iGEM 2024 — Team szu-china

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Notebook

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![](https://static.igem.wiki/teams/5160/arrow.png)

Menu

* [Overview](#section_1)
* [Team Log](#section_2)

  [Brainstrom](#log1)
  [Wet Lab](#log2)
  [Products](#log3)
  [Dry Lab](#log4)
  [Hardware](#log5)
  [Human Practices](#log6)
  [Art Desigining](#log7)
  [Wiki Develop](#log8)

# **Overview**

#### 2023.12.3 Team Formation

We set up a team with 12 members.

####

#### 2024.12.16-2024.2.6 Experimental Skills and Literature Review Training

#### 2024.1.16-2024.3.16 Brainstorming

Brainstorming sessions were conducted in groups, with each group working on independent projects. Feasibility assessments led to the elimination of impractical proposals during meetings.

####

#### 2023.3.20 Project Identification

The interest in our project was piqued by a sugar content visualization display in a hospital, an encounter that occurred when one of our members was admitted due to irregular dietary habits.

####

#### 2024.3.16-2024.4.1 Background Research and Preliminary Experiments

The preliminary design of the entire project system was completed, along with extensive background and literature research. Preliminary experiments were initiated.

####

#### 2024.5.12-2024.9.18 Mid-term Experiments and Product Iteration

#### 2024.9.19-2024.10.1 Completion of Experiments and Wiki Upload

All experiments were completed and the results were uploaded to our wiki.

####

#### 2024.10.23 iGEM Jamboree

Participation in the iGEM Jamboree, an event showcasing our project to the synthetic biology community.

####

# **Team log**

## 2024.1.16-2024.3.10 Brainstrom

#### **1.16 Start Our Brainstorming**

**1. Discussed and determined the complete process of team brainstorming and established the "three-steps" strategy:**
Step 1. Release your thoughts and record them.
Step 2. Search and organize a lot of data.
Step 3. Initially select the project with the most development potential for further advancement, and finally select the most suitable one as our project after reviewing each pre-project from multiple perspectives.
**2. Created a brainstorming summary sheet to record our interesting ideas.**

#### **1.16-3.10 Some Brainstorming was Constructed**

##### **Climate Crisis:**

Jiachun Chen and Yuanrun Liu —— Methanol bioabsorption pigment
Qizheng Liu, Zhengyue Hong and Haosheng Deng —— Biological feed to reduce carbon monoxide emissions from livestock
Qizheng Liu and Zhengyue Hong —— Quorum quenching method for treating water blooms

##### **Bioremediation:**

Kexin Li and Siyuan Guo —— Bioremediation of petroleum-contaminated soil
Haosheng Deng —— Bioremediation of heavy metal pollution
Yuntian Wu —— Bioremediation of cultural relics and ancient buildings

##### **Agriculture:**

Haosheng Deng —— Research and development of targeted fertilizers for multi-fruit eggplant trees

##### **Food and Nutrition:**

Qizheng Liu and Zhengyue Hong —— Sugar substitute fruits

##### **Fashion and Cosmetics:**

Zejia Wei —— Biological production of carmine cochinea

##### **Diagnostics:**

Yitong Li —— Biological detection of parvovirus in puppies
Jiachun Chen —— Intestinal colonizing flora to improve obesity
Ruohan Chen —— Using antibodies to reduce drug addiction in people

##### **Therapeutics:**

Zhengyue Hong and Zejia Wei —— Hydrogel for treating dental caries and dental diseases
Yuntian Wu —— Biological glue instead of medical tension reducer
Yitong Li —— Mass production of albumin

##### **Biomanufacturing:**

Yitong Li —— Biological moisture-proof pigment
Yuanrun Liu —— Biological smoke-proof wall
Qizheng Liu, Zhengyue Hong and Guangbin An —— Biological production of badminton feathers.

#### **2024.3.10 Project Selection from Brainstorming Session**

##### We have selected three projects for further exploration:

**1. Production of Sugar Substitute Fruits:** Utilizing grapes as a biological chassis, we aim to engineer a reduction in the production of free sugars and to enhance the production of natural sugar substitutes.
**2. Fruit Preservation:** We plan to modify antagonistic yeasts to secrete a gelatinous substance that can be sprayed onto fruits to serve as a preservative.
**3. Production of Carmine Red:** We intend to harness microbial fermentation for the production of carmine red pigment.

#### **2024.3.12 Changes in Brainstorming**

When one of our members was hospitalized due to irregular diet and lifestyle, the display cabinet visualizing the sugar content of beverages in the hospital aroused our interest.

#### **2024.3.14 Project Confirmation:**

 After consulting with our Principal Investigator (PI) and advisors, we commenced research on the production of sugar substitutes.

## 2024.3.16-2024.9 Wet Lab

#### **3.16-3.30 Initial Phase Attempts** Prokaryotic Expression Vector Construction and Transformation into *E. coli*; Detection of Prokaryotic System Expression Levels:

##### 3.16

Yitong Li —— Retrieved *E. coli* BL21 from the -80°C freezer and inoculated it onto solid culture media for overnight incubation.

##### 3.18

Yitong Li —— Observed the growth status of *E. coli*.

##### 3.19

Yitong Li —— Selected appropriate single colonies and inoculated them into liquid culture media for scaled-up cultivation.

##### 3.22

Yitong Li —— Prepared competent *E. coli*, transformed the constructed plasmid into *E. coli*, and then inoculated it into antibiotic-containing culture media for selection and cultivation.

##### 3.20

Yitong Li —— Selected appropriate single colonies and inoculated them into liquid culture media for scaled-up cultivation.

##### 3.23-3.30

Yitong Li —— Performed colony PCR and agarose gel electrophoresis to detect *E. coli*, and used Western Blot (WB) to examine the expression of the target protein in *E. coli*.

#### **3.31-6.6 Second Phase** Construction of Eukaryotic Expression Plasmids, Transformation into Tomato Plants via Viral Methods; Detection of Plant Expression Levels:

##### **Cultivation of the First Batch of Tomato Plants**

##### 3.31

Siyuan Guo, Yitong Li, Guangbin An, Qizheng Liu —— Cleaned and disinfected the seeds of the first batch of plants, then sowed them into nutrient soil.

##### 4.1-4.6

Yitong Li, Guangbin An —— Continuously observed and recorded the germination of the seeds, and transplanted the successfully germinated seedlings into individual pots.

##### 4.7-4.14

Yitong Li, Guangbin An —— Continuously observed and recorded the growth of the plants, applying fertilizer and pesticides as appropriate.

##### 4.15-6.18

Siyuan Guo, Yitong Li, Guangbin An, Qizheng Liu —— Continuously observed and recorded the growth of the plants, applying fertilizer and pesticides as appropriate.

##### ***Agrobacterium* Cultivation**

##### 4.1-4.3

Siyuan Guo, Qizheng Liu —— Retrieved *Agrobacterium* GV3101 from the -80°C freezer, activated it, and inoculated it onto solid culture media for overnight incubation.

##### 4.4

Siyuan Guo, Qizheng Liu —— Observed the growth status of *Agrobacterium*.

##### 4.5

Siyuan Guo, Qizheng Liu —— Selected appropriate single colonies and inoculated them into liquid culture media for scaled-up cultivation.

##### 4.6

Siyuan Guo, Qizheng Liu —— Prepared competent *Agrobacterium*, transformed the constructed plasmid into *Agrobacterium*, and then inoculated it into antibiotic-containing culture media for selection and cultivation.

##### 4.10

Siyuan Guo, Qizheng Liu —— Selected appropriate single colonies and inoculated them into liquid culture media for scaled-up cultivation.

##### 4.12

Siyuan Guo, Qizheng Liu —— Performed colony PCR and agarose gel electrophoresis to detect *Agrobacterium*, confirming the success of the transformation.

##### **Infection and Detection of Plants**

##### 4.14

Siyuan Guo, Qizheng Liu —— Infected the first batch of tomato plants with *Agrobacterium* using leaf infiltration.

##### 4.28

Siyuan Guo, Qizheng Liu —— Collected leaves from the first batch of plants and quickly froze them with liquid nitrogen, then crushed them.

##### 4.29

Siyuan Guo, Qizheng Liu —— Extracted protein samples from the plant leaf samples, and stored the remaining samples at -80°C.

##### 4.30

Siyuan Guo, Qizheng Liu —— Performed PCR detection on the leaf tissue samples.

##### 5.1-5.2

Siyuan Guo, Qizheng Liu —— Performed Western Blot (WB) detection on the extracted leaf proteins.

##### 5.14

Siyuan Guo, Qizheng Liu —— Collected flowers from the first batch of plants and quickly froze them with liquid nitrogen, then crushed them.

##### 5.15

Siyuan Guo, Qizheng Liu —— Extracted protein samples from the plant flower samples, and stored the remaining samples at -80°C.

##### 5.16

Siyuan Guo, Qizheng Liu —— Performed PCR detection on the flower tissue samples.

##### 5.17-5.18

Siyuan Guo, Qizheng Liu —— Performed Western Blot (WB) detection on the extracted flower proteins.

##### 6.14

Siyuan Guo, Qizheng Liu —— Collected mature fruits from the first batch of plants and quickly froze them with liquid nitrogen, then crushed them.

##### 6.15

Siyuan Guo, Qizheng Liu —— Extracted protein samples from the plant fruit samples, and stored the remaining samples at -80°C.

##### 6.16

Siyuan Guo, Qizheng Liu —— Performed PCR detection on the fruit tissue samples.

##### 6.17-6.18

Siyuan Guo, Qizheng Liu —— Performed Western Blot (WB) detection on the extracted fruit proteins.

#### **3.31-9.20 Third Phase** Conduct of Transgenic Experiments Cultivation of the Second Batch of Tomato Plants

##### 4.31

Siyuan Guo, Qizheng Liu, Yitong Li, Guangbin An —— Cleaned and disinfected the seeds of the second batch of plants, then sowed them into nutrient soil.

##### 5.1-5.8

Siyuan Guo, Qizheng Liu, Yitong Li, Guangbin An —— Observed and recorded the growth conditions of the plants, applying fertilizer and pesticides as appropriate.

#### *Agrobacterium* Cultivation

##### 5.3

Siyuan Guo, Qizheng Liu, Yitong Li, Guangbin An —— Retrieved *Agrobacterium* GV3101 from the -80°C freezer, activated it, and inoculated it onto solid culture media for overnight incubation.

##### 5.4

Siyuan Guo, Qizheng Liu —— Observed the growth status of *Agrobacterium*.

##### 5.5

Siyuan Guo, Qizheng Liu —— Selected appropriate single colonies and inoculated them into liquid culture media for scaled-up cultivation.

##### 5.6

Siyuan Guo, Qizheng Liu —— Prepared competent *Agrobacterium*, transformed the constructed pBWA(V)HS and pCAMBIA1301 plasmids into different groups of *Agrobacterium*, and then inoculated them into antibiotic-containing culture media for selection and cultivation.

##### 5.10

Siyuan Guo, Qizheng Liu —— Selected appropriate single colonies and inoculated them into liquid culture media for scaled-up cultivation.

##### 5.12

Siyuan Guo, Qizheng Liu —— Performed colony PCR and agarose gel electrophoresis to detect *Agrobacterium*, verifying the success of the transformation.

#### Infection and Detection of Plants and Subsequent Experiments

##### 5.9-5.12

Siyuan Guo, Qizheng Liu, Yitong Li, Guangbin An —— After the leaves fully expanded, excised the hypocotyl and cotyledon tips with a scalpel, leaving the middle part to be cut into 2 to 3 segments and inoculated into pre-cultivation medium.

##### 5.13-5.15

Siyuan Guo, Qizheng Liu, Yitong Li, Guangbin An —— Infected the explants with the transformed *Agrobacterium* and then co-cultivated.

##### 5.16-6.1

Siyuan Guo, Qizheng Liu, Yitong Li, Guangbin An —— Transferred the callus tissue from the recovery culture of the second batch of plant explants to the selection medium for cultivation.

##### 6.2-7.2

Siyuan Guo, Qizheng Liu, Yitong Li, Guangbin An —— After selecting suitable callus tissue, inoculated it onto the differentiation medium for cultivation.

##### 7.3-7.14

Siyuan Guo, Qizheng Liu, Yitong Li, Guangbin An —— Transferred the differentiated seedlings to the rooting medium for cultivation.

##### 7.15-7.26

Qizheng Liu, Yitong Li, Guangbin An —— Transplanted the seedlings into water for cultivation and continuously observed and recorded their growth.

##### 7.27

Qizheng Liu, Yitong Li, Guangbin An —— Transplanted the plants into soil for cultivation and continuously observed and recorded their growth.

##### 8.10

Qizheng Liu, Yitong Li, Guangbin An —— Collected leaves from the first batch of plants and quickly froze them with liquid nitrogen, then crushed them.

##### 8.11

Qizheng Liu, Yitong Li, Guangbin An —— Extracted protein from the plant leaf samples, and stored the remaining samples at -80°C.

##### 8.12

Qizheng Liu, Yitong Li, Guangbin An —— Performed PCR testing on the leaf tissue samples.

##### 8.13-8.14

Qizheng Liu, Yitong Li, Guangbin An —— Performed Western Blot (WB) testing on the extracted leaf proteins.

##### 8.22

Qizheng Liu, Yitong Li, Guangbin An —— Collected flowers from the first batch of plants and quickly froze them with liquid nitrogen, then crushed them.

##### 8.23

Siyuan Guo, Qizheng Liu —— Extracted protein from the plant flower samples, and stored the remaining samples at -80°C.

##### 8.24

Qizheng Liu, Yitong Li, Guangbin An —— Performed PCR testing on the flower tissue samples.

##### 8.24-8.25

Qizheng Liu, Yitong Li, Guangbin An —— Performed Western Blot (WB) testing on the extracted flower proteins.

##### 9.5

Qizheng Liu, Yitong Li, Guangbin An —— Collected mature fruits from the first batch of plants and quickly froze them with liquid nitrogen, then crushed them.

##### 9.5

Qizheng Liu, Yitong Li, Guangbin An —— Extracted protein from the plant fruit samples, and stored the remaining samples at -80°C.

##### 9.6

Qizheng Liu, Yitong Li, Guangbin An —— Performed PCR testing on the fruit tissue samples.

##### 9.7-9.8

Qizheng Liu, Yitong Li, Guangbin An —— Performed Western Blot (WB) testing on the extracted fruit proteins.

##### 9.10

Guangbin An —— Used the Thaumatin ELISA kit to measure the concentration of sweet protein Thaumatin in the experimental group of tomatoes, achieving the expected results.

##### 9.11

Guangbin An —— Detected the concentration of citric acid and glucose in the transgenic tomato fruits using colorimetric assay kits.

##### 9.12

Qizheng Liu, Guangbin An —— Used a combination of human sweet receptor protein T1R2 and HA tag ELISA kits to determine whether the sweet protein in the transgenic tomatoes could interact with the human sweet receptor protein T1R2.

##### 9.14-9.18

Yuanrun Liu, Guangbin An —— Used the control group of tomato fruits to establish a database by adding a concentration gradient of Thaumatin sweet protein.

##### 9.18-9.26

Yuanrun Liu —— Used the established database to test the sweetness of the experimental group of tomato samples and achieved the expected results.

##### 9.14-9.20

Yitong Li, Qizheng Liu —— Constructed the pGD\_SPS-NTPP-Thaumatin-EGFP plasmid.

##### 9.21-9.24

Yitong Li, Qizheng Liu —— The constructed pGD plasmid was transformed into Agrobacterium.

##### 9.25-9.29

Yitong Li, Qizheng Liu —— The aforementioned Agrobacterium was used to infect the leaves of commercially available tobacco and wild-type tomatoes.

##### 9.30

Yitong Li, Qizheng Liu —— The tobacco and tomato leaves that had been infected were observed under a laser scanning confocal microscope.
 Thaumatin sweet protein.

#### **9.9-9.23 Fourth Phase** Conduct of Vacuolar Localization Experiments:

##### 9.9

Yitong Li, Guangbin An, Qizheng Liu —— Retrieved *Agrobacterium* GV3101 from the -80°C freezer and inoculated it onto solid culture media for shaking culture for 3 hours, then transferred it to liquid culture media for overnight cultivation.

##### 9.9-9.10

Yitong Li, Guangbin An, Qizheng Liu —— Constructed a plasmid pGD carrying a vacuolar targeting peptide and transformed it into competent *Agrobacterium* GV3101 , then inoculated it onto solid culture media containing resistance.

##### 9.12

Yitong Li, Guangbin An, Qizheng Liu —— Selected appropriate single colonies and inoculated them into liquid culture media for further cultivation.

##### 9.14

Yitong Li, Guangbin An, Qizheng Liu —— Infected commercial Nicotiana benthamiana plants with four leaves using the aforementioned *Agrobacterium*.

##### 9.23

Yitong Li, Guangbin An, Qizheng Liu —— Performed confocal microscopy observation on Nicotiana benthamiana leaves.

## Products

##### 8.14

All team members —— After a team meeting, we considered that our initial product was a sweet tomato, which, however, had several drawbacks, such as inconvenience for carrying, transportation, and utilization. Considering our original intention was to produce a sugar substitute, our initial idea was to provide our sugar substitute product to factories for the replacement of sugar at the source.

##### 8.20

Yitong Li, Ruohan Chen, Kexin Li —— After visiting the CHI Forest factory, we learned about the compressed form of sugar substitute materials they use, which inspired us. Upon returning and discussing, we decided to design our product as a concentrated liquid form.

##### 9.4

Jiachun Chen, Zejia Wei, Kexin Li —— We visited a food exhibition held in our city and noticed that many vendors were selling products in very small packaging. They mentioned this was to allow customers to easily try or carry the products. Considering simplicity and cost-effectiveness, after obtaining the fruit from the tomato plants, we wished to use a simple procedure to experiment with the manufacture of the product. Therefore, our final strategy was to use concentrated tomato fruit juice as a sweetener, enclosed in a small packet that is convenient for carrying and adding.

##### 9.20

Siyuan Guo —— Designed the cover for the product's small packaging.

## Dry Lab

##### 6.25-7.15

Ruohan Chen —— Utilized AlphaFold software for the visualization analysis of Thaumatin protein, Brazzein protein, and the human sweet receptor protein T1R2.

##### 7.16-8.1

Ruohan Chen —— Predicted the binding models of the human sweet receptor protein and sweet proteins (Protein-Protein Interaction, PPI).

##### 8.2-8.20

Ruohan Chen —— Identified the wilting peptides carried by Thaumatin and analyzed three protein sites responsible for bitterness.

##### 8.21-9.5

Ruohan Chen —— Employed computational algorithms to perform random mutations based on these three sites, simulating the process of directed evolution.

##### 9.6-9.23

Ruohan Chen —— Utilized the mutated Thaumatin and reapplied the PPI protein interaction principle for analysis within the computer, and selected the optimal sweet protein sequence.

## Hardware

##### 4.25-5.15

Haosheng Deng —— After reviewing extensive literature, proposed the preliminary concept of a bio-electronic detection device for detecting sweet proteins and regular sugars.

##### 5.16-6.10

Haosheng Deng —— Constructed a specific model, but after discussion with our Principal Investigator (PI), it was concluded that the bio-electronic detection device still had many unavoidable drawbacks. Therefore, Haosheng Deng abandoned the experiments related to this device and began researching other detection hardware related to sugar detection.

##### 6.11-7.20

Yuanrun Liu, Haosheng Deng —— Studied the specific mechanisms and usage methods of commercial electronic tongues.

##### 7.22-7.26

Haosheng Deng —— In the process of integrating human practices, he discovered that current detections of free sugars are all one-to-one detection methods, which gave Haosheng Deng great inspiration.

##### 7.26-8.10

Haosheng Deng —— After reviewing a vast amount of literature, Haosheng Deng proposed SweetGlow, a common sugar detector based on lanthanide metal-ligand fluorescence, hoping to detect three or more types of sugars within ten minutes and accurately measure their concentrations. Recognizing the substantial workload required to observe the effects of protein detection directly, Haosheng Deng proposed the concept of a lateral flow immunoassay strip for detecting the concentration of sweet proteins in our tomato plants. After team discussion, we began working on the conceptual model design and principle verification of SweetGlow.

##### 8.11-9.20

Haosheng Deng —— Using 3D printing, successfully created a conceptual model of SweetGlow. After contacting Professor Yujuan Chai, an expert in lateral flow immunoassay technology, and with Professor Chai's guidance and assistance, began to develop a lateral flow immunoassay strip for detecting thaumatin, and successfully detected it, creating a specific hardware model. Successfully produced the prototype of SweetGlow.

## Human Practices

##### 2023.12.27

Zejia Wei, Siyuan Guo, Yuanrun Liu, Guangbin An, Haosheng Deng —— Discussed synthetic biology and the use of cell factories with Xbiome company. This meeting inspired the idea of using cell factories for our project.

##### 2024.1.26

Zejia Wei —— The new team SMU-GDMU-China visited Shenzhen University to learn and gain experience.

##### 2024.1.29

Zejia Wei —— Returned to her high school alma mater, the High School Affiliated to SUSTech (HSAS), to give a presentation. At the meeting, she introduced iGEM and encouraged interested students to contact her for further information.

##### 2.27

All team members —— Successfully held an iGEM competition exchange meeting, inviting Vice Chairman of the iGEM Organizing Committee, Miss Zhang Nan, and teams from Southern University of Science and Technology (SUSTech), South China University of Technology (SCUT), Beijing Normal University Zhuhai Campus (BNUZH), Shenzhen Polytechnic University (SZPU), Southern Medical University (SMU), and Guangdong Medical University (GDMU).

##### 3.31

Zejia Wei, Yuanrun Liu, Qizheng Liu, Jiachun Chen —— The iGEM teams from the University of Macau and Beijing Normal University, Zhuhai Campus, came to Shenzhen University to exchange the outcomes of brainstorming sessions.

##### 4.12

All team members —— Hosted students from the Yantian District Committee. We introduced synthetic biology in layman's terms to the students and led them an activity for creatively designing synthetic biology projects.

##### 4.26

Team members Zejia Wei, Qizheng Liu, Zhengyue Hong, Haosheng Deng, Yuntian Wu, Guangbin An —— Produced a safety propaganda video and posted it on a video platform, using an entertaining approach to educate the public about laboratory safety.

##### 5.18

All team members —— Organized the 8th iGEM Southern China Regional Meeting. The meeting brought together teams from the South China region, providing a platform for interactive displays, collaborative communication, and mutual learning, fostering an atmosphere conducive to exploring synthetic biology.

##### 6.8

Team members Zejia Wei, Jiachun Chen, Qizheng Liu, Zhengyue Hong, Yitong Li, Guangbin An, Ruohan Chen, Yuanrun Liu —— During the project advancement phase, we met with the team leader from CAU-China to discuss some issues related to the project.

##### 6.11

Zejia Wei, Yuntian Wu, Kexin Li, Qizheng Liu —— Conducted a street interview activity to gather public opinions on sugar substitutes and genetically modified foods.

##### 6.14

Zejia Wei, Kexin Li, Zhengyue Hong, Yuntian Wu —— Visited the Futian Foreign Language School to give a biology lecture, introducing basic biological knowledge to students and sparking their interest in biology.

##### 6.22

All team members —— Opened our laboratory to the Shenzhen community for a visit, teaching citizens to identify laboratory equipment, learn how to use basic laboratory equipment such as pipettes, and popularize knowledge about synthetic biology.

##### 6.23

Zejia Wei, Zhengyue Hong, Haosheng Deng —— Exchanged offline with students from HKUST-GZ, lending them a DNA reagent.

##### 7.9

All team members —— Held an online meeting with AIS-China to exchange ideas and solve mutual doubts.

##### 7.12

All team members —— Attended the CCiC meeting, which brought together teams from different regions, providing a platform for interactive displays, collaborative communication, and mutual learning.

##### 7.18

Siyuan Guo, Yuanrun Liu —— Provided opportunities for children in remote areas to learn about advanced knowledge, introduced them to synthetic biology, and conducted several simple experiments.

##### 7.18

All team members —— Held an offline meeting with members of BASIS-China to discuss experimental design and HP.

##### 7.19

Kexin Li, Yuntian Wu —— Consulted Professor Tengbo Huang on issues related to sugar storage in tomatoes.

##### 7.22

Yuanrun Liu, Siyuan Guo, Qizheng Liu, Zhengyue Hong —— Several high school leagues came to Shenzhen University to exchange ideas on project design with us.

##### 7.22

Yuanrun Liu, Yuntian Wu —— Attended a plant science lecture at Shenzhen University, learning about organelle localization.

##### 7.23-7.24

Zejia Wei, Kexin Li, Yuntian Wu, Ruohan Chen, Haosheng Deng —— Visited a large number of manufacturers related to sugar substitutes to explore the urgent problems that need to be solved in the sugar substitute market.

##### 7.25-7.26

Zejia Wei, Kexin Li, Yuntian Wu, Ruohan Chen, Haosheng Deng —— Visited large planting sites to understand the current issues in plant cultivation.

##### 7.27

Zejia Wei, Yuntian Wu —— Participated in Synbio Talk organized by CityU as a guest speaker, which popularized the knowledge of synthetic biology to high school students and emphasized safety issues.

##### 7.28

Zhengyue Hong, Qizheng Liu, Kexin Li, Yuntian Wu —— Consulted Professor Mo on the feasibility of tomato chassis and received a positive response.

##### 7.30

Zejia Wei, Yuntian Wu —— Shared our project with Professor Liu Yun from Shenzhen University, who pointed out issues related to the gentamicin gene, which drew our attention.

##### 7.31

Kexin Li, Yitong Li —— Inquired about issues related to genetically modified food and diabetes, which increased our confidence in the feasibility of sugar substitutes.

##### 8.1 - 8.3

Zejia Wei, Kexin Li, Yuntian Wu —— Attended the 2nd iGEM Greater Bay Area Synthetic Biology Industry-Academia-Research Forum, exploring new ways of cooperation among iGEM teams, and emphasizing cooperation between academia and industry.

##### 8.2

Zejia Wei, Yuntian Wu —— Visited the Shenzhen Guangming New District Synthetic Biology Industrial Park for study and learning. Had a brief conversation with the heads of some synbio companies, understanding the importance of food safety in synthetic biology.

##### 8.2

Zejia Wei, Kexin Li, Yuntian Wu —— Collaborated with university teams such as Jilin University to write a white paper. We were responsible for the interpretation and analysis of domestic and international laws on synthetic biology food.

##### 8.5

Kexin Li, Yuntian Wu —— To define the safety of genetically modified food and ensure the stability of proteins, we consulted expert Liqing Zhao.

##### 8.15

Zejia Wei, Yuntian Wu —— Inquired about the market launch process for synthetic biology food in Shenzhen from the Shenzhen Market Supervision Administration.

##### 8.17

Kexin Li, Yuntian Wu —— Discussed safety issues with the iGEM team from Hubei University.

##### 8.19

Zejia Wei, Yuntian Wu —— Hosted youth from all over the world who came to Shenzhen University to study, and explained the development history of synthetic biology to them.

##### 8.20

Kexin Li, Ruohan Chen, Yitong Li —— Visited the CHI Forest Factory in Zhaoqing to understand the company's use of sugar substitutes.

##### 8.21-8.22

Zejia Wei, Yuntian Wu, Yuanrun Liu, Qizheng Liu, Zhengyue Hong, Jiachun Chen —— Attended the Asia-Pacific Mini Jamboree at the University of Hong Kong.

##### 8.23

Haosheng Deng, Yuanrun Liu —— Had a brief discussion with the electronic tongue business.

##### 8.27

Zejia Wei, Jiachun Chen, Kexin Li —— Visited the Shenzhen Agricultural Promotion Center and talked with Chief Researcher Ms. Li about tomato-related issues.

##### 8.31

Zejia Wei, Ruohan Chen, Qizheng Liu —— Participated in the biosafety propaganda activity organized by the Southern University of Science and Technology (SUSTech), popularizing the safety of sugar substitutes for local residents.

##### 9.2

Zejia Wei, Yuntian Wu —— Introduced iGEM to the freshmen of the Collage of Life Sciences and Oceanography, Shenzhen University.

##### 9.4

Zejia Wei, Kexin Li, Jiachun Chen —— Attended the SIAL Food Exhibition to understand how other food companies view sugar substitutes and synthetic biology food.

##### 9.6

Jiachun Chen, Kexin Li, Yuntian Wu, Yuanrun Liu, Guangbin An, Zhengyue Hong, Yitong Li —— Visited the Shenzhen University Longhua Transgenic Base to learn about the cultivation standards and restrictions of genetically modified crops.

##### 9.13

All team members —— Promoted iGEM at the school's club carnival.

## Art Desigining

##### 4.10-5.10

Siyuan Guo —— Designed the venue and layout for the 8th iGEM Southern China Regional Meeting, designed the team uniform styles, and created patterns for peripheral gift items.

##### 5.11-5.19

Siyuan Guo —— Designed the logo for the 2024 SZU-China team.

##### 5.20-6.24

Siyuan Guo, Kexin Li, DingCheng Qi —— Produced a summary video for the 8th iGEM Southern China Regional Meeting.

##### 6.25-7.15

Siyuan Guo, Kexin Li, Jiachun Chen, Guangbin An, Yitong Li, Qizheng Liu —— Designed posters, PPTs, and promotional products for the CCiC Exchange Meeting.

##### 7.16-9.30

Siyuan Guo, Kexin Li —— Designed patterns and graphics for the wiki webpage.

##### 8.10-9.6

Siyuan Guo —— Created a propaganda picture book about sugar substitutes.

##### 8.15-9.4

Kexin Li, Siyuan Guo, DingCheng Qi —— Provided illustrations or animations for the promotional video and edited the promotional video.

##### 10.3-10.9

Kexin Li, Siyuan Guo —— Designed illustrations and special effects for the team's presentation video.
DingCheng Qi —— Edited the presentation video.

## Wiki Develop

##### 6.28-7.15

Zhengyue Hong — Familiarized with the official iGEM Wiki editing operations.

##### 7.15-8.25

Zhengyue Hong — Learned HTML, CSS, and JavaScript.

##### 8.26-9.1

Zhengyue Hong — Constructed the Wiki framework for our SZU iGEM 2024.

##### 9.2-10.2

Zhengyue Hong — Completed the homepage and gradually finished other necessary pages.
**With Deepest Appreciation:**
 We acknowledge the assistance of advisors. We are also very thankful for the other members of the SZU 2024 iGEM team and our close collaboration, which has enabled us to successfully complete the creation of this Wiki!

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Engineering

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![](https://static.igem.wiki/teams/5160/arrow.png)

Menu

* [Overview](#section_1)
* [Production of sweet protein](#section_2)

  [Expression in prokaryotic system](#pro1)
  [Expression in tomato](#pro2)
* [Sweet protein
   storage system](#section_3)
* [References](#section_4)

# **Overview**

Failure and progress go hand in hand. Only by learning from failure can we achieve progress. Undeniably, this applies to work in any field, especially in biological science. Working in the iGEM team, we experienced the cycles of "Design-Build-Test-Learn (DBTL)", making progress through continuous failures.

![](https://static.igem.wiki/teams/5160/project/plant11/engineering-dbtl.png)

We develop two modules to produce Thaumatin steadily and efficiently in tomato fruits in our project. Our Thaumatin was optimized to remove bitterness and retain sweetness based on the directed evolution. And our production system combines a stable synthesis module with an automatic storage module, both working together to form a complete production line. Finally, we get nutritional sweetener SWEETEIN from the juice of tomato fruits straight, providing a nutritious, safe and cheap sugar substitute for the public.

In our project, we follow the principle of DBTL cycle, testing the functions of protein design, production system and sweetness analysis in the project cycle. Then to achieve the expected effect, improve and iterate based on the results.

# **Production of sweet protein**

## Expression in prokaryotic system

#### Design

Although Thaumatin and Brazzein are small molecule proteins[1], the protein structure of Thaumatin contains eight disulfide bonds. We can't ensure protein will be folded correctly in the prokaryotic system, but we still made an initial attempt in *E. coli*. We chose the most common strain BL21(DE3) to realize fermentation production. Our designed target sequence is controlled by T7 promoter, and assembled to pET28a to get expression vector.

#### Build

We pick colonies from the culture medium containing kanamycin to enlarge cultivation. To prove the success of transformation, we use specific primer to amplify the target sequence from the plasmid template extracted from *E. coli* in PCR. Agarose gel electrophoresis proved that our expression vector successfully entered *E. coli* BL21 (DE3) (Fig 1).

![](https://static.igem.wiki/teams/5160/wet-lab/outline/image.png)

**Fig 1. The genes for Thaumatin and Brazzein were constructed on the pET28a vector**

#### Test

Test
In our experiment, the proteins expression was induced by IPTG. Then, we extracted proteins from *E. coli* for Western Blot verification (Fig 2). In this process, we found that the protein was very viscous when extracted, so we suspected that it was caused by the formation of inclusion bodies due to the limitations of the prokaryotic expression system of *E. coli*. We then broke the cells and centrifuged to get the supernatant, and found that the expression of Thaumatin and Brazzein was still very low.

![](https://static.igem.wiki/teams/5160/wet-lab/outline/engineering-renew/en1-1.webp)

**Fig 2. SDS-PAGE and Western blot analyses of Thaumatin and Brazzein expression in transgenic tomato flowers. SDS-PAGE results for Thaumatin (A) and Brazzein (C), and corresponding Western blot results for Thaumatin (B) and Brazzein (D).**

#### Learn

Yeast is one of the commonly used representatives of eukaryotic organisms in biological research and is regarded as the preferred microorganism for fermentation processes. However, through an investigation of comprehensive human practices, it has been observed that the fermentation production requires a protein purification process that is highly complex and may pose increased risks to safety. Although some studies suggest that modifying yeast can partially alter this situation, the modification of yeast strains necessitates prolonged selection and evolution to achieve stability, with the time and financial costs involved being incalculable. Clearly, yeast is not a suitable choice. Therefore, we have shifted our focus to plant-based systems, considering the practical application scenarios of sugar substitutes as food additives. We aim to save on purification steps and reduce safety risks in the process of obtaining Thaumatin. Consequently, we have chosen to conduct trials on a fruit commonly consumed in everyday life: tomatoes.

## Expression in tomato

### I. Transient expression

#### Design

Transient infection is an effective way to verify the availability of plant expression system. Many studies show transient infection can be achieved through *Agrobacterium* immerse or by leaf injection using a viral vector.

*Agrobacterium* [2] attaches to the surface of plant cells through its external pili. It then secretes a protein called virulence protein, which forms channels on the plant cell surface, allowing the transfer of *Agrobacterium* DNA into the plant cell. Considering that our chasis is tomato fruit, it is obviously not feasible to immerse the fruit in *Agrobacterium* to achieve instantaneous infection.

A viral infectious clone vector refers to the process where, under the action of reverse transcriptase, the RNA viral genome is transcribed in vitro to synthesize cDNA, and the cDNA is specifically modified and cloned to enable both self-replication and the expression of foreign genes. In comparison, plant viral expression vectors can remain for a longer period after leaf injection, which facilitates the detection of proteins in fruit.

Therefore, we selected the modified TRV virus for cloning. The target sequences Thaumatin and Brazzein were cloned onto the TRV2 gene of the TRV virus. The TRV vector was injected into *Agrobacterium* for amplification, followed by injection into tomato leaves.

![](https://static.igem.wiki/teams/5160/wet-lab/result/trv-pl.png)

**Fig 3. The genes for Thaumatin and Brazzein were constructed on the pTRV2 vector**

#### Build

We used the TRV (Tobacco rattle virus) to construct the TRV2-CaMV 35S-Thaumatin and TRV2-CaMV 35S-Brazzein plasmids and infected tomato plants. First, the plasmids were transferred into *Agrobacterium* GV3101 for amplification. To confirm successful transformation, we performed PCR with specific primers and verified through agarose gel electrophoresis that our expression vector had successfully entered *Agrobacterium* GV3101 (Fig 4,5).

![](https://static.igem.wiki/teams/5160/wet-lab/outline/image-3.png)

**Fig 4. The colonies PCR results of Thaumatin. M is the Marker. Numbers 1 to 20 are experimental groups. C is the control group. The position in the white box in the figure is the area where the target band is located.**

![](https://static.igem.wiki/teams/5160/wet-lab/outline/trv-2.png)

**Fig 5. The colonies PCR results of Brazzein. M is the Marker. Numbers 1 to 20 are experimental groups. C is the control group. The position in the white box in the figure is the area where the target band is located.**

#### Test

We chose Micro-Tom as our chassis in many varieties of tomatoes. When the tomato plants reached the four-leaf stage, we injected *Agrobacterium* into the tomato leaves. After waiting for a period of time, we extracted RNA from tomato fruits and leaves for RT-PCR validation (Fig 6).

![](https://static.igem.wiki/teams/5160/project/engineering/engineering-6.png)

**Fig 6. RT-PCR results of Thaumatin and Brazzein.
A: Shows the RT-PCR results of Thaumatin. M is the marker. Numbers 1 to 4 are the experimental groups. CTR is the leaf control group of wild-type tomato plants. The position in the white box in the figure is the area where the target band is located.
 B: Shows the RT-PCR results of Brazzein.CTR is the leaf control group of wild-type tomato plants. Numbers 1 to 6 are the experimental groups. M is the marker. The position in the white box in the figure is the area where the target band is located.**

We detected the protein expression of Thaumatin and Brazzein in positive plants. We found that Thaumatin and Brazzein can be successfully transcribed and translated in tomato plants (Fig 7).

![](https://static.igem.wiki/teams/5160/wet-lab/outline/engineering-renew/en2-1.webp)

**Fig 7. Western Blot results of Thaumatin and Brazzein extracted from Micro-Tom leaves and fruits.
A: The WB result diagram of Thaumatin expression in Micro-Tom leaves. M is the Marker. Numbers 1 to 8 are the experimental groups. CTR is the WB result of wild-type tomato leaves.
 B: The WB result diagram of Brazzein expression in Micro-Tom leaves. M is the Marker. Numbers 1 to 7 are experimental groups. CTR is the WB result of wild-type tomato leaves.
 C: The Western Blot (WB) result diagram of Thaumatin expression in Micro-Tom fruits. M is the Marker. Numbers 1 to 5 are the experimental groups. CTR is the WB result of wild-type tomato fruits.
 D: The WB result diagram of Brazzein expression in Micro-Tom fruits. M is the Marker. Numbers 1 to 5 are the experimental groups. CTR is the WB result of wild-type tomato fruits.**

#### Learn

In the tomato expression system, Thaumatin and Brazzein were successfully synthesized, demonstrating that the plant chassis indeed has advantages for protein expression. However, from a practical application perspective, viral transient infection still has drawbacks. Even though some studies have used viral vectors for transient expression to produce various recombinant proteins, in practical production, this method still requires purification, otherwise consumers' concern cannot be alleviated. On the other hand, transient expression cannot achieve stable inheritance, making it far from suitable for long-term production. Therefore, we decided to change the method and use transgenic techniques to stably express sweet proteins.

### II. GMO plant expression

#### Design

Transgenic technology can introduce foreign gene sequences into the host genome, causing heritable changes in biological traits. This method has long been used not only as a traditional breeding approach but also plays a crucial role in producing products through bioreactors. To ensure the sustainable production of Thaumatin and Brazzein, we decided to adopt transgenic methods. We utilized the integration ability of the *Agrobacterium* Ti plasmid to introduce the target gene into tomato callus tissue, achieving heterologous expression.

#### Build

We initially selected the eukaryotic promoter CaMV 35S for preliminary expression attempts and used the binary vector pBWA(V)HS to construct the plasmids pBWA(V)HS\_Thaumatin and pBWA(V)HS\_Brazzein, which were transformed into *Agrobacterium* GV3101. To confirm successful transformation, we performed PCR with specific primers and verified through agarose gel electrophoresis that our expression vector had successfully entered *Agrobacterium* GV3101 (Fig 8).

![](https://static.igem.wiki/teams/5160/project/engineering/engineering-8.png)

**Fig 8. Colonies PCR with hygromycin gene as the target sequence. (A) *Agrobacterium* tumefaciens transformed with the 35S-Thaumatin-3x HA construct. (B) *Agrobacterium* tumefaciens transformed with the 35S-Brazzein-3x HA construct.**

#### Test

We infected the callus tissue with engineered *Agrobacterium* GV3101, placed the dried explants onto co-cultivation medium, and after the differentiated seedlings grew to a certain size, cut them into sections and inoculated them onto rooting medium. The seedlings were then cultured at 23°C and finally transplanted into soil for cultivation. We collected leaf, flower and fruit samples, extracted proteins for WB analysis, and obtained positive results, indicating that transgenic tomatoes capable of stably producing sweet proteins have been successfully cultivated (Fig 9, 10, 11).

![](https://static.igem.wiki/teams/5160/project/engineering/engineering-9.png)

**Fig 9. Western Blot results from 35S promoter transgenic tomato leaves. (A) The target band is Thaumatin-3x HA. (B) The target band is Brazzein-3x HA(B).**

![](https://static.igem.wiki/teams/5160/project/engineering/engineering-10-1.png)

**Fig 10. Western Blot results from 35S promoter transgenic tomato flowers. Western Blot results for (A) Thaumatin flower, (B) Brazzein flower.**

![](https://static.igem.wiki/teams/5160/project/engineering/engineering-11.png)

**Fig 11. Western Blot results from 35S promoter transgenic tomato fruits. Western Blot results for (A) Thaumatin fruit and (B) Brazzein fruit.**

#### Learn

In this part of the experiment, we further validated the feasibility of utilizing the tomato chassis and successfully cultivated transgenic tomato plants. Therefore, we can confirm that tomatoes modified through transgenic methods can produce Thaumatin and Brazzein for use as sweeteners. However, to meet application demands, we must ensure the stability and yield of the expression process in these transgenic tomatoes. This requires us to increase expression levels and reduce degradation. As a result, we further designed a expression system and storage localization system for the next iteration.

### III. Fruit-specific expression

#### Design

To achieve stability in the tomato production system, we considered two aspects and designed a expression system. On one hand, humans have always favored the fruit of tomato crops, with little use of leaves, stems, and other parts. This is not only because of the fruit's appealing color and high nutritional value but also due to its unique flavor and versatile applications. Therefore, we chose a fruit-specific expression promoter to reduce the metabolic burden on other parts of the plant. On the other hand, to avoid and alleviate expression inhibition caused by DNA methylation modification in tomatoes, we decided, after reviewing several studies, to use the tomato's endogenous E8 promoter[3]. The E8 promoter is one of the most well-known fruit ripening-specific promoters and shows strong conservation across different tomato varieties. We hope that this design will enable efficient and specific expression of sweet proteins in the fruit.

#### Build

We inserted the Thaumatin and Brazzein genes respectively at the downstream of the E8 promoter to construct the plasmids pCAMBIA1301\_Thaumatin and pCAMBIA1301\_Brazzein, which were transformed into *Agrobacterium* GV3101. Subsequently, we performed PCR with specific primers and verified through agarose gel electrophoresis that our expression vectors had successfully entered *Agrobacterium* GV3101 (Fig 12).

![](https://static.igem.wiki/teams/5160/wet-lab/result/e8-bra-eb-thau-a-gv3101.webp)

**Fig 12. Colonies PCR result(With the hygromycin resistance gene as the target band). B1-3:E8-Brazzein-1x HA. T1-3:E8-Thaumatin-1x HA.**

#### Test

The callus tissue was infected by engineered *Agrobacterium* GV3101, and we used the same method and condition to cultivate transgenic tomatoes. Then, we collected samples of leaves, flowers, and fruits, extracted proteins for WB analysis, and obtained positive results. We found that Thaumatin and Brazzein were not detected in the leaves and flowers of the transgenic tomatoes but were expressed only in the fruit. This indicates that the E8 promoter can achieve the specific production of sweet proteins in tomato fruit (Fig 13).

![](https://static.igem.wiki/teams/5160/project/engineering/engineering-13.png)

**Fig 13. Western Blot results from E8 promoter transgenic tomato leaves and flowers.
Western Blot results for Thaumatin leaf (A), Thaumatin flower (C), Thaumatin fruits (E), Brazzein leaf (B) Brazzein flower (D) and Brazzein fruits (F).**

To ensure that Thaumatin synthesis induced by the E8 promoter is efficient, we further compared the expression effects of the E8 promoter and the 35S promoter. The team measured the concentration of Thaumatin in tomato fruit using a Thaumatin Enzyme-Linked Immunosorbent Assay (ELISA). A standard curve was established using Thaumatin standards, and positive plant samples for both promoters were randomly selected from WB results as the experimental group. The results showed no significant difference in expression levels between the 35S and E8 promoters, indicating that Thaumatin synthesis induced by the E8 promoter is efficient (Fig 14).

![](https://static.igem.wiki/teams/5160/wet-lab/result/cthuamatin-of-fruit-all.png)
![](https://static.igem.wiki/teams/5160/wet-lab/result/cthuamatin-of-fruit-35s.png)
![](https://static.igem.wiki/teams/5160/wet-lab/result/cthuamatin-of-fruit-e8.png)

**Fig 14. The expression level of Thaumatin under different promoters.**

#### Learn

Based on the above results, we can conclude that the E8 promoter we designed has demonstrated excellent specific expression. We can ensure that Thaumatin and Brazzein are efficiently expressed only in the fruit. In the future, this production model has the potential for industrialization.

# **Sweet protein storage system**

#### Design

The protein levels depend on the balance between synthesis and degradation[4]. To increase protein accumulation, we need to ensure high synthesis and low degradation. Therefore, we must pay attention to the degradation of the protein. We chose to provide a protective area within the tomato fruit for the storage of Thaumatin, reducing the likelihood of its degradation.

In the integrated human practices, we reviewed a large number of publications and discussed our storage localization system with plant expert Prof. Mo Beixin and tomato research expert Prof. Huang Tengbo, who has years of experience in tomato studies. Based on their suggestions, we decided to use the vacuole as the storage compartment for Thaumatin, as the vacuole offers the following advantages:

* In tomato fruit cells, most of the space is occupied by vacuoles, which are often used for storing various nutrients[5].
* The pH environment in vacuoles is lower than 7.0, and the acidic environment suits Thaumatin's acid stability characteristics.
* The environment inside the fruit vacuole is relatively more stable than the cytoplasm and apoplast during the fruit ripening process.

#### Build

Therefore, we chose to use the sweet potato sporamin N-terminal propeptide (SPS-NTPP) sequence for fusion expression with the sweet protein sequence. SPS-NTPP is a kind of vacuole transform signal molecule. SPS-NTPP is a vacuolar transport signal molecule that can accurately and efficiently direct Thaumatin to the vacuole for storage. After completing its targeting function, SPS-NTPP self-degrades without causing any effects.

![](https://static.igem.wiki/teams/5160/wet-lab/result/pgd-sps-ntpp.png)

**Fig 15. Plasmid carrying the Thaumatin gene with the plant vacuole localization peptide SPS-NTTP and fluorescent protein.**

#### Test

We transformed pGD\_SPS-NTPP-Thaumatin-EGFP and pGD\_Thaumatin-EGFP separately into *Agrobacterium* GV3101. Considering time constraints and our immediate objectives, we chose to use Nicotiana benthamiana for transient infection, and then used confocal microscopy to compare the distribution and aggregation of the fluorescence focus[6]. The Thaumatin with EGFP guided by the vacuole localization peptide SPS-NTPP was infected into tobacco leaves by using an *Agrobacterium* vector. The observation results of confocal microscopy imaging showed that there were clustered green spots at the vacuole positions of tobacco leaf cells, proving that our SPS-NTPP protein can be normally expressed and function in tobacco cells (Fig 16).

In order to further verify the feasibility of the SPS-NTPP localization peptide in tomato fruits, and considering the complexity of transgenic plant cultivation, after discussion by the team, we decided to first conduct a pre-experiment of infecting leaves in tomatoes. We used *Agrobacterium* to infect tomato leaves with Thaumatin carrying the SPS-NTPP localization peptide and green fluorescent protein, and then continued to observe the feasibility of the localization peptide under confocal microscopy. From the figure, it can be analyzed that green fluorescent spots are clustered in the cells, and red spots are widely distributed in various positions of the cells. The green spots are caused by green fluorescent protein, while the red spots are caused by chloroplasts. Because when we designed the plasmid, we co-expressed and combined green fluorescent protein with Thaumatin, so the green fluorescent spots we see now represent the aggregation of Thaumatin. Secondly, we see that the chloroplasts of red spots are clustered around the green spots without mixing to produce yellow light, which proves that they are stored separately. Because chloroplasts exist in the cell matrix, and most of the remaining part is vacuoles, we can preliminarily infer that the heterologously expressed SPS-NTPP localization peptide in tomatoes is effective. This verifies the rationality of expressing and localizing Thaumatin in tomato fruits.

![](https://static.igem.wiki/teams/5160/project/plant11/all.png)

**Fig 16. Confocal result diagram of verifying the vacuole localization function of SPS-NTPP in tobacco and tomatoes.**

#### Learn

Based on the above experimental results, we can ensure that the SPS-NTPP sequence has complete functionality for targeting the vacuole. This design allows Thaumatin to be stored in the vacuole, reducing interference with Thaumatin and increasing the total yield. Therefore, we can prove that sweetein has reached the expectation of our final product, which can efficiently, stably, and specifically synthesize Thaumatin and store them in the vacuole. We can achieve the direct acquisition of Thaumatin as a nutritional sweetener in tomato juice, which demonstrates good safety and convenience in application, significantly reducing the cost of Thaumatin and providing consumers with a better sweet taste experience and convenience.

[ ]

References

[1]Okunogbe et al., “Economic Impacts of Overweight and Obesity.” 2nd Edition with Estimates for 161 Countries. World Obesity Federation, 2022.
[2]Wang Cheng,LI Dongyang. White Paper on Healthy China's Beverage and Food Sugar Reduction Action (2021) released[N]. China Food Safety News,2021-09-09(B01).DOI:10.28737/n.cnki.nspzl.2021.001858.
[3]Li Tao, NIU Chunan, PENG Donghai, et al. Suggestions to support and promote the development of natural sugar substitute industry[N]. China Food Safety News,2024-03-06(B03).DOI:10.28737/n.cnki.nspzl.2024.000522.
[4]FAO, IFAD, UNICEF, WFP and WHO. 2023. Aspartame hazard and risk assessment results released
[5]Joseph JA, Akkermans S, Nimmegeers P and Van Impe JFM (2019) Bioproduction of the Recombinant Sweet Protein Thaumatin: Current State of the Art and Perspectives. Front. Microbiol.
[6]Quinet M, Angosto T, Yuste-Lisbona FJ, Blanchard-Gros R, Bigot S, Martinez J-P and Lutts S (2019) Tomato Fruit Development and Metabolism. Front. Plant Sci. 10:1554.

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Proof of concept

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![](https://static.igem.wiki/teams/5160/arrow.png)

Menu

* [Prologue](#section_1)
* [Targeted storage of
   sweet proteins in tomatoes](#section_2)
* [Product Design
   and Advancement](#section_3)
* [References](#section_4)

# **Prologue**

This year, SZU-China will introduce a healthier and more natural sweet sauce product, "Sweetein," to everyone. After a series of experiments, we have successfully obtained transgenic tomatoes for the expression of sweet proteins and verified their expression efficacy. On this page, we will present to you the continued product upgrades, iterations, and corresponding experimental plans.

# **Targeted storage of sweet proteins in tomatoes**

In our project to genetically modify Micro-Tom tomatoes to produce the sweet protein Thaumatin, we aim for successful expression and targeted aggregation of the sweet protein into the vacuoles of fruit cells for storage (for details, see [**Outline**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27outline%27%29%20%7D%7D))[1]. Therefore, we plan to conduct a proof-of-concept test on our transgenic Micro-Tom.

At the end of the [**Results**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page, we used tobacco leaves as experimental material and preliminarily demonstrated that our SPS-NTPP targeting peptide can guide sweet proteins to the vacuoles in tobacco cells, indicating that the plasmid construction idea of SPS-NTPP-Thau-EGFP is feasible and that the targeting peptide [2] itself is not problematic. However, differences in the chassis may also affect the function of the targeting peptide. Therefore, we intend to repeat the tobacco confocal experiment when verifying in tomatoes and if the results show that the targeting peptide can play its role correctly, we will also conduct further tests on the impact of the targeting peptide on tomato metabolism and its targeting efficiency.

Currently, we have completed the *Agrobacterium* infection and confocal experiments on tomatoes. The results showed that green fluorescence appeared within the contours of the vacuoles, preliminarily proving that SPS-NTPP has the function of directing protein transport to the vacuoles(Figure 1).

![](https://static.igem.wiki/teams/5160/wet-lab/result/laser4x2tomato2.png)

**Figure 1. Confocal microscopy results image of tomato. red:ChlorophyII autofluorescence(to indicate cytoplasmic location), green:EGFP**

Our ultimate goal is to store sweet proteins in the vacuoles of fruit cells to indirectly increase their expression levels by reducing degradation. To verify this hypothesis, we plan to continue with experiments, constructing the Thau-EGFP\_pGD plasmid and introducing it into tomatoes as a control group, while the experimental group will still be introduced with the SPS-NTPP-Thau-EGFP\_pGD plasmid. Subsequent confocal microscopy will be used to count EGFP foci in cells for comparative verification. Additionally, after team discussion, we also plan to perform mass spectrometry identification to explore whether the use of the SPS-NTPP targeting peptide will have an impact on the tomato's own metabolism.

If the outcomes are favorable, we plan to incorporate the SPS-NTPP targeting peptide into our future products to ensure the production of sweet proteins, offering a unique transgenic tomato from SZU-China as an efficient and natural platform for sweet protein production!

# **Product Design and Advancement**

Our HP team members observed during a visit to the SIAL food exhibition that many companies offer their products in packaging that comes in more than one size. We interviewed some vendors about their product philosophies and found that the use of different package sizes aims to meet the needs of a broader audience. For individual consumers, there is a preference for smaller, more portable packaging options[3], while factories or shops often opt for larger sizes to reduce costs on retail packaging and enhance the cost-effectiveness of the raw materials. Subsequently, our team visited the Yuanqi Forest company and noticed that many of the raw materials they use are in concentrated forms, which also reduces costs to a certain extent.

In our team meeting, we reached a consensus that the production of a product must consider the needs of its target audience. Consequently, we have envisioned our product to be produced in the form of tomato sweet puree, and we plan to employ drying technology to concentrate it for different usage forms and audiences, ultimately yielding a series of sweet puree products named "Sweetein" (Figure 2). On this page, we will validate the feasibility of producing Sweetein and the ease of its processing.

![](https://static.igem.wiki/teams/5160/hp/eps/longterm2.png)
![](https://static.igem.wiki/teams/5160/hp/eps/longterm.png)

**Figure 2. Tomato sweet puree product**

Our products are divided into three different levels of sweetness, corresponding to the use of sweet protein tomatoes of different weights as raw materials. This means that products of different sweetness levels contain different equivalents of sweet proteins and therefore have different uses(Figure 3).

![](https://static.igem.wiki/teams/5160/wet-lab/outline/a1.webp)

**Figure 3. The corresponding sweet protein equivalents for different sweetness levels (the sweet protein equivalents in the picture serve only as a reference, please refer to the actual product for specifics).**

A. The sweet pulp of grade A is uncondensed juice and can be directly consumed as a beverage.
B. The sweet pulp of grade B is juice that has been concentrated 10 times and can be diluted by families for making beverages or used as a sweet dipping sauce.
C. The sweet pulp of grade C is juice that has been concentrated 20 times and can be provided to dessert shops or beverage stores as a sweetener (Figure 4).

![](https://static.igem.wiki/teams/5160/wet-lab/proof/product-forms.png)

**Figure 4. Possible product forms of sweet pulps of grades A, B, and C.**

Next, we conducted a simple simulation of the production process (The production process is for reference only).

![](https://static.igem.wiki/teams/5160/1212121212.png)

**Figure 5. A,B. Use a juicer to crush the fruit. C,D. Filter our tomato pulp.**

![](https://static.igem.wiki/teams/5160/wet-lab/outline/a.gif)
![](https://static.igem.wiki/teams/5160/wet-lab/outline/b.gif)
![](https://static.igem.wiki/teams/5160/wet-lab/outline/c.gif)

**Figure 6. Left and middle: Put the obtained clarified liquid into a dryer for concentration and drying. right: Package the concentrated sweet pulp.**

First, tomatoes are crushed and then filtered. The resulting juice becomes Grade A sweet pulp. Subsequently, the materials for Grade B and Grade C sweet pulp are placed into a dehumidifying dryer for dehydration treatment. According to the required sweetness level, they are concentrated to different multiples of concentrated liquid. Finally, the end product is obtained (Figure 5,6).

Through this simulated experiment, we have verified the feasibility and simplicity of producing Sweetein sweet pulp. By adopting a series of straightforward operations, we are able to produce healthy Sweetein sweet pulp with rich nutritional value. Undoubtedly, this reduces production costs and enhances the cost-effectiveness of the product. In the future, we plan to extend this technology to more crops and products, aiming to create a world of low-calorie sweetness.

[ ]

References

[1] Marin Viegas VS, Ocampo CG, Petruccelli S. Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields. Bioengineered. 2017;8(3):203-211. doi:10.1080/21655979.2016.1222994
[2] Jha S, Agarwal S, Sanyal I, Jain GK, Amla DV. Differential subcellular targeting of recombinant human α₁-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants. Plant Sci. 2012;196:53-66. doi:10.1016/j.plantsci.2012.07.004
[3] Paurav Shukla, Jaywant Singh, Weisha Wang,The influence of creative packaging design on customer motivation to process and purchase decisions,Journal of Business Research,Volume 147,2022,Pages 338-347,

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Results

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![](https://static.igem.wiki/teams/5160/arrow.png)

Menu

* [Overview](#section_1)
* [Protein Expression](#section_2)

  [*E. coli* Experiment](#ab_1)
  [TRV-Mediated Delivery](#ab_2)
  [Transgenic tomato 1.0](#ab_3)
  [Transgenic tomato 2.0](#ab_4)
* [Expression Assessment](#section_3)

  [Metabolism test](#de_1)
  [Expression Assessment](#de_2)
  [Sweetness Detection](#de_3)
* [Vacuole Localization](#section_4)

  [Localization Peptide](#co_1)
  [Application on Tomatoes](#co_2)
* [References](#section_5)

# **Overview**

This year, the SZU-China team decided to undertake a project that combines classical molecular biology techniques, foodomics, and innovative approaches based on synthetic biology to successfully express two sweet proteins, Thaumatin and Brazzein, in tomatoes. To achieve this, knowledge from different scientific fields was employed, including molecular biology, biochemistry, food science, and computer science.

On this page, you can find all the results for the production, testing, and localization of our sweet protein product, while also noting that the experimental page provides analytical information on the strategies used to carry out each step.

# **Protein Expression**

## *E. coli* Experiment

*Escherichia coli*, as one of the most common prokaryotes in the research field, has many advantages suitable for protein expression. Therefore, we first chose *E. coli* as the tool for the trial expression of sweet proteins.

Initially, our team inserted the Thaumatin(~23.02 kDa) and Brazzein(~8.67 kDa) genes into the pET28a[6] vector to construct a plasmid, and placed the strong promoter 35S in front of the open reading frame. The hope was to enhance the expression level of the plasmid.

Subsequently, we used whole-genome synthesis technology to transform the aforementioned expression vector into *E. coli* BL21 (DE3), picked single colonies in a Kan (kanamycin)-containing medium for expansion and extracted bacterial proteins for Western Blot (WB) experiments to detect sweet proteins. Since expressing recombinant proteins in *E. coli* tends to form inclusion bodies[1], we added protein extraction buffer to 4 mL of bacterial culture for lysis, and after centrifugation, we took both the supernatant and the pellet for Western Blot. It was found that both Thaumatin and Brazzein were expressed in *E. coli*, but the expression levels were low (Figure 1), which may be due to the limitations of the *E. coli* prokaryotic expression system and the formation of inclusion bodies. This experiment can prove that the sweet proteins expressed by *E. coli* are not effective, hence we need to optimize the choice of the chassis.

![](https://static.igem.wiki/teams/5160/wet-lab/outline/sigongge-1.webp)

**Figure 1. SDS-PAGE and Western blot analyses of Thaumatin and Brazzein expression in transgenic tomato flowers. SDS-PAGE results for Thaumatin (A) and Brazzein (C), and corresponding Western blot results for Thaumatin (B) and Brazzein (D).**

At the same time, in the *E. coli* experiments, considering the time and difficulty, we chose a crude extraction method. However, the challenges in protein purification operations made us realize that using microorganisms as a chassis not only increases production costs but also raises safety risks[2]. This prompted us to consider using a safer model organism as a sweet protein factory. If an organism is already commonly consumed in people's daily lives, then this chassis would undoubtedly save us the trouble of purification and dispel safety concerns[3]. Thus, it naturally occurred to us to further attempt using the widely loved cherry tomatoes(For more reasons to choose tomatoes, please see the [**Design**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) page).

## TRV-Mediated Delivery

The most common method of genetic modification in plants is *Agrobacterium* infection, so we only need to select the appropriate plasmid vector and a suitable tomato variety as a chassis to achieve the addition of sweet protein genes in tomatoes. We ultimately chose the Micro-Tom and TRV vectors (for details, see the [**Outline**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27outline%27%29%20%7D%7D) page).

In order to utilize the TRV vector to introduce the sweet protein gene into Micro-Tom for expression, we first constructed the TRV2\_Thaumatin and TRV2\_Brazzein plasmids using the TRV2 vector.

Subsequently, we transformed the well-constructed plasmids, along with TRV1, mCherry, and P19 (for details, see the [**Outline**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) webpage), into *Agrobacterium* GV3101 as the experimental group. The control group differed only by the absence of the TRV2 plasmid, and *Agrobacterium* PCR was also performed, yielding positive results (Figure 2), indicating that our plasmids had been successfully transferred into *Agrobacterium* GV3101.

![](https://static.igem.wiki/teams/5160/wet-lab/result/1.png)

**Figure 2. The colonies PCR results of Thaumatin and Brazzein. (A) The colonies PCR results of Thaumatin. M is the Marker. Numbers 1 to 20 are experimental groups. C is the control group. The position in the white box in the figure is the area where the target band is located.
(B) The colonies PCR results of Brazzein. M is the Marker. Numbers 1 to 20 are experimental groups. C is the control group. The position in the white box in the figure is the area where the target band is located.**

We selected Micro-Tom among numerous varieties of cherry tomatoes as the chassis system (for details, see the [**Outline**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27outline%27%29%20%7D%7D) page). After inducing *Agrobacterium*-mediated expression, we infected tomato leaves with *Agrobacterium* solution, making small incisions on the abaxial side of the leaves with a syringe needle, taking care to avoid the veins, and then injected the bacterial solution using a needleless syringe. Subsequently, to verify the expression of sweet proteins in the tomato leaves, we harvested the fifth leaf from the bottom of the plant, extracted RNA from a portion of the leaf tissue for RT-PCR, and obtained positive results (Figure 3).

![](https://static.igem.wiki/teams/5160/wet-lab/result/2.png)

**Figure 3. RT-PCR results of Thaumatin and Brazzein. (A) The RT-PCR results of Thaumatin. M is the marker. Numbers 1 to 4 are the experimental groups. CTR is the leaf control group of wild-type tomato plants. The position in the white box in the figure is the area where the target band is located.
(B) The RT-PCR results of Brazzein.CTR is the leaf control group of wild-type tomato plants. Numbers 1 to 6 are the experimental groups. M is the marker. The position in the white box in the figure is the area where the target band is located.**

Another part was subjected to WB assay to detect the expression at the protein level, and bands of Thaumatin with a 3x Flag tag (28.10 kDa) and Brazzein with a 3x Flag tag (9.48 kDa) were successfully detected near 28 kDa and 9 kDa, respectively (Figure 4). This suggests that our sweet protein genes can be expressed in Micro-Tom plants.

![](https://static.igem.wiki/teams/5160/wet-lab/result/3.png)

**Figure 4. Western Blot results of Thaumatin and Brazzein extracted from Micro-Tom leaves. (A) The WB result diagram of Thaumatin expression in Micro-Tom leaves. M is the Marker. Numbers 1 to 8 are the experimental groups. CTR is the WB result of wild-type tomato leaves.
(B) The WB result diagram of Brazzein expression in Micro-Tom leaves. M is the Marker. Numbers 1 to 7 are experimental groups. CTR is the WB result of wild-type tomato leaves.**

To ensure that our sweet proteins can also be expressed in the fruit of the tomato, we collected red fruits at the appropriate time for protein extraction and performed Western blot analysis, obtaining the same positive results (Figure 5). These experiments indicated that our sweet proteins can not only be expressed in Micro-Tom plants but also successfully expressed in the fruit. This outcome is exhilarating and represents the second milestone in our experimental progress, laying the foundation for our subsequent experiments. It proves that expressing sweet proteins in plant chassis is a feasible approach. The results encouraged us to continue constructing a more stable expression system in tomatoes.

![](https://static.igem.wiki/teams/5160/wet-lab/result/4.png)

**Figure 5. Western Blot results of Thaumatin and Brazzein extracted from Micro-Tom fruits. (A) The WB result diagram of Thaumatin expression in Micro-Tom fruits. M is the Marker. Numbers 1 to 5 are the experimental groups. CTR is the WB result of wild-type tomato fruits.
(B) The WB result diagram of Brazzein expression in Micro-Tom fruits. M is the Marker. Numbers 1 to 5 are the experimental groups. CTR is the WB result of wild-type tomato fruits.**

## Transgenic tomato 1.0

Genetic modification technology, as a method of introducing target genes into the genome of an organism to cause heritable changes in biological traits, has long been used not only as a traditional breeding method but also plays a significant role in the production of products by bioreactor systems.

In order to make our protein expression system heritable, thereby enabling the production of sweet proteins on a larger scale to meet potential societal demands, we continued to update and iterate, using *Agrobacterium* transformation to make Micro-Tom a stable sweet protein factory[4].

We initially attempted expression using the eukaryotic promoter 35S, constructing two plasmids, pBWA(V)HS\_Thaumatin and pBWA(V)HS\_Brazzein. These were transformed into *Agrobacterium* GV3101 and subjected to PCR screening for the neomycin resistance gene within the bacteria (Figure 6).

![](https://static.igem.wiki/teams/5160/wet-lab/result/5.png)

**Figure 6. Colonies PCR with hygromycin gene as the target sequence. (A) Colonies PCR with hygromycin gene as the target sequence. Agrobacterium tumefaciens transformed with the 35S-Thaumatin-3x HA construct. (B) Agrobacterium tumefaciens transformed with the 35S-Brazzein-3x HA construct.**

Positive bacteria were then used to infect wounded tissue (Figure 7A), and the excised explants were placed on co-cultivation medium and incubated in the dark for 2 days. Once the differentiated seedlings grew to approximately 2-3 cm, the recultured callus was transferred to selection medium and cultivated under the same conditions for 15-30 days. The selected callus was then inoculated onto differentiation medium and cultured under the same conditions for 30-40 days. When the differentiated seedlings reached a height of about 2-3 cm, they were excised from the callus and inoculated onto root induction medium, where they were cultured under the same conditions for an additional 10-15 days to encourage root formation (The process from pre-cultivation of tomato seedlings to post-infection differentiation and rooting is illustrated in Figure 8).

![](https://static.igem.wiki/teams/5160/wet-lab/result/louis/yushangzuzhi11.png)

**Figure 7. Callus tissue infection and screening.
(A) Callus tissue infection.
(B) Select positive seedlings with hygromycin.**

![](https://static.igem.wiki/teams/5160/wet-lab/result/louis/yushangzuzhi22.png)

**Figure 8. The process from pre-cultivation of tomato seedlings
 to post-infection differentiation and rooting**

Then we sampled the leaves to extract DNA for PCR, successfully obtaining positive results(Figure 9). Subsequently, we selected some plant leaves to extract proteins for WB detection and successfully obtained positive results near 28 kDa and 10 kDa(Figure 10), indicating that Thaumatin (28.36 kDa) and Brazzein (9.75 kDa) were successfully expressed in the transgenic Micro-Tom. This marks the second milestone in our experimental process, suggesting that the transgenic tomato with the ability to stably inherit and produce sweet proteins has been successfully cultivated.

![](https://static.igem.wiki/teams/5160/wet-lab/result/6.png)

**Figure 9. DNA PCR result.
(A) 35S-Thaumatin-3x HA,performing PCR with hygromycin as the
 target sequence.
Screening 1-33, positive: 1-16, 20, 22-25, 28-33. Positive rate: 27/33 = 82%;
(B) 35S-Brazzein-3x HA,performing PCR with hygromycin as the target sequence.
Screening 1-30, positive: 1-14, 16-23, 25, 27-30. Positive rate: 27/30 = 90%.**

![](https://static.igem.wiki/teams/5160/wet-lab/result/7.png)

**Figure 10. Western Blot results from 35S promoter transgenic tomato leaves. (A) Western blot results from 35S promoter transgenic tomato leaves. The target band is Thaumatin-3x HA. (B) The target band is Brazzein-3x HA.**

Although we are well aware that the characteristic of the 35S promoter to express in any part of the plant is not conducive to the production efficiency of sweet proteins. And due to time constraints, we have already begun to conceive and design a fruit-specific expression module. But this does not mean that these 35S transgenic tomatoes have lost their significance. Firstly, the ability to express in leaves does not guarantee successful expression in flowers and fruits. Secondly, these tomatoes not only allow us to become familiar with the operations of a series of experiments on the expression and detection of transgenic plant proteins in advance, but also serve as very suitable control groups, providing a standard for comparison with the results of our subsequent fruit-specific expression experiments. Considering the above factors and time constraints, we still took meticulous care of these plants, closely monitored the plants with positive leaf WB results. At the appropriate time, we collected and flowers and fruits to extract proteins for WB experiments and screen the results (Figure 11), preserving the samples corresponding to the positive bands at -20°C for use as positive controls in subsequent experiments.

![](https://static.igem.wiki/teams/5160/wet-lab/result/8.png)

**Figure 11. Western blot results from 35S promoter transgenic tomato flowers and fruits. Western blot results: (A) Thaumatin flower,(B) Brazzein flower,(C) Thaumatin fruit,(D) Brazzein fruit.**

## Transgenic tomato 2.0

The E8 promoter is one of the most widely characterized and mature fruit-specific tomato promoters, exhibiting strong conservation across different tomato varieties[5][7][9][14]. To achieve efficient and specific expression of sweet proteins in the fruit, we inserted the genes thaumatin and brazzein downstream of the E8 promoter, constructing two plasmids, pBWA(V)HS\_Thaumatin and pBWA(V)HS\_Brazzein. These were then transformed into *Agrobacterium* GV3101 and subjected to PCR experiments and obtained positive results(Figure 12) .

![](https://static.igem.wiki/teams/5160/wet-lab/result/e8-bra-eb-thau-a-gv3101.webp)

**Figure 12. Colonies PCR result (With the hygromycin resistance gene as the target band). B1-3:E8-Brazzein-1x HA. T1-3:E8-Thaumatin-1x HA.**

We then conducted a tomato transgenic experiment following the same steps as Transgenic Tomato 1.0, and after a period of cultivation, we extracted DNA from the leaves for PCR. The majority of the results were positive, indicating that the target sequence has been successfully integrated into the Micro-Tom plant (Figure 13).

![](https://static.igem.wiki/teams/5160/wet-lab/result/9.png)

**Figure 13. DNA PCR result.
(A) E8-Thaumatin,performing PCR with hygromycin as the target sequence.
Screening 1-10, positive: 2, 5-10. Positive rate: 7/10 = 70%.
(B) E8-Brazzein,performing PCR with hygromycin as the target sequence.
Screening 1-37, positive: 1-3, 5-7, 9-11, 13, 15-21, 23-34, 36. Positive rate: 30/37 = 81%.**

Additionally, we extracted protein from the leaves to verify the fruit-specific expression function of the E8 promoter. We performed WB with E8-Thaumatin (26.20 kDa) and E8-Brazzein (7.58 kDa) and compared the results with those obtained from the 35S promoter in Transgenic Tomato 1.0 (Figure 16,17). The results showed that the target genes downstream of the E8 promoter were not expressed in the leaves.

To make this difference more pronounced, we specifically tried Western blot with samples of transgenic tomato leaves containing 35S-Thaumatin (28.36 kDa) and E8-Thaumatin (26.20 kDa) together, and the results showed that the 35S samples had a clear band near 28 kDa, while the E8 samples did not show a clear band near 26 kDa (Figure 14A,B), again indicating that the E8 promoter does not initiate the expression of the target genes downstream in the leaves .

Flowers are another major important organ of plants. Thus, to ensure our experiment is more comprehensive and the results are convincing and valuable for reference, we also collected tomato flowers at the appropriate time to extract protein for WB experiments(Figure 14C,D), reaching the same conclusion as the leaf experiments .

![](https://static.igem.wiki/teams/5160/wet-lab/result/10.png)

**Figure 14. Western blot results from E8 promoter transgenic tomato leaves and flowers. Western blot results: (A) Thaumatin leaf,(B) Brazzein leaf,(C) Thaumatin flower,(D) Brazzein flower.**

Since the fruit is our ultimate goal, we naturally collected the red fruits expressing Thaumatin and Brazzein, and similarly extracted protein for WB experiments. Due to the growth cycle of tomatoes, our E8-Thaumatin fruits are still scarce, resulting in a small sample size. However, the results showed that both had distinct bands at around 26-30 kD and 10 kD(Figure 15), respectively, which could already indicate that the E8 promoter can effectively initiate the expression of downstream genes in the fruit.

![](https://static.igem.wiki/teams/5160/wet-lab/result/11.png)

**Figure 15. Fruit WB result.
(A) E8-Thaumatin-1x HA. The target genes downstream
 of the E8 promoter were expressed in the fruits.
(B) E8-Brazzein-1x HA. The target genes downstream
 of the E8 promoter not expressed in the flowers.**

Through comprehensive experiments, after multiple validations and comparisons, we began with the simple recombination expression of sweet proteins, achieved a more natural and safer production environment through the replacement of chassis, optimized the stable and heritable expression of sweet proteins through transgenic methods and improved the utilization rate of sweet proteins by iterating the promoter to achieve fruit-specific expression. Each step has been systematically detected at the molecular level, ultimately achieving the natural, heritable, and specific expression of sweet proteins in Micro-Tom!

# **Sweetness Detection**

To further test the performance of the sweet proteins produced by our natural plant chassis, tomatoes, we conducted the following validation experiments focusing on the concentration, sweetness, and biological functions of the recombinant sweet proteins:

## Metabolism test

#### Macroscopic Phenotypic Metabolism

An indispensable part of producing proteins through heterologous expression is to clarify whether heterologous expression has an impact on the metabolism of the chassis organism and what the impact is[12]. Following the research principle from macro to micro, we first conducted a statistical study on the macroscopic phenotypic metabolism of tomato plants. The figure below (Figure 16) shows a comparison of the phenotypic traits (plant appearance and leaves) of tomato plants in different groups:

![](https://static.igem.wiki/teams/5160/wet-lab/result/166.png)

**Figure 16. Comparison of plant appearance and leaves of tomato plants in different groups
(From left to right: Wild; Transgenic tomato 35S promoter group; Transgenic tomato E8 promoter group.)**

Using statistical methods, we collected phenotypic data from each group of tomatoes and analyzed the differences in three of the most representative characteristics: fruit weight, leaf length, and plant height (Figure 16), where the fruit weight was measured for all ripe tomatoes in each group of plants, leaf length was measured on the fifth leaf of similar growth stage and condition tomato plants in the same group, and plant height was recorded for all tomato plants in the same group.

Based on the figure below (Figure 17), it can be seen that there are no significant differences between the three phenotypic traits (fruit weight, leaf length, and plant height) of tomato plants in different groups, indicating that the use of tomato chassis for heterologous expression to produce sweet proteins does not have a significant negative impact on tomato plant metabolism[10].

![](https://static.igem.wiki/teams/5160/wet-lab/result/apparent-all.png)

**Figure 17. Data on fruit weight, leaf length, and plant height of tomato plants in different groups
(A) Data on fruit weight of tomato plants with different expression vectors;(B) Data on leaf length of tomato plants with different expression vectors;(C) Data on plant height of tomato plants with different expression vectors. (ns P > 0.05; \* P < 0.05; \*\* P < 0.01; \*\*\* P < 0.001; \*\*\*\* P < 0.0001).**

#### Glucose Colorimetric Assay

In our macroscopic phenotypic metabolism analysis, we have determined that the expression of the sweet protein Thaumatin does not significantly affect the macroscopic phenotypic traits of tomato organs. Following the principle of gradually delving deeper into the molecular level of molecular biology research, we have designed experiments for a more detailed metabolic analysis at the molecular level. Considering the limitations of experimental time and the representativeness of target substances, we chose to first test glucose. For more details, please refer to our [**Design**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) page. The figure below (Figure 18) shows the results of our detection of glucose content using the Glucose (Glu) Colorimetric Assay Kit (GOD-POD Method)(Elabscience).

![](https://static.igem.wiki/teams/5160/wet-lab/result/cglucose-of-fruit4.png)

**Figure 18. The glucose content in the tomato fruits of different groups
TRV-Thau: TRV virus Thaumatin group; TRV-Bra: TRV virus Brazzein group; 35S-Thau: Transgenic tomato 35S promoter Thaumatin group; 35S-Bra: Transgenic tomato 35S promoter Brazzein group; E8-Thau: Transgenic tomato E8 promoter Thaumatin group; E8-Bra: Transgenic tomato E8 promoter Brazzein group**

From the figure above, it can be observed that there are no significant differences in glucose content among the various groups of tomato fruits, and the glucose content of all groups falls within the range reported in the literature. However, we have noticed that the glucose content in the experimental groups is slightly but not significantly higher than that in the wild-type and control groups. This could be due to the fact that the growth stages of the control and experimental groups of tomato plants are not entirely aligned, with the experimental group tomatoes being slightly more mature. The conversion of organic acids to glucose may result in a higher content of it.

## Expression detection

To further test the capability of our natural plant chassis, tomato, to produce sweet proteins, we conducted the following validation experiments targeting the concentration of recombinant sweet proteins:

#### Detecting the concentration of sweet proteins produced (ELISA)

Our team used Thaumatin enzyme-linked immunosorbent assay (ELISA) (Shanghai hnybio) to determine the concentration of Thaumatin in tomato fruits. A standard curve was established using Thaumatin standards, and positive plant samples from the Western Blot results were selected as the experimental group. The results are shown in Figure 19.

![](https://static.igem.wiki/teams/5160/wet-lab/result/cthuamatin-of-fruit4.png)

**Figure 19. ELISA detection of Thaumatin sweet protein concentration in tomato fruits.
TRV: TRV virus; 35S: Transgenic tomato 35S promoter group; E8: Transgenic tomato E8 promoter group; CK: control check group**

It can be seen from the above figure that the Thaumatin in the fruits of each experimental group of tomatoes is significantly higher than that in the control group. By comparison, it can be clearly observed that the average expression amount of Thaumatin in the Transgenic tomato E8 promoter group is better than that in the Transgenic tomato 35S promoter group, indicating that the use of the E8 promoter as a promoter will not have a negative impact on the expression content of Thaumatin[7].

## Sweetness detection

#### TAS1R2-sweet protein binding in an innovative assay

We have innovatively put forward a method similar to Enzyme-Linked Immunosorbent Assay (ELISA) for the specific detection of the binding affinity between sweet proteins and human sweet taste receptors[8](Figure 20). (For detailed experimental principles and methods, please refer to the [**Outline**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27outline%27%29%20%7D%7D) webpage.)

![](https://static.igem.wiki/teams/5160/wet-lab/result/louis/elisa-1.png)

**Figure 20. Detection of the binding between human receptor protein TAS1R2 and Thaumatin sweet protein based on ELISA, the more yellow the color, the greater the amount of Thaumatin bound in the sample**

The first column was set as the blank control group. In the first row (except for the first well; the same applies to the second and third rows), it represented the positive control group. The second row corresponded to the experimental group with 35S-Thaumatin fruit protein samples, and the third row was designated for the experimental group with E8-Thaumatin fruit protein samples. As can be seen from the Figure 20, all wells in the experimental groups exhibit color development, indicating that the mature fruits of both transgenic lines contain correctly expressed Thaