until samples are removed | 1 |

Expression of His-Tagged Fusion PBP Proteins

1.Material and Apparatus

|  |
| --- |
| IPTG |
| LB medium |
| Kanamycin |
| Shaker Incubator |

2.Procedure

1) Inoculate a single colony of BL21 E. coli containing the plasmid into 10 mL of LB medium with the 50 μg/mL kanamycin.
2) Incubate overnight at 37°C with shaking (200-250 rpm).
3) Dilute the overnight culture 1:100 into 50 mL fresh LB medium containing the 50 μg/mL kanamycin.
4) Incubate at 37°C with shaking until the OD600 reaches 0.4-0.6.
5) Add IPTG to a final concentration of 1 mM to induce protein expression.
6) Incubate at 16°C for 18 hours with shaking.
7) Harvesting Cells: centrifuge the culture at 4,000 ×g for 15 min at 4°C to pellet the cells. Discard the supernatant and proceed to protein purification.

Purification of His-Tagged Fusion PBP Proteins

1.Material and Apparatus

|  |
| --- |
| IPTG |
| Beyotime His-tag Protein Purification Kit (Denaturant-resistant), P2229S |
| Sonicator |
| 50 mL Centrifuge Tubes |
| Centrifuge Tubes |
| Pipettes and Tips |

2.Procedure

1) Cell Lysis

a.Resuspend the cell pellet in 10 mL lysis buffer provided in the Purification Kit. Use non-denaturing condition for tailspike protein and denaturing condition for plyV12 and gp17 protein.

b.Lyse the cells by sonication on ice (30 sec on, 30 sec off, for a total of 15 min).

c.Centrifuge the lysate at 12,000 ×g for 30 min at 4°C to remove cell debris. Take 100 µL of supernatant as a sample for subsequent analysis, and transfer the remaining supernatant to a new, clean centrifuge tube.

2) Protein Binding

a.Take 1 mL of well-mixed 50% BeyoGold™ His-tag Purification Resin and add it to the column. Add 0.5 mL of lysis buffer, mix to equilibrate, and discard the liquid. Repeat the equilibration 1-2 times and discard the liquid.

b.Add the cell lysis supernatant to a column containing Purification Resin

c.Incubate the mixture at 4℃ with gentle shaking for 1 hour to allow binding.

3) Protein Wash

a.Open the cap at the bottom of the purification column and let the liquid flow out by gravity. Collect approximately 100 µL of the flow-through for subsequent analysis.

b.Wash the resin 5 times, each time adding 0.5-1 mL wash buffer. Collect approximately 100 µL of the flow-through wash buffer each time for subsequent analysis.

4) Protein Elution: Elute the target protein 6-10 times, each time using 0.5 mL elution buffer. Collect each elution fraction into different centrifuge tubes. The collected elution fractions are the purified His-tagged protein samples.

SDS-PAGE Gel Electrophoresis

1.Material and Apparatus

|  |
| --- |
| 15% SDS-PAGE Color Preparation kit, C671104 |
| Tris-Glycine-SDS buffer |
| Protein Ladder |
| Loading Buffer |
| SDS-PAGE Gel Casting System |

2.Procedure

1) Prepare a 15% SDS-PAGE gel according to the instructions provided with the 15% SDS-PAGE Color Preparation kit, C671104
2) Mix protein samples with 2× loading buffer and heat at 95°C for 10 min.
3) Load the samples and protein ladder into the wells of the SDS-PAGE gel.
4) Place the gel in the electrophoresis tank and add 1×Tris-Glycine-SDS running buffer.
5) Run the gel at 100 V for the stacking gel and then increase to150 V for the resolving gel until the dye front reaches the bottom.

