iGEM 2025 — Team banz-sza

{% extends "layout.html" %}
{% set table\_data = [{
"content": '

|  |  |
| --- | --- |
| Date | 2025.7 |
| Participants | Wu Wenran, Du Xiaolu, Kang Qinhao, Chen Ziqi, Zhang Fengshang, Pan Yanxi, Li Xinyang, Wang Youjie, Wang Zibing, Che Kaiyue, Zhou Yuanrui |
| Contents | 1.Prepare LB solid and liquid media, followed by high-temperature sterilization.  2.Perform PCR amplification of the target genes PDI and Thaumatin-A/B/C/D, and retain the amplified products for subsequent use after a 2-hour amplification reaction.  3.Prepare agarose gel and set it aside after solidification.  4.Inoculate the pRSFDuet plasmid into competent cells for shake culture. |
| Results | ![descript](https://static.igem.wiki/teams/5743/notebook/1.webp)![descript](https://static.igem.wiki/teams/5743/notebook/2.webp)  ![](https://static.igem.wiki/teams/5743/notebook/3.webp)  Agarose gel electrophoresis results of PCR for five clones |
| Date | 2025.7 |
| Participants | Wu Wenran, Du Xiaolu, Kang Qinhao, Chen Ziqi, Zhang Fengshang, Pan Yanxi, Li Xinyang, Wang, Youjie, Wang Zibing, Che Kaiyue, Zhou Yuanrui, Wu Xinke |
| Contents | 1.Extraction of pRSFDuet Vector Plasmid  2.Restriction Enzyme Double Digestion  3.DNA Gel Electrophoresis Verification  4.Gel Extraction of Linearized Vector DNA  5.Homologous Recombination pRSFDuet-PDI  6.Heat-Shock Transformation *E.coil* DH5α  7.Make kan+ Antibiotic-Resistant Plate  8.DNA Gel Dissolution and Purification |
| Results | ![descript](https://static.igem.wiki/teams/5743/notebook/4.webp)  ![descript](https://static.igem.wiki/teams/5743/notebook/5.webp)  ![descript](https://static.igem.wiki/teams/5743/notebook/6.webp) |
| Date | 2025.7 |
| Participants | Du Xiaolu, Wu Wenran, Kang Qinhao, Chen Ziqi, Pan Yanxi, Wang Youjie, Wu Xinke, Zhou Yuanrui |
| Contents | 1.prepare LB liquid/solid (solid:4×100ml) (liquid:4×150ml)  2.Colony PCR identification (picking 8 groups of colony, PCR identification  3.Identify the correct group of colony and prepare for expanding cultivation  4.Agarose gel electrophoresis determine the molecular weight of nucleic acid  5.shaking culture let the pRSF-PDI to grow |
| Results | ![descript](https://static.igem.wiki/teams/5743/notebook/7.webp)  ![descript](https://static.igem.wiki/teams/5743/notebook/8.webp)  ![descript](https://static.igem.wiki/teams/5743/notebook/9.webp)  ![descript](https://static.igem.wiki/teams/5743/notebook/10.webp) |
| Date | 2025.7 |
| Participants | Wu Wenran, Chen Ziqi, Wu Xinke, Zhou Yuanrui, XU Yihan, Huang Yunni, Che Kaiyue |
| Contents | 1.Extraction of PRSF-PDI Vector Plasmid  2.Restriction Enzyme Double Digestion  3.Prepare agarose gel and set it aside after solidification.  4.alidation of PRSF-PDI linear by agarose gel electrophoresis  5.Agarose gel recovery  6.Homologous Recombination  7.Heat-Shock Transformation |
| Results | ![descript](https://static.igem.wiki/teams/5743/notebook/11.webp)  ![descript](https://static.igem.wiki/teams/5743/notebook/12.webp) |
| Date | 2025.8 |
| Participants | Du XiaoLu, Chen Ziqi, Wu Xinke, Zhou YuanRui, XU Yihan, Huang Yunni, Che Kaiyue |
| Contents | 1.pRSFDuet-Thaumatin-A/B/C/D colony PCR identification  2.Prepare agarose gel and set it aside after solidification. (15%PAGE Gel)  3. Enlarging cultvate of *E. coli* DH5a and Origami2(DE3) |
| Results | ![](https://static.igem.wiki/teams/5743/notebook/13.webp)  Agarose gel electrophoresis results of Colony PCR identification for five clones  ![](https://static.igem.wiki/teams/5743/notebook/14.webp)  Agar plate showing BL21 colonies after transformation |
| Date | 2025.8 |
| Participants | Du XiaoLu, Chen Ziqi, Wu Xinke, Zhou YuanRui, XU Yihan, Huang Yunni, Che Kaiyue, Kang Qinhao, Zhang Fengshang |
| Contents | 1.Plasmid extraction and identification  2.Bacterial culture  3.Agarose gel and electrphoresis |
| Results | ![descript](https://static.igem.wiki/teams/5743/notebook/15.webp) |
| Date | 2025.8 |
| Participants | Du Xiaolu, Kang Qinhao, Zhang Fengshang, Pan Yanxi, Li Xinyang, Wang Zibing, Che Kaiyue, Zhou Yuanrui, Pan Yanxi, Wu Xinke |
| Contents | 1.Extract 4 kinds of crude proteins by ultrasonic disruption  2.Extract pure proteins by nickel column affinity purification  3.Perform SDS-PAGE gel electrophoresis to detect whether the protein size meets expectations  4.Conduct Coomassie brilliant blue decolorization and staining, and leave it overnight |
| Results | ![](https://static.igem.wiki/teams/5743/notebook/16.webp)  SDS-PAGE analysis of crude protein  ![](https://static.igem.wiki/teams/5743/notebook/17.webp)  SDS-PAGE analysis After Protein Elution |
| Date | 2025.8 |
| Participants | Du Xiaolu, Kang Qinhao, Chen Ziqi, Wu Xinke, Zhang Fengshang, Pan Yanxi, Wang Youjie, Che Kaiyue, Zhou Yuanrui, Huang Yunni, Xu Yihan |
| Contents | 1.Enzyme assay  2.Principles of DNA testing |
| Results | ![descript](https://static.igem.wiki/teams/5743/notebook/18.webp)![descript](https://static.igem.wiki/teams/5743/notebook/19.webp) |

'
}] %}
{% block title %}Notebook{% endblock %}
{% block lead %}NOTEBOOK{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [
{
"title": "Overview",
"content": '

Currently, many people suffer from health issues like obesity and diabetes due to excessive sucrose intake. To reduce sugar
consumption, many turn to sugar substitutes such as aspartame or erythritol; however, consuming these traditional sugar
substitutes often leads to side effects like dizziness and nausea.

In nature, there exists a protein that produces sweetness by binding to human receptors. We aimed to express the gene
encoding this protein in a bacterial plasmid. We used bacterial homologous genes that are highly similar to the plant
gene—specifically, four genes named Thaumatin A/B/C/D. Additionally, we included a fragment of the PDI (Protein Disulfide
Isomerase) gene to promote proper protein folding by facilitating the correct formation of disulfide bonds, thereby
enhancing proper protein expression.

'
},
{
"title": "Part 1: pRSFDuet-PDI",
"content": '

**Design**

We selected the pRSFDuet plasmid with dual promoters to achieve the simultaneous expression of two proteins (Thaumatin
homologous protein and PDI protein). The pRSFDuet plasmid vector contains two independent multiple cloning sites, each
equipped with a T7 promoter, a ribosomal binding site, and a transcription terminator, enabling efficient regulation of the
synchronous expression of two target genes in the same host cell. Additionally, it has a kanamycin resistance marker, which
facilitates the screening and identification of positive clones.

![](https://static.igem.wiki/teams/5743/engineering/1.webp)

Figure 1 The map of pRSFDuet plasmid

We successfully inserted the PDI gene fragment into the pRSFDuet plasmid via double digestion and homologous recombination
techniques. The PDI protein catalyzes the formation of protein disulphide bonds, which results in the correct folding of the
protein, allowing the Thaumatin A/B/C/D proteins to successfully bind to the human sweetness receptor and generate a sweet
taste.

![](https://static.igem.wiki/teams/5743/engineering/2.webp)

Figure 2 The map of pRSFDuet-PDI

**Build**

The PDI gene fragment (1568 bp) was amplified using PCR. Figure 3 showed the agarose gel electrophoresis results of PCR. We
can find out that the DNA fragment of PDI is approximately 1568 bp in length, which is consistent with the predicted size.
This indicates that the target gene was successfully amplified.

![](https://static.igem.wiki/teams/5743/engineering/3.webp)

Figure 3 Gel electrophoresis of PCR for PDI gene

Both the gene and the pRSFDuet vector were digested with BamHI and HindIII restriction enzymes to generate compatible sticky
ends, and then assembled using the homologous recombination enzyme. The ligation product was transformed into*E. coli*
DH5α competent cells via heat shock to obtain the recombinant plasmid. Colonies were screened using colony PCR, and the band
sizes were confirmed through gel electrophoresis and DNA sequencing.

![](https://static.igem.wiki/teams/5743/engineering/4.webp)

Figure 4 Colony growth, gel electrophoresis result and DNA sequencing of pRSFDuet-PDI

',
},
{
"title": "Part 2: pRSFDuet-PDI-Thaumatin-A",
"content": '

**Design**

We selected the pRSFDuet-PDI recombinant plasmid as the base vector, and inserted the Thaumatin-A gene fragment via double
digestion and homologous recombination techniques to construct a new recombinant plasmid.

![0cea13e5c2532c8db0d550f68872629](https://static.igem.wiki/teams/5743/engineering/5.webp)

Figure 5 The map of pRSFDuet-PDI-Thaumatin-A

**Build**

The Thaumatin-A gene (1011 bp) was amplified by PCR. Figure 6 showed the agarose gel electrophoresis results of PCR. We can
find out that the DNA fragment of Thaumatin-A gene is approximately 1011 bp in length, which is consistent with the
predicted size. This indicates that the target gene was successfully amplified.

![8fda1e6eebdfa58e5bf46d5956fd0fc8_](https://static.igem.wiki/teams/5743/engineering/6.webp)

Figure 6 Gel electrophoresis of PCR for Thaumatin-A

The pRSFDuet-PDI plasmid was linearized and assembled with the gene fragment of Thaumatin-A by
homologous recombination. The ligation product was transformed into *E. coli* DH5α competent cells. Colonies were
screened using colony PCR, and the band sizes were confirmed through gel electrophoresis and DNA sequencing.

![](https://static.igem.wiki/teams/5743/engineering/7.webp)

Figure 7 Colony growth, gel electrophoresis result and DNA sequencing of pRSFDuet-PDI-Thaumatin-A

'
},
{
"title": "Part 3: pRSFDuet-PDI-Thaumatin-B",
"content": '

**Design**

The design is pragmatically like that of Part 2, except we used Thaumatin-B, in place of Thaumatin-A.

![c7b2230c2ae7ee3a33510687aea2fc5](https://static.igem.wiki/teams/5743/engineering/8.webp)

Figure 8 The map of pRSFDuet-PDI-Thaumatin-B

**Build**

The Thaumatin-B gene (759 bp) was amplified by PCR. Agarose gel electrophoresis was also used to
determine whether the PCR-amplified gene fragment was Thaumatin-B. As Figure 9 showed, the DNA fragment of Thaumatin-B gene
is approximately 759 bp in length, which is consistent with the predicted size. This indicates that the target gene was
successfully amplified.

![a12e31eb180192c795da4448483505b2_](https://static.igem.wiki/teams/5743/engineering/9.webp)

Figure 9 Gel electrophoresis of PCR for Thaumatin-B

The pRSFDuet-PDI plasmid was linearized and assembled with the gene fragment of Thaumatin-B by
homologous recombination. The ligation product was transformed into *E. coli* DH5α competent cells via heat shock to
obtain the recombinant plasmid. Colonies were screened using colony PCR, and the band sizes were confirmed through gel
electrophoresis and DNA sequencing. As shown in Figure 10, the Thaumatin-B gene fragment was successfully ligated to the
pRSFDuet-PDI vector without any apparent mutations, confirming the successful construction of the pRSFDuet-PDI-Thaumatin-B
plasmid.

![](https://static.igem.wiki/teams/5743/engineering/10.webp)

Figure 10 Colony growth, gel electrophoresis result and DNA sequencing of pRSFDuet-PDI-Thaumatin-B

'
},
{
"title": "Part 4: pRSFDuet-PDI-Thaumatin-C",
"content": '

**Design**

The design is pragmatically like that of Part 2, except we used Thaumatin-C, in place of Thaumatin-A.

![图示  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5743/engineering/11.webp)

Figure 11 The map of pRSFDuet-PDI-Thaumatin-C

**Build**

The Thaumatin-C gene (1062 bp) was amplified by PCR. Figure 12 showed the agarose gel
electrophoresis results of PCR. We can find out that the DNA fragment of Thaumatin-C gene is approximately 1062 bp in
length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.

![图形用户界面, 应用程序  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5743/engineering/12.webp)

Figure 12 Gel electrophoresis of PCR for Thaumatin-C

The pRSFDuet-PDI plasmid was linearized and assembled with the gene fragment of Thaumatin-C by homologous recombination. The
ligation product was transformed into *E. coli* DH5α competent cells via heat shock to obtain the recombinant
plasmid. Colonies were screened using colony PCR, and the band sizes were confirmed through gel electrophoresis and DNA
sequencing. As shown in Figure 13, the Thaumatin-C gene fragment was successfully ligated to the pRSFDuet-PDI vector without
any apparent mutations, confirming the successful construction of the pRSFDuet-PDI-Thaumatin-C plasmid.

![](https://static.igem.wiki/teams/5743/engineering/13.webp)

Figure 13 Colony growth, gel electrophoresis result and DNA sequencing of pRSFDuet-PDI-Thaumatin-C

'
},
{
"title": "Part 5: pRSFDuet-PDI-Thaumatin-D",
"content": '

**Design**

The design is pragmatically like that of Part 2, except we used Thaumatin-D, in place of Thaumatin-A.

![图表, 图示  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5743/engineering/14.webp)

Figure 14 The map of pRSFDuet-PDI-Thaumatin-D

**Build**

The Thaumatin-D gene (1125 bp) was amplified by PCR. Figure 15 showed the agarose gel
electrophoresis results of PCR. We can find out that the DNA fragment of Thaumatin-D gene is approximately 1125 bp in
length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.

![图形用户界面, 应用程序  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5743/engineering/15.webp)

Figure 15 Gel electrophoresis of PCR for Thaumatin-D

The pRSFDuet-PDI plasmid was linearized and assembled with the gene fragment of Thaumatin-D by homologous recombination. The
ligation product was transformed into *E. coli* DH5α competent cells via heat shock to obtain the recombinant
plasmid. Colonies were screened using colony PCR, and the band sizes were confirmed through gel electrophoresis and DNA
sequencing. As shown in Figure 16, the Thaumatin-D gene fragment was successfully ligated to the pRSFDuet-PDI vector without
any apparent mutations, confirming the successful construction of the pRSFDuet-PDI-Thaumatin-D plasmid.

![](https://static.igem.wiki/teams/5743/engineering/16.webp)

Figure 16 Colony growth, gel electrophoresis result and DNA sequencing of pRSFDuet-PDI-Thaumatin-D

'
},
{
"title": "Test",
"content": '',
"children": [
{
"title": "1.Protein expression",
"content": '

The confirmed plasmids were transformed into *E.coli* Origami2(DE3) for protein expression. IPTG was added to induce
expression, and the culture was harvested after 20 hours. Cell lysis was performed using a sonicator under low-temperature
conditions to prevent protease denaturation. Purification was carried out using nickel affinity chromatography. The column
was washed with buffer A (low imidazole) and eluted with buffer B (high imidazole). Collected fractions including lysate,
flow-through, and elution were analyzed via SDS-PAGE.

As shown in Figure 17, distinct protein bands were observed at approximately 38 kDa for all target
proteins. This result is consistent with the expected molecular weight, indicating that these Thaumatin proteins were
successfully expressed and purified in the elution fractions under the current experimental conditions.

![](https://static.igem.wiki/teams/5743/engineering/17.webp)

Figure 17 SDS-PAGE results of all target proteins

',
},
{
"title": "2.TLC Detection of Monosaccharide Expression",
"content": '

TLC was employed to analyze the monosaccharide expression resulting from the action of Thaumatin-A
and Thaumatin-B. As shown in Figure 18, distinct bands were observed in the lanes, within the region marked by the red box.
This TLC-based detection demonstrated that Thaumatin-A and Thaumatin-B can mediate the breakdown of polysaccharide
substrates to produce monosaccharide-related products, successfully achieving the functional verification of monosaccharide
expression at the enzymatic activity level.

![](https://static.igem.wiki/teams/5743/engineering/18.webp)

Figure 18 TLC Detection of Monosaccharide Expression

',
},
{
"title": "3. DNS detection of glucose",
"content": '

To quantify glucose levels, the DNS method was used. A standard curve was constructed by measuring the absorbance (at 540
nm) of DNS-reacted glucose solutions with known concentrations. As shown in Figure 19, the linear equation is
**y = 0.9596x-0.0189** with a high correlation coefficient **R2 = 0.9907,** indicating
a good linear relationship between absorbance and glucose concentration in the tested range.

![](https://static.igem.wiki/teams/5743/engineering/19.webp)

Figure 19 Glucose Standard Curve

The Single-enzyme activity results showed that Thaumatin-A had the highest enzyme activity, followed by Thaumatin-B,
Thaumatin-D, and Thaumatin-C.

![](https://static.igem.wiki/teams/5743/engineering/20.webp)

Figure 20 Graphs of single-enzyme activity comparison

In the dual-enzyme combinations, the mixture of Thaumatin-C&D displayed the highest activity, followed by
Thaumatin-A&C, Thaumatin-A&B, Thaumatin-A&D, Thaumatin-B&D, and Thaumatin-B&C.

![](https://static.igem.wiki/teams/5743/engineering/21.webp)

Figure 21 Graphs of dual-enzyme activity comparison

In the triple-enzyme combinations, the mixture of Thaumatin-A&B&D displayed the highest activity, followed by
Thaumatin-A&B&C, and Thaumatin-B&C&D.

![](https://static.igem.wiki/teams/5743/engineering/22.webp)

Figure 22 Graphs of triple-enzyme activity comparison

Finally, the above three combinations with the best activity were selected and compared with the mixed system containing all
four enzymes. As shown in Figure 23, the mixture of Thaumatin-A&B&C&D displayed the highest activity, followed
by Thaumatin-C&D, Thaumatin-A&B&D, and Thaumatin-A.

![](https://static.igem.wiki/teams/5743/engineering/23.webp)

Figure 23 Graphs of optimal combinations comparison

',
},
{
"title": "4. Electronic tongue detection",
"content": '

To characterize the taste properties of samples, electronic tongue detection was performed.The differences in taste
characteristics among different samples were shown in Figure 24A, with each sample group forming distinct clusters,
indicating significant differences in taste profiles between samples. Sweetness values for different samples were shown in
Figure 24B. The 4% Sucrose group served as a control with extremely low sweetness. Thaumatin-A had the highest sweetness,
followed by Thaumatin-C, Thaumatin-D, and Thaumatin-B. These results indicate that Thaumatin proteins can significantly
enhance the sweetness of the system, with varying degrees of sweetness-enhancing effects among different Thaumatin variants.

![](https://static.igem.wiki/teams/5743/engineering/24.webp)

Figure 24 Result of electronic tongue detection

',
},
{
"title": "5. Molecular docking of sweet proteins with sweet taste receptor protein",
"content": '

To investigate the binding mode between sweet proteins and sweet taste receptor proteins, we performed AlphaFold modeling
and protein-protein molecular docking. As shown in Figure 25, the sweet protein TLP-A formed 5 sets of hydrogen bonds with
TAS1R2. The amino acid residues in TLP-A form hydrogen bonded with S87, D406, and S408 of TAS1R2, respectively.

![](https://static.igem.wiki/teams/5743/engineering/25.webp)

Figure 25 The Interaction between the Sweet Taste Receptor and TLP-A

As shown in Figure 26, the sweet protein TLP-B forms 3 sets of hydrogen bonds with TAS1R2. The amino acid residues in TLP-B
form hydrogen bonded with W341, N368, and D364 of TAS1R2, respectively. The TAS1R2 region involved in the interaction is the
key binding site for the combination of sweet ligand proteins and sweet molecules.

![](https://static.igem.wiki/teams/5743/engineering/26.webp)

Figure 26 The Interaction between the Sweet Taste Receptor and TLP-B

',
},
]
},
{
"title": "Learn",
"content": '

1. Different PCR and DNA assembly conditions can affect fragment amplification and DNA assembly. We conducted multiple
troubleshooting steps by adjusting the amounts of primers and templates, as well as the PCR reaction program. All the
target genes were successfully amplified, and the successful construction of the recombinant plasmids were confirmed by
sequencing and electrophoresis analyses.

2. Clear bands were observed at ~38 kDa in the supernatant lanes of Thaumatin-C/D, indicating that the target protein is
expressed in a soluble form. For Thaumatin-A/B, it is recommended to improve their solubility by adjusting the expression
conditions: methods such as lowering the temperature (e.g., 30°C/25°C) with a slight extension of induction time, or
optimizing the inducer (testing different IPTG concentrations or switching to lactose) can be adopted. Ultimately,
verification by SDS-PAGE should be performed to obtain stronger ~38 kDa bands in their supernatants.

3. Among the enzyme activity assays, the Thaumatin-A&B&C&D combination exhibited the highest activity. This
demonstrates the synergistic effect formed through functional complementation between different Thaumatin proteins, which
can enhance the catalytic capacity for substrates, highlighting the significant advantages of multi-component combined use
in improving enzymatic reaction efficiency.

'
},
] %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}ENGINEERING{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "1. Disposition of LB Culture",
"content": '

**Goal:** To create a functional and nutritional liquid environment for bacteria to grow. Liquid media used for
various purposes such as propagation of microorganisms or other tests.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Sterilized Duran bottles, with caps |
|  | Autoclave |
|  | Lab Refrigerator |
| Chemicals and Reagents | Tryptone |
|  | Yeast Extract |
|  | Sodium Chloride |
|  | Double distilled H2O |

**1L of LB medium was prepared in-house with the following components:**

|  |  |
| --- | --- |
| Chemicals | Mass |
| Tryptone | 10 g |
| Yeast Extract | 5 g |
| Sodium Chloride | 10 g |
| Double distilled H2O | 1 L |
| Agar (to prepare solid medium) | Add 1.5 g per 100 ml of ddH₂O |

**Procedure:**

After accurately weighing and dissolving, all prepared LB media were sterilized
by autoclaving at 121 °C for 15 minutes. The cooled medium was stored at 4°C for subsequent experiments.

',
},
{
"title": "2. The construction of plasmid",
"content": '',
"children": [
{
"title": '2.1 PCR amplification',
"content": '

**Goal:** Amplify the complete coding genes of PDI and its isoforms to obtain target fragments with homologous
recombination sites.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Centrifuge tube |
|  | Centrifuge |
|  | PCR thermal cycler |
|  | Lab refrigerator |
| Chemicals and Reagents | 2×PrimeMixStar |
|  | Primer |
|  | Template DNA |
|  | ddH2O |

50 μL PCR amplification reaction system:

|  |  |
| --- | --- |
| Component | Volume (μL) |
| 2×PrimeMixStar | 25 |
| Primer F | 2 |
| Primer R | 2 |
| Template DNA | 2 |
| ddH2O | 19 |
| Total | 50 |

**PCR amplification reaction procedure:**

|  |  |  |  |
| --- | --- | --- | --- |
| Step | Temperature (°C) | Time | Number of cycles |
| Initial Denaturation | 95 | 5 minutes | 1 |
| Denaturation | 95 | 30 seconds | 30 |
| Annealing | 55 | 30 seconds |
| Extension | 72 | 1 minute |
| Final Extension | 72 | 10 minutes | 1 |
| Hold | 4 |  | — |

**Procedure:**

Mix all components in labeled PCR tubes, followed by brief centrifugation. Load samples into a pre-programmed thermal cycler
running through the PCR amplification reaction procedure above.

',
},
{
"title": '2.2 Agarose gel construction, deployment and recycle',
"content": '

**Goal:** To produce a functional and well-shaped gel for electrophoresis.

Materials:

|  |  |
| --- | --- |
| Instruments and Equipment | Casting Tray |
|  | Well comb |
|  | Microwave |
|  | Erlenmeyer flask |
|  | Electrophoresis chamber |
|  | UV Transilluminator / Gel Imager |
| Chemicals and Reagents | Agarose |
|  | 1× TAE Buffer |
|  | ddH2O |
|  | Nucleic acid gel stain |

**Procedure:**

Dissolve 0.3 g Agarose in 30 mL erlenmeyer flask. Add 1× TAE buffer to reach the target volume. Heat the material with
microwave at short intervals until the agarose dissolves completely. Add 3μl nucleic acid gel stain when it cool. Pour the
mixture into a casting tray, insert a well comb, wait 7-10 minutes for the gel to solidify. Remove the comb and place the
gel in the gel box.

',
},
{
"title": '2.3 Agarose gel electrophoresis',
"content": '

**Goal:** To verify the DNA bands’ lengths and thus preparing for the next step of gel extraction to extract
the bands that are of the correct length, eliminating the genetic fragments that didn’t successfully undergo PCR.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Casting tray |
|  | Electrophoresis chamber |
| Chemicals and Reagents | Prepared gel |
|  | 1× TAE Buffer |
|  | DNA ladder |
|  | DNA sample |
|  | Loading Buffer |

**Procedure:**

Mix 5 μl of PCR product with 1μl of loading buffer, followed by loading into the wells alongside the DNA marker. Perform
electrophoresis at a constant voltage of 180V for 20 minutes in 1×TAE buffer. After electrophoresis, place the gel in a UV
gel imaging system for observation.

',
},
{
"title": '2.4 Bacterial inoculation',
"content": '

**Goal:** To abtain a sufficient quantity of engineered strains with stable activity for subsequent
experiments.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Laminar flow hood |
|  | Shaking incubator |
|  | Shaking culture tube |
| Chemicals and Reagents | LB liquid medium |
|  | antibiotic(kan+) |
|  | glycerol stock(pRSFDuet） |

**Procedure:**

Add 5 mL of LB medium, 5 μL of pRSFDuet glycerol stock, and 5 μL of kanamycin (Kan⁺) antibiotic to labeled tubes. Place the
tubes in a shaking incubator at 37°C and 220 rpm. Allow the bacteria to grow overnight (12-16 hours).

',
},
{
"title": '2.5 Extraction of Plasmids',
"content": '

**Goal:** To obtain high-purity plasmid DNA from host cells, including empty vectors or recombinant vectors.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Centrifuge |
|  | Centrifugate tube |
|  | FastPure DNA Mini Column |
|  | Collection Tubes |
| Chemicals and Reagents | Buffer P1 |
|  | Buffer P2 |
|  | Buffer P3 |
|  | Buffer PW1 |
|  | Buffer PW2 |
|  | ddH2O |

**Procedure:**

1. Transfer 2 mL of the overnight culture into a 1.5 mL microcentrifuge tube. Centrifuge at 12000 rpm for 1 min, and drop the
   solution.
2. Add 250 μL Buffer P1 containing RNase A, and mix thoroughly by vortexing.
3. Add 250 μL Buffer P2, and gently invert the tube up and down 10 times to fully lyse the bacterial cells.
4. Add 350 μL Buffer P3, immediately invert gently up and down 10 times to mix thoroughly, then centrifuge at 12,000 rpm for
   2 minutes.
5. Place the FastPure DNA Mini Columns spin column into the Collection Tubes, carefully pipette the supernatant into the
   adsorption columns, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid, and put the adsorption column back
   into the collection tube.
6. Add 500 μL of Buffer PW1, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.
7. Add 600 μL of Buffer PW2, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.
8. Repeat step 7.
9. Put the spin column back into the collection tube, and centrifuge at 12,000 rpm for 1 minute.
10. Place the spin column into a new clean 1.5 mL centrifuge tube. Add 40 μL of ddH2O to the middle part of the
    adsorption membrane, let it stand at room temperature for 1 minute, then centrifuge at 12,000 rpm for 1 minute to obtain
    the plasmid DNA solution.

',
},
{
"title": '2.6 Restriction Enzyme Double Digestion',
"content": '

**Goal**：Double-digest the plasmid vector to linearize it, providing a backbone for recombination and
preventing self-ligation.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Centrifuge tube |
|  | Centrifuge |
|  | PCR thermal cycler |
|  | Lab Refrigerator |
|  | Water bath pot |
| Chemicals and Reagents | plasmid |
|  | Restriction Enzyme A and B |
|  | 10×cutsmart Buffer |
|  | ddH2O |

**20 μL double digestion reaction system:**

|  |  |
| --- | --- |
| Component | Volume (μL) |
| plasmid | 10 |
| Restriction Enzyme A | 1 |
| Restriction Enzyme B | 1 |
| 10×cutsmart Buffer | 2 |
| ddH2O | 6 |
| Total | 20 |

**Procedure:**

Add all the components to PCR tubes, followed by brief centrifugation at 12,000 rpm for 10 seconds. Incubate the reaction
system in a 37 ℃ water bath for 20 minutes to ensure complete digestion of the vector plasmid into linearized fragments.

',
},
{
"title": '2.7 Gel Extraction of Linearized Vector DNA',
"content": '

**Goal:** To isolate and purify linearized vector DNA fragments from agarose gel, while removing impurities
such as uncut circular vectors, other restriction-generated fragments, proteins, and agarose.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Centrifuge tube |
|  | Centrifuge |
|  | Scalpel |
|  | Lab Refrigerator |
|  | Water bath pot |
| Chemicals and Reagents | Buffer GDP |
|  | Buffer GW |
|  | ddH2O |

**Procedure:**

1. After completing agarose gel electrophoresis, precisely excise the gel slice containing linearized vector DNA under UV
   illumination. Transfer the excised gel slice to a 1.5 mL centrifuge tube.
2. Add an equal volume of Buffer GDP. Incubate at 65°C in a metal bath until the gel becomes completely transparent.
3. Transfer the solution to the spin column and centrifuge at 12000 rpm for 60 seconds.
4. Discard the filtrate. Place the spin column in the collection tube. Add 300 μL Buffer GDP to the spin column, and let it
   stand for 1 minute. Centrifuge at 12000 rpm for 60 seconds.
5. Discard the filtrate. Place the spin column in the collection tube. Add 700 μL Buffer GW to the spin column. Centrifuge at
   12000 rpm for 60 seconds.
6. Repeat Step 5.
7. Discard the filtrate. Place the spin column in the collection tube. Centrifuge at 12000 rpm for 2 minutes.
8. Place the spin column into a new clean 1.5 mL centrifuge tube. Add 30 μL of ddH2O to the middle part of the
   adsorption membrane, let it stand for 2 minute. Centrifuge at 12,000 rpm for 1 minute. Store the DNA solution at -20℃.

',
},
{
"title": '2.8 Homologous recombination cloning',
"content": '

**Goal：**To construct a recombinant vector by ligating the target gene to the linearized vector via homologous
recombination.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | PCR thermal cycler |
|  | Centrifuge |
|  | Incubator |
|  | Water bath pot |
| Chemicals and Reagents | 2×CE enzyme |
|  | target gene |
|  | vector |

**Procedure:**

1. Prepare the system:

|  |  |
| --- | --- |
| 2×CE enzyme | 5 μL |
| vector | 4 μL |
| target gene | 1 μL |

1. Place the system in 50°C water bath for 15 minutes, then cool to 4℃ immediately

',
},
{
"title": '2.9 Heat-shock transformation into E. coli DH5α competent cells',
"content": '

**Goal：**To introduce the recombinant vector into DH5α for amplification, followed by verification of the
target gene sequence, insertion orientation, and vector integrity via sequencing.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Centrifuge |
|  | Incubator |
|  | Water bath pot |
|  | Ice-making machine |
| Chemicals and Reagents | pRSFDuet-PDI plasmid |
|  | *E. coli* DH5α competent cells |
|  | LB liquid medium (K⁺) |

**Procedure:**

1. Put 10 μL pRSFDuet-PDI plasmid into 100 μL *E. coli* DH5α competent cells, then place on ice for 20 minutes.
2. Heat-shock at 42℃ for accurate 45 seconds, then immediately put on ice for 2 minutes.
3. Add 800 μL of LB liquid medium, then incubate at 37℃ for an hour.
4. Centrifugate at 12000 rpm for 1 minute.
5. Remain 100 μL supernatant, spreading on kan+ plate, then incubate at 37℃ for 12-16 hours.

',
},
]
},
{
"title": "3.Protein Expression and Purification",
"content": "",
"children": [
{
"title": '3.1 Transformation intoE. coli Origami 2(DE3) Expression Strain',
"content": '

**Goal:** To introduce the target plasmid into the expression strain for protein production.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Centrifuge |
|  | Incubator |
|  | Water bath pot |
|  | Ice-making machine |
| Chemicals and Reagents | pRSFDuet-PDI-Thaumatian A/B/C/D plasmid |
|  | *E. co*li Origami 2(DE3) competent cells |
|  | LB liquid medium (K⁺) |

**Procedure:**

1. Thaw competent cells on ice for 10 minutes.
2. Add 1-5 µL plasmid DNA to 50 µL cells, mix gently, then incubate on ice for 30 min.
3. Heat-shock at 42°C for accurate 45 seconds, then immediately put on ice for 2 minutes.
4. Add 500 µL LB medium (no antibiotic), incubate at 37°C for 1 hour.
5. Plate 100 µL of the transformed cells on LB agar plates (antibiotic), and incubate at 37℃ for 12-16 hours.

',
},
{
"title": '3.2 Scale-up Cultivation and IPTG Induction',
"content": '

**Goal:** To optimize protein expression under inducible conditions.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Centrifuge |
|  | Shaking incubator |
| Chemicals and Reagents | Single colony from transformed strain |
|  | TB/Autoinduction medium |
|  | IPTG (1 M stock) |
|  |  |

**Procedure:**

1. Inoculate 5 mL LB (antibiotic) with single colony, incubate at 37°C, 220 rpm for 12–16 hours.
2. Transfer 1 mL culture to 100 mL TB/autoinduction medium. Incubate at 37°C until OD₆₀₀ ≈ 0.6 (3–4 h).
3. Add IPTG to final concentration (e.g., 0.5 mM).
4. Induce at:
   1. 37°C, 4–6 h for soluble proteins
   2. 16–18°C, 16–20 h for complex proteins

',
},
{
"title": '3.3 Ultrasonic Cell Disruption for Crude Protein Extraction',
"content": '

**Goal:** To lyse cells to extract soluble proteins.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Centrifuge |
|  | Ultrasonic Cell Disruptor |
|  | Ice-making machine |
| Chemicals and Reagents | Bacterial pellet |
|  | Lysis buffer |

**Procedure:**

1. Resuspend the pellet in 5 mL lysis buffer.
2. Sonicate on ice for 3 cycles: 30 seconds pulse, 30 seconds rest at 40% amplitude.
3. Centrifuge at 15,000 ×g, for 30 minutes at 4°C.
4. Collect the supernatant as the crude extract.

',
},
{
"title": '3.4 Ni-NTA Affinity Chromatography for Protein Purification',
"content": '

**Goal:** To isolate His-tagged protein via immobilized metal affinity.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Gravity column |
|  | Centrifuge tube |
| Chemicals and Reagents | Ni-NTA resin |
|  | Wash buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 40 mM imidazole) |
|  | Elution buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole) |

**Procedure:**

1. Equilibrate 1 mL of Ni-NTA resin with lysis buffer.
2. Load the crude extract onto the column and collect the flow-through.
3. Wash the column with 10 column volumes (CV) of wash buffer.
4. Elute the target protein with 5 CV of elution buffer, collecting 0.5 mL fractions.
5. Analyze the collected fractions using SDS-PAGE.

',
},
{
"title": '3.5 SDS-PAGE Analysis for molecular weight verification',
"content": '

**Goal:** To confirm protein size and purity.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Vertical electrophoresis system |
|  | Microcentrifuge |
|  | Water bath |
|  | Vortex mixer |
| Chemicals and Reagents | 12.5% SDS-PAGE gel |
|  | Running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) |
|  | Protein samples |
|  | Protein ladder |
|  | Coomassie Blue stain |
|  | Destaining solution |

**Procedure:**

1. Mix 10 µL protein sample with 2× Laemmli buffer in clean centrifuge tubes, Vortex for 10 seconds. Boil for 5 minutes.
2. Load the ladder and samples into the lanes.Run at 120 V for 90 min.
3. Stain with Coomassie Blue stain for 30 minutes. Incubate the gel in destaining solution overnight (12-16 hours).

',
},
]
},
{
"title": '4. Functional Verification',
"content": '',
"children": [
{
"title": '4.1 TLC for Qualitative Monosaccharide Detection',
"content": '

**Goal:** To screen monosaccharide expression profiles.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Silica gel TLC plates |
| Chemicals and Reagents | Solvent system (e.g., butanol:acetic acid:water = 12:3:5) |
|  | Sample solution |
|  | Sugar standards (glucose, fructose) |
|  | Staining reagent (anisaldehyde-sulfuric acid) |

**Procedure:**

1. Spot samples and standards 1 cm from the bottom of the TLC plate.
2. Develop the plate in the solvent system until the solvent front reaches 8 cm from the starting line.
3. Air-dry the plate completely. Spray with staining reagent, and heat at 105°C for 10 minutes to visualize the spots.
4. Calculate and compare the Rf values of sample spots with those of standard sugars for identification.

',
},
{
"title": '4.2 DNS Assay for Quantitative Glucose Determination',
"content": '

**Goal:** To measure reducing sugar content via colorimetry.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Microplate reader |
|  | Centrifuge tube |
|  | Water bath pot |
| Chemicals and Reagents | DNS reagent (3,5-dinitrosalicylic acid) |
|  | Glucose standard (0–10 mM) |
|  | ddH2O |
|  | Sample solution |

**Procedure:**

1. Mix 50 µL of sample with 100 µL of DNS reagent in centrifuge tubes.
2. Incubate in a boiling water bath for 5 minutes, then immediately cool the mixture on ice to stop the reaction.
3. Add 100 µL of ddH₂O to the tube and mix thoroughly.
4. Measure OD₅₄₀, calculate concentration using standard curve.

',
},
{
"title": '4.3 Electronic Tongue Analysis for Sweetness Evaluation',
"content": '

**Goal:** To assess sweetness profile of expressed proteins.

**Materials:**

|  |  |
| --- | --- |
| Instruments and Equipment | Electronic tongue system |
| Chemicals and Reagents | Reference solutions (sucrose, aspartame) |
|  | Sample buffer (neutral pH) |

**Procedure:**

1. Dialyze samples into buffer.
2. Calibrate sensors with reference solutions.
3. Measure samples in triplicate.
4. Analyze data via PCA (Principal Component Analysis).

',
},
{
"title": '4.4 Molecular docking of sweet proteins with sweet taste receptor protein',
"content": '

**Goal:** To investigate the binding mode between sweet proteins and sweet taste receptor proteins.

**Procedure:**

1. Download the structural file of the sweet taste receptor protein from the Protein Data Bank (PDB), and use AutoDock Tools
   software to preprocess the structure.
2. Use the AlphaFold tool to predict the three-dimensional structures of the sweet proteins TLP-A and TLP-B.
3. Perform molecular docking between the sweet protein TLP and the sweet taste receptor using the Z-dock online tool.
4. Analyze the docking results by combining visualization tools and interaction analysis methods.

',
},
]
}
]%}
{% block title %}Experiments{% endblock %}
{% block lead %}EXPERIMENTS{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [
{
"title": "Overview",
"content": '

The use of saccharides in the production and processing of food has proliferated as people’s demand for food flavor has
increased. Excessive intake of saccharides may lead to increase in blood glucose level, which may threaten health; sugar
substitutes such as aspartame may lead to intestinal flora disorders. As a new type of sugar substitute product, Thaumatin
has the advantages of high sweetness and good stability. However, the traditional Thaumatin is mainly extracted from plants,
which faces the problems of low yield and high production cost. This project proposes to improve the production efficiency
of Thaumatin by inserting the Thaumatin protein gene into *E. coli* for bioculture.

**![01](https://static.igem.wiki/teams/5743/result/1.webp)**

Figure 1 Overview of Experimental Steps

',
},
{
"title": "1. Plasmid construction",
"content": '',
"children": [
{
"title": "1.1 PCR amplification",
"content": '

We first configured LB liquid and solid medium. Then a large number of genes of PDI and Thaumatin-A/B/C/D were obtained by
PCR amplification respectively. We obtained the fragments of the target genes by performing agarose gel electrophoresis.
Figure 2 shows the agarose gel electrophoresis results of PCR for five clones. We can find out that the DNA fragment of
*PDI* is approximately 1586 bp in length, *Thaumatin-A* is 1011 bp, *Thaumatin-B* is 759 bp,
*Thaumatin-C* is 1062 bp and *Thaumatin-D* is 1125 bp, which is consistent with the predicted size. This
indicates that the target gene was successfully amplified.

![D:/LYJ/2025-iGEM/秦臻- 甜蜜”零”负担/紫竹-英澳美实验结果/组图1_画板 1.jpg组图1_画板 1](https://static.igem.wiki/teams/5743/result/2.webp)Figure 2 Agarose gel electrophoresis results of PCR for five clones

',
},
{
"title": "1.2 Homologous recombination",
"content": '

We used two different restriction endonucleases BamhI and HindIII to cleave the two cleavage sites on the plasmid, avoiding
plasmid closure before insertion of the gene, and facilitating the insertion of the gene at a later stage. Then we performed
DNA electrophoresis to make gel plates, and then DNA recovery to obtain the pRSFDuet-Linear Vector. We used homologous
recombination technology to insert two different homologous recombinases (F1 and R1) at each end of the PDI gene and the
plasmid, and then inserted the target gene into the plasmid to allow the homologous recombinases to bind to each other.
Finally we got the plasmid with four Thaumatin genes and PDI respectively.

After homologous recombination, the recombinant plasmids were transformed into chemically competent *E. coli* DH5α
cells via the heat shock method. Following overnight incubation on LB agar plates containing the appropriate antibiotic,
several well-isolated colonies were observed (Figure 3), indicating successful transformation.

![](https://static.igem.wiki/teams/5743/result/3.webp)

Figure 3 Agar plate showing DH5α colonies after transformation

To verify the presence of the target gene, five individual colonies were randomly selected for colony PCR. As shown in
Figure 4, each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct
insertion of the target gene.

![](https://static.igem.wiki/teams/5743/result/4.webp)

Figure 4 Agarose gel electrophoresis results of Colony PCR identification for five clones

',
},
{
"title": "1.3 DNA sequencing",
"content": '

We extracted the plasmid and sent it to a biotechnology company for sequencing to verify the accuracy of the target gene
sequences. As shown in Figure 5, the sequencing results are completely consistent with the target sequences, confirming the
successful construction of our plasmid.

![](https://static.igem.wiki/teams/5743/result/5.webp)

Figure 5 DNA sequencing results of recombinant plasmids

',
},
]
},
{
"title": "2. Protein Expression and Purification",
"content": '',
"children": [
{
"title": "2.1 Transformation of E. coli Origamiz2(DE3)",
"content": '

After LB media preparation and PCR-based amplification/recovery, *E. coli* Origami2(DE3) was transformed with five
recombinant plasmids. After incubation, as shown in Figure 6, numerous single colonies of *E. coli* Origami2(DE3)
grew on the medium. These well-separated, round, and milky-white colonies indicated successful transformation and stable
propagation of the engineered bacteria. This laid a foundation for subsequent large-scale liquid culture expansion and
induced osmotic expression of the target protein, ensuring sufficient bacterial biomass for subsequent protein expression
experiments.

![](https://static.igem.wiki/teams/5743/result/6.webp)

Figure 6 Agar plate showing BL21 colonies after transformation

',
},
{
"title": "2.2 Ultrasonic lysis",
"content": '

After ultrasonic lysis of *E. coli* Origami2(DE3) expressing Thaumatin-A/B/C/D, the samples were separated into
supernatant and precipitate fractions, which were then analyzed by SDS-PAGE (Figure 7). In the supernatant lanes (left of
Marker M), distinct protein bands corresponding to the expected molecular weight (~38 kDa) were observed for
Thaumatin-A/B/C/D, indicating that the target proteins were successfully extracted into the soluble fraction via ultrasonic
lysis. The precipitate lanes (right of Marker M) showed relatively weaker and less prominent bands at the ~38 kDa position,
suggesting that most of the target proteins remained in the soluble supernatant after lysis. The clear ~38 kDa bands in the
supernatant confirmed that ultrasonic lysis effectively released the target proteins, providing soluble crude protein
samples for subsequent purification and analysis.

![](https://static.igem.wiki/teams/5743/result/7.webp)

Figure 7 SDS-PAGE analysis of crude protein

',
},
{
"title": "2.3 SDS-PAGE verification",
"content": '

After ultrasonic lysis and centrifugation, the supernatant containing crude proteins was subjected to Ni-NTA affinity
purification. The resulting elution fractions were analyzed by SDS-PAGE. The results are shown in Figure 8. In the
supernatant lanes (left of Marker M), complex protein bands were observed, indicating a mixture of target proteins and other
impurities prior to purification. In the elution lanes (right of Marker M), distinct bands at ~38 kDa, corresponding to
Thaumatin-A/B/C/D, were detected.

This result confirmed that Ni-NTA affinity purification effectively enriched the target proteins. The clear, concentrated
~38 kDa bands in the elution fractions demonstrated successful separation of the target proteins from most impurities,
providing purified protein samples for subsequent experiments.

![](https://static.igem.wiki/teams/5743/result/8.webp)

Figure 8 SDS-PAGE analysis After Protein Elution

',
},
]
},
{
"title": "3. Functional verification",
"content": '',
"children": [
{
"title": "3.1 TLC Detection of Monosaccharide Expression",
"content": '

TLC was employed to analyze the monosaccharide expression resulting from the action of Thaumatin-A
and Thaumatin-B. As shown in Figure 9, distinct bands were observed in the lanes, within the region marked by the red box.
This TLC-based detection demonstrated that Thaumatin-A and Thaumatin-B can mediate the breakdown of polysaccharide
substrates to produce monosaccharide-related products, successfully achieving the functional verification of monosaccharide
expression at the enzymatic activity level.

![](https://static.igem.wiki/teams/5743/result/9.webp)

Figure 9 TLC Detection of monosaccharide expression

',
},
{
"title": "3.2 DNS detection of glucose ",
"content": '

To quantify glucose levels, the DNS method was used. A standard curve was constructed by measuring the absorbance (at 540
nm) of DNS-reacted glucose solutions with known concentrations. As shown in Figure 10, the linear equation is**y = 0.9596x-0.0189**
with a high correlation coefficient **R2 = 0.9907**, indicating a good linear relationship between
absorbance and glucose concentration in the tested range.

β-1,3-glucanase hydrolyzes laminarin into reducing sugars. These sugars react with DNS reagent (3,5-dinitrosalicylic acid)
in boiling water, forming a colored complex. For the samples (reaction systems containing Thuam proteins), their absorbance
values were measured and substituted into the standard curve equation to calculate glucose concentrations. This allowed for
the quantification of glucose production, facilitating the analysis of the ability of Thuam proteins to catalyze substrate
degradation and glucose production, thus completing the functional verification from the perspective of glucose content.

![DNS标准曲线](https://static.igem.wiki/teams/5743/result/10.webp)

Figure 10 Glucose standard curve

The Single-enzyme activity results showed that Thaumatin-A had the highest enzyme activity, followed by Thaumatin-B,
Thaumatin-D, and Thaumatin-C.

![](https://static.igem.wiki/teams/5743/result/11.webp)

Figure 11 Graphs of single-enzyme activity comparison

In the dual-enzyme combinations, the mixture of Thaumatin-C&D displayed the highest activity, followed by
Thaumatin-A&C, Thaumatin-A&B, Thaumatin-A&D, Thaumatin-B&D, and Thaumatin-B&C.

![](https://static.igem.wiki/teams/5743/result/12.webp)

Figure 12 Graphs of dual-enzyme activity comparison

In the triple-enzyme combinations, the mixture of Thaumatin-A&B&D displayed the highest activity, followed by
Thaumatin-A&B&C, and Thaumatin-B&C&D.

![](https://static.igem.wiki/teams/5743/result/13.webp)

Figure 13 Graphs of triple-enzyme activity comparison

Finally, the above three combinations with the best activity were selected and compared with the mixed system containing all
four enzymes. As shown in Figure 14, the mixture of Thaumatin-A&B&C&D displayed the highest activity, followed
by Thaumatin-C&D, Thaumatin-A&B&D, and Thaumatin-A.

![](https://static.igem.wiki/teams/5743/result/14.webp)

Figure 14 Graphs of optimal combinations comparison

',
},
{
"title": "3.3 Electronic tongue detection",
"content": '

To characterize the taste properties of samples, electronic tongue detection was performed. The differences in taste
characteristics among different samples were shown in Figure 15A, with each sample group forming distinct clusters,
indicating significant differences in taste profiles between samples. Sweetness values for different samples were shown in
Figure 15B. The 4% Sucrose group served as a control with extremely low sweetness. Thaumatin-A had the highest sweetness,
followed by Thaumatin-C, Thaumatin-D, and Thaumatin-B. These results indicate that Thaumatin proteins can significantly
enhance the sweetness of the system, with varying degrees of sweetness-enhancing effects among different Thaumatin variants.

![](https://static.igem.wiki/teams/5743/result/15.webp)

Figure 15 Result of electronic tongue detection

',
},
]
},
{
"title": "4. Molecular Docking Experiments",
"content": '

To investigate the binding mode between sweet proteins and sweet taste receptor proteins, we performed AlphaFold modeling
and protein-protein molecular docking. The model of the sweet taste receptor protein was downloaded from the Protein Data
Bank (PDB). The model of the sweet protein TLP was predicted using AlphaFold2. And the molecular docking between the sweet
protein TLP and the sweet taste receptor protein was performed using the Z-DOCK online tool.

For TLP-A, the predicted structure contained 12 β-sheets and 6 α-helices, presenting an overall compact globular
conformation. The conformation of surface flexible loop regions was reasonable, and the predicted local distance difference
test (pLDDT) scores were all > 85, indicating high structural reliability. For TLP-B, the predicted structure consisted
of 12 β-sheets and 2 α-helices, also showing an overall compact globular conformation. The surface flexible loop regions had
a reasonable conformation, and the pLDDT scores were equally > 85, confirming the reliability of its structural
prediction.

As shown in Figure 16, the sweet protein TLP-A formed 5 sets of hydrogen bonds with TAS1R2. The amino acid residues in TLP-A
form hydrogen bonded with S87, D406, and S408 of TAS1R2, respectively.

![](https://static.igem.wiki/teams/5743/result/16.webp)

Figure 16 The interaction between the sweet taste receptor and TLP-A

As shown in Figure 17, the sweet protein TLP-B forms 3 sets of hydrogen bonds with TAS1R2. The amino acid residues in TLP-B
form hydrogen bonded with W341, N368, and D364 of TAS1R2, respectively. The TAS1R2 region involved in the interaction is the
key binding site for the combination of sweet ligand proteins and sweet molecules.

![](https://static.igem.wiki/teams/5743/result/17.webp)

Figure 17 The interaction between the sweet taste receptor and TLP-B

',
},
{
"title": "5. Future plans",
"content": '

To optimize taste, we will improve potential aftertaste issues through protein structure modification or compounding
techniques. In terms of safety control, we will conduct specific allergen testing to identify the sources of potential
allergens.

We will test solubility and thermal stability, specifically measure solubility and dissolution rate under different
solvents, temperatures, and pH conditions. Meanwhile, we will also test taste stability in different food matrices.

',
},
] %}
{% block title %}Results{% endblock %}
{% block lead %}RESULTS{% endblock %}