Coomassie Brilliant Blue Staining

1.Material and Apparatus

|  |
| --- |
| Coomassie Brilliant Blue R-250 Stain Solution |
| Destaining Solution |
| Gel Imaging Systems |

2.Procedure

1) After electrophoresis, carefully remove the gel from the glass plates and place it in a staining tray.
2) Cover the gel with the staining solution and gently agitate on a shaker for 30 min at room temperature
3) Replace the staining solution with the destaining solution and gently agitate on a shaker.
4) Change the destaining solution periodically until the background is clear and protein bands are distinctly visible.
5) Once the gel is adequately destained, place it in distilled water to remove any residual destaining solution. Visualize the gel using a gel imaging system.

Synthesis of AuNPs and Preparation of AuNPs@PBP Conjugation

1.Material and Apparatus

|  |
| --- |
| Aurochlorohydric acid (HAuCl4) Solution: 10 mg/mL |
| Sodium Citrate Solution: 10 mg/mL |
| Potassium Carbonate Solution: 100 mM |
| 1% Bovine Serum Albumin (BSA) |
| Phosphate Buffered Saline (PBS) |
| Round-Bottom Flask |
| Magnetic Stirrer and Stir Bar |
| Shaker |
| Spectrophotometer |
| Microplate |

2.Procedure

1) Preparation of Gold Nanoparticles (AuNPs)

a.Add 100 mL of ultra-pure water to a round-bottom flask.

b.Add 1 mL of 10 mg/mL HAuCl4 solution to the flask. And place the flask on a magnetic stirrer and heat the mixture to boiling, allowing it to reflux.

c.After the mixture reaches boiling, add 1 mL of 10 mg/mL sodium citrate solution to the flask.

d.Continue stirring and heating until the solution turns a deep purple-red color, indicating the formation of gold nanoparticles.

e.Continue heating and stirring for an additional 10 min to ensure complete reaction.

f.Remove the flask from the heat and allow the solution to cool to room temperature. Store the solution at 4°C in the dark for future use.

2) Protein Conjugation with AuNPs

a.Adjusting pH of AuNPs Solution: Take 1 mL of the AuNPs solution prepared in the previous steps. Then gradually add 7-8 μL of 100 mM potassium carbonate solution to adjust the pH to 8.0. Monitor pH carefully.

b.Add 100 μg of PBP protein to the pH-adjusted AuNPs solution. Incubate the mixture on a shaker at room temperature for 30 min to allow protein binding.

c.Centrifuge the mixture at 12,000 rpm for 5 minutes at 4°C. Then carefully discard the supernatant to remove unbound protein.

d.Add 1% BSA solution to resuspend the pellet and incubate at room temperature for 30 min to block any remaining binding sites on the AuNPs.

e.Centrifuge again at 12,000 rpm for 5 min at 4°C and discard the supernatant.

f.Resuspend the AuNPs@PBP pellet in 333 μL of PBS buffer.

g.Store the conjugated AuNPs@PBP solution at 4°C for future use.

3) Measurement of AuNPs@PBP Absorbance

a.Scan the full wavelength range to determine the maximum absorption peak of the AuNPs@PBP solution using a spectrophotometer.

b.Note that AuNPs typically have a maximum absorption peak at 530 nm. Successful conjugation with PBP protein should result in a red shift of 2-3 nm in the absorption peak.

TEM for Detection of Gold Nanoparticles (AuNPs)

1.Material and Apparatus

|  |
| --- |
| AuNP suspension |
| Carbon-coated copper grids (300 mesh) |
| Potassium Carbonate Solution: 100 mM |
| Ultrapure water |
| Micropipette (0.5-10 μL) |
| Forceps |
| Filter paper |
| Desiccator |
| Talos F200x TEM (Thermo Fisher, USA) |

2.Procedure

1)Prepare the TEM grids:

Hold the carbon-coated copper grid with fine-tipped forcepsand ensure the carbon-coated side is facing up.
2)Sample preparation:

a.Sonicate the AuNP suspension for 5 min to ensure homogeneous dispersion.

b.Dilute the AuNP suspension with ultrapure water (concentration should be approximately 0.1-1 mg/mL).

c.Place a 5 μL droplet of the prepared AuNP suspension onto the carbon-coated side of the grid.

d.Allow the sample to adsorb for 60 sec.

e.Gently touch the edge of the grid with a piece of filter paper to remove excess liquid.

f.Be careful not to touch the center of the grid where the sample is deposited.

g.Place the grid in a desiccator for 30 min to ensure complete drying.

3)TEM imaging:

a.Load the prepared grid into the TEM according to the instrument's guidelines.

b.Operate the TEM at an accelerating voltage of 80-200 kV, depending on the instrument and sample requirements.

c.Capture images at various magnifications to observe the size, shape, and distribution of the AuNPs.

Bacterial Detection Using AuNPs @ PBP and Smartphone-Based Colorimetric Assay

1.Material and Apparatus

|  |
| --- |
| Phosphate Buffered Saline (PBS) |
| Bacterial culture |
| Smartphone with camera |
| RGB analysis software or app |
| Fresh lettuce and apples from a local supermarket |

2.Procedure

1) Preparation of Bacterial Suspension:

a.Cultivate the target bacteria to achieve an OD600 of 1 (approximately 10^9 CFU/mL).

b.Collect the bacterial cells by centrifuging the culture and discard the supernatant.

c.Resuspend the bacterial pellet in PBS and adjust the suspension to an OD600 of 1 (approximately 10^9 CFU/mL).

d.Perform serial dilutions in PBS to achieve concentrations ranging from 10^3 to 10^9 CFU/mL.

2) Add 200 μL of each bacterial dilution to 100 μL of AuNPs @ PBP solution and incubate the mixtures at room temperature for 30 min. Include appropriate controls, such as pET28a empty vector controls, to validate the assay specificity and accuracy.
3) After incubation, Transfer 200 μL of the supernatant into new 1.5 mL centrifuge tubes for imaging.
4) Imaging and Data Collection:

a.Assemble the smartphone and lightbox setup. Ensure the lightbox is equipped with a white LED light tube and black curtains to minimize external light interference.

b.Place the centrifuge tubes containing the supernatant in the lightbox. Capture images of the samples using the smartphone camera.

5) Data Analysis

a.Extract the RGB values from the captured images using the RGB analysis software or app.

b.Generate a standard curve based on the RGB values corresponding to known bacterial concentrations.

c.Import the standard curve into the app for subsequent analysis.

Photothermal sterilization of AuNP@PBP

1.Material and Apparatus

|  |
| --- |
| LB solid medium |
| near-infrared light (808 nm) |
| thermal imaging camera (TIC) |

2.Procedure

a.The bacterial solution was diluted to a concentration of 10^6 CFU/mL and reacted with the above AuNP@PBP detection method for bacteria.

b.After the above detection part was reacted for 30 min at room temperature, the sample tube was irradiated under near infrared light (NIR) with a wavelength of 808 nm and a power of 2 W for 10 min (at a distance of 8 cm).

c.The temperature rise of the samples after NIR treatment was monitored using a photothermal camera.

d.Plates were coated with the irradiated samples and incubated at 37°C overnight

e.Bacterial survival was determined by plate counting method.

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* Overview
* Plasmid construction
* Protein expression and purification
* Synthesis of AuNPs and Preparation of AuNPs@PBP Conjugation
* Bacterial Detection Using AuNPs @ PBP and Smartphone-Based Colorimetric Assay
* Photothermal sterilisation test
* Conclusion
* Future Plan

Overview

This project focuses on developing a biosensor for rapid detection and sterilization of *Escherichia coli (E. coli)*, *Salmonella*, and *Staphylococcus aureus (S. aureus)*. We selected three phage-derived bacterial-binding proteins (PBPs) — T3 phage tail fiber protein (TFP), *Salmonella* phage CKT1 tailspike protein (TSP), and *S. aureus* phage plyV12 cell wall binding domain (CBD). These proteins were synthesized using synthetic biology and conjugated to gold nanoparticles (AuNPs) via electrostatic adsorption, resulting in AuNPs@TFP, AuNPs@TSP, and AuNPs@CBD. AuNPs@TFP and AuNPs@TSP showed strong binding affinity and induced a color change upon bacterial detection. The biosensor can rapidly detect these bacteria within 30 min using a smartphone app to analyze RGB values for real-time results. Additionally, under 808 nm NIR light for 10 min, the biosensor exhibited potent antibacterial activity.

Plasmid construction

Goal: To express and purify three distinct proteins: T3 phage tail fiber protein (TFP), the tailspike protein (TSP), and the cell wall binding domain (CBD) of the endolysin plyV12, we aim to construct three expression plasmids: pET28a-gp17, pET28a-Tailspike and pET28a-plyV12. These plasmids will be designed to facilitate the efficient expression of the respective proteins in BL21(DE3) bacterial, thereby enabling subsequent purification and functional studies.

1) Gene and Linearized Vector Amplification

To construct the pET28a-Tailspike, pET28a-gp17, and pET28a-plyV12 plasmids, we first amplified the target genes and linearized pET28a vector fragments using Polymerase Chain Reaction (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 target genes and the linearized vector fragments. Distinct bands corresponding to the expected sizes of the tailspike (2067 bp), gp17(1689 bp), and plyV12(525 bp) genes were observed, confirming the presence of the desired PCR products (Figure 1). Additionally, the linearized pET28a vector fragment (5854 bp) was also clearly visible, indicating successful linearization.

These results indicate that the PCR amplification of the target genes and the linearized pET28a vector fragments were successful, providing the necessary components for subsequent cloning steps.

![](https://static.igem.wiki/teams/5532/wiki/results/fihure-1.png)
Figure 1. Agarose Gel Electrophoresis of PCR Amplified Products for Plasmid Construction
Agarose gel electrophoresis (1% agarose) of PCR amplified products used for the construction of plasmids. PCR product of the (A) gp17 gene and corresponding linearized pET28a vector. (B) gp17 gene and corresponding linearized pET28a vector. (C) Tailspike gene and corresponding linearized pET28a vector. Note: M indicated DNA ladder.

2) Gibson Assembly and Bacterial Transformation

Following the successful amplification of the target genes and linearized vector fragments by PCR, we proceeded with the Gibson Assembly to recombine the DNA fragments into the desired plasmids: pET28a-Tailspike, pET28a-gp17, and pET28a-plyV12. The Gibson Assembly reaction mixtures were then used to transform DH5α competent cells.

After transformation, the DH5α cells were plated on LB agar plates containing kanamycin, for selection. Numerous single colonies were observed after the plates were incubated overnight at 37°C (Figure 2), indicating successful transformation.

![](https://static.igem.wiki/teams/5532/wiki/results/fihure-2.png)
Figure 2: Bacterial Colony Formation on LB Agar Plates Post-Transformation
LB agar plates containing kanamycin selection were used to assess the success of the Gibson Assembly and transformation into DH5α cells. The plates were incubated overnight at 37°C. Plate showing colonies resulting from the transformation with (A) the pET28a-gp17 plasmid. (B) the pET28a-plyV12 plasmid. (C) the pET28a-Tailspike plasmid.

3)Colony PCR Screening

To identify potential positive clones, colony PCR was performed on several bacterial colonies from each transformation plate. The colony PCR products were analyzed via agarose gel electrophoresis. The gel images revealed distinct bands corresponding to the expected sizes of the Tailspike, gp17, and plyV12 gene inserts (Figure 3A), indicating the presence of the desired recombinant plasmids in several colonies.

These colonies will be further verified by sequencing. The sequencing results confirmed that the gene sequences in the plasmids were correct and matched the expected sequences for gp17, plyV12, and Tailspike (Figure 3B-D). This confirmed that the plasmid constructs pET28a-gp17, pET28a-plyV12, and pET28a-tailspike were successfully constructed without any mutations.

![](https://static.igem.wiki/teams/5532/wiki/results/fihure-3.png)
Figure 3: Colony PCR and Sequencing Results for the Identification of Positive Clones
(A) Agarose gel electrophoresis showing colony PCR results for the identification of positive clones containing the recombinant plasmids. Distinct bands at the expected sizes for the gp17, Tailspike and plyV12 inserts confirm the presence of the desired recombinant plasmids.
(B-D) Sequencing chromatograms of representative positive clones. The chromatograms show high-quality sequences with no mutations or insertions.

Protein expression and purification

To express and purify the tail fiber protein (TFP) from bacteriophage T3, the tailspike protein (TSP) from Salmonella phage CKT1, and the cell wall binding domain (CBD) of the endolysin plyV12 from Staphylococcus phage, the constructed the corresponding expression plasmids: pET28a-gp17, pET28a-Tailspike, and pET28a-plyV12 were transformed 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 shows distinct bands at the expected molecular weights for plyV12, gp17 and Tailspike protein (Figure 4A), confirming successful IPTG-induced expression of the target proteins. After 24 hours of induction, the cells were harvested, lysed, and the His-tagged proteins were purified using nickel-affinity chromatography. The purified proteins were analyzed by SDS-PAGE to assess their purity and molecular weight (Figure 4B, C, D). The results demonstrated successful expression and purification of the target proteins.

![](https://static.igem.wiki/teams/5532/wiki/results/fihure-4.png)
Figure 4. SDS-PAGE Analysis of Expression and Purification of target Protein
SDS-PAGE Analysis of Protein Expression and Purification. (A) Total cell lysate of BL21(DE3) cells carrying pET28a-gp17, pET28a-Tailspike, and pET28a-plyV12 plasmid before/after IPTG induction were used as sample. SDS-PAGE Analysis of Purified Proteins, Different purified fraction of (B) purified TSP from BL21(DE3) cells carrying pET28a-Tailspike, (C) purified CBD from BL21(DE3) cells carrying pET28a-plyV12, (D) purified TFP from BL21(DE3) cells carrying pET28a-gp17.S indicated the supernatant of cell lysis. P indicated the pellet of cell lysis. FT indicated the Flow-through fraction. Wash indicated the wash solution fraction. Elution indicated the elution buffer of purified protein.

Synthesis of AuNPs and Preparation of AuNPs@PBP Conjugation

The synthesis of gold nanoparticles was achieved through the reduction of chloroauric acid (HAuCl₄) using sodium citrate as the reducing agent. Initially, the chloroauric acid solution was colorless (Figure 5A). Upon addition of trisodium citrate and subsequent heating, a distinct color change was observed. The solution gradually turned purple-red, indicating the successful formation of gold nanoparticles (Figure 5B). This color change is attributed to the specific surface plasmon resonance (SPR) of the gold nanoparticles, which confirms their synthesis. We used transmission electron microscopy to observe the prepared AuNP, and the results showed that our prepared AuNP were uniform spheres with a particle size between 40-50 nm, which further confirmed the success of AuNP preparation(Figure 5C).

![](https://static.igem.wiki/teams/5532/wiki/results/figure-r5.png)
Figure 5. Synthesis of Gold Nanoparticles.
(A) Initial colorless chloroauric acid (HAuCl₄) solution before the addition of sodium citrate. (B) The solution after the addition of trisodium citrate and heating, showing a purple-red color. (C) TEM image of AuNP.

Following the synthesis of gold nanoparticles (AuNPs), we aimed to conjugate PBP proteins (TFP, TSP, and CBD) to AuNPs via electrostatic physical adsorption. This process was conducted in a mildly alkaline pH environment. Specifically, 100 μg of each protein TFP, TSP, and CBD was added to 1 mL of AuNPs solution. Additionally, pET28a empty vector protein was used as a control. The AuNPs solution was initially purple and exhibited an absorption peak at 530 nm. Upon successful protein conjugation, the maximum absorption peak was expected to shift from 530 nm to 533 nm.

After adding 100 μg pET28a empty vector protein to 1 mL of AuNPs, the absorption peak shifted from 530 nm to 533 nm. This shift in the absorption peak indicates successful conjugation (Figure 6A). Similar to pET28a empty vector protein, after adding 100 μg of TFP and TSP proteins to 1 mL of AuNPs, the absorption peak shifted from 530 nm to 533 nm. This shift in the absorption peak indicates successful conjugation of both TFP and TSP proteins to the AuNPs (Figure 6B，C). During the conjugation process of CBD protein to AuNPs, the solution changed from purple-red to colorless upon the addition of 100 μg CBD to 1 mL of AuNPs. Additionally, no significant 530 nm or 533 nm absorption peak was observed in the spectrum. These observations suggest that the conjugation of CBD protein to AuNPs was failed (Figure 6D).

We consulted the literature and found that the pH of the solution affects the degree of protonation/deprotonation of the chemical groups (i.e. amines and carboxyl groups) that confer electrostatic charge on proteins and AuNPs [1]. The pH of the buffer is critical because it must be above the isoelectric point of the protein so that the protein can displace the negatively charged citrate groups adsorbed on the surface of the AuNPs to form the AuNP@PBP [2]. Thus, we used an online tool (https://web.expasy.org/protparam/) to predict the theoretical isoelectric points (pI) of the three proteins. We found that the theoretical pI of TFP (gp17) and TSP (Tailspike) proteins are 5.74 and 5.0, respectively, which are smaller than pH 8.0, and thus can bind well to AuNP. On the other hand, the theoretical pI of CBD (plyV12) was 9.84, which was larger than pH 8.0, which might lead to the failure of AuNP@CBD preparation as CBD could not bind well to AuNP.

![](https://static.igem.wiki/teams/5532/wiki/results/fihure-6.png)
Figure 6. UV-Vis Absorption Spectra of AuNPs Conjugated with PBP
UV-Vis absorption spectrum of synthesized AuNPs and AuNPs after conjugation with (A) pET28a empty vector protein (B) TFP protein (C) TSP protein showing a shift in the absorption peak from 530 nm to 533 nm, indicating successful conjugation. (D) UV-Vis absorption spectrum of AuNPs after attempted conjugation with plyV12 protein, no significant absorption peak was observed, indicating unsuccessful conjugation.

Bacterial Detection Using AuNPs @ PBP and Smartphone-Based Colorimetric Assay

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 (Figure 7A). 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 (Figure 7B, 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 (Figure 7D).

![](https://static.igem.wiki/teams/5532/wiki/results/fihure-7.png)
Figure 7. Colorimetric Analysis of Bacteria Detection Using AuNP@PBP Conjugates
(A) Smartphone images of the supernatant after incubation with AuNP@pET28a (control) and E. coli, Salmonella, and S. aureus. (B) Smartphone images of the supernatant after incubation with AuNP@TFP and varying concentrations of E. coli ((10^3) to (10^9) CFU/mL). (C) Smartphone images of the supernatant after incubation with AuNP@TSP and varying concentrations of Salmonella ((10^3) to (10^9) CFU/mL). (D) Smartphone images of the supernatant after incubation with AuNP@CBD and S. aureus.

Subsequently, we performed an RGB colour analysis of the results of AuNPs@TFP and AuNPs@TSP, and found a correspondence between the increase in bacterial concentration and the corresponding B/R values. As a result, we obtained a standard curve between the logarithmic values of bacterial concentration and B/R. (Figure 8) For AuNP@TFP, the standard curve was y=0.0125x+1.0404 (R2=0.9583), and for AuNP@TSP, the standard curve was y=0.00098x+1.0684 (R2=0.9804).

![](https://static.igem.wiki/teams/5532/wiki/results/figure-8.png)
Figure 8. Standard curve between the logarithmic values of bacterial concentration and B/R
(A) AuNP@TFP detection of E. coli; (B) AuNP@TSP detection of Salmonella

In order to detect bacterial concentration by smartphone, we built the standard curve obtained from the experiment into the smartphone APP. After taking a picture to record the incubation supernatant of bacteria with AuNP@TFP or AuNP@TSP, we uploaded the picture to the APP, which will automatically read the RGB value of the picture and calculate the B/R value, and finally show the corresponding bacterial concentration according to the standard curve (Figure 9).

![](https://static.igem.wiki/teams/5532/wiki/results/figure-9.png)
Figure 9. Bacteria detection results based on smartphone APP

Photothermal sterilisation test

AuNPs have significant near-infrared absorption capabilities that enable them to conduct light energy into heat and make them promising candidates for targeted photothermal therapies [3]. After the above experiments, we successfully prepared novel colorimetric biosensors, including AuNPs@TFP and AuNPs@TSP. After detecting the bacteria, we irradiated the sensors with near-infrared light at a wavelength of 808 nm with a power of 2 W.

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 (Figure 10).

However, after 10 min of NIR light irradiation, there were still a few colonies growing, and in the subsequent experiments, we could adjust the distance between the NIR light and the sample, which allowed the temperature to be further increased for better bactericidal effect.

![](https://static.igem.wiki/teams/5532/wiki/results/figure-10.png)
Figure 10. Photothermal sterilisation results of (A)AuNP@TFP and (B)AuNP@TSP

Conclusion

In this project, we identified three phage-derived bacterial-binding proteins (PBPs) that specifically bind to E. coli, Salmonella, and S. aureus. We successfully constructed plasmids containing the corresponding protein genes and expressed and purified these proteins in BL21(DE3) cells. Concurrently, we synthesized gold nanoparticles (AuNPs) and conjugated the PBPs to the AuNPs through electrostatic adsorption. Analysis of the absorption peaks indicated that AuNPs@TFP and AuNPs@TSP were successfully prepared, while AuNPs@CBD was not. The successfully prepared AuNPs@TFP and AuNPs@TSP demonstrated strong binding affinity to E. coli and Salmonella, respectively, causing a color change in the detection system. This biosensor allows for rapid colorimetric detection of these bacteria within 30 minutes, providing a promising approach for bacterial detection in the food industry.

Future Plan

In this experiment, the preparation of AuNPs@CBD was unsuccessful, likely due to an inappropriate conjugation method. Due to time constraints, we were unable to explore alternative conjugation methods. Therefore, in future work, we will attempt various conjugation strategies to ensure efficient binding of the protein to AuNPs. Additionally, we will optimize the amount of AuNPs@PBP added to bacterial samples to enhance detection efficiency. We also plan to compare this method with other existing bacterial detection methods to confirm its accuracy and reliability. This will help in establishing a robust and efficient biosensor for bacterial detection in various applications, particularly in the food industry.

Reference

[1] Capocefalo A, Bizien T, Sennato S, Ghofraniha N, Bordi F, Brasili F. Responsivity of Fractal Nanoparticle Assemblies to Multiple Stimuli: Structural Insights on the Modulation of the Optical Properties. Nanomaterials (Basel). 2022;12(9):1529.
[2] Waldeisen JR, Wang T, Ross BM, Lee LP. Disassembly of a core-satellite nanoassembled substrate for colorimetric biomolecular detection. ACS Nano. 2011; 5(7):5383-9.
[3] Addae E, Dong X, McCoy E, Yang C, Chen W, Yang L. Investigation of antimicrobial activity of photothermal therapeutic gold/copper sulfide core/shell nanoparticles to bacterial spores and cells. J. Biol. Eng. 2014; 8: 11.

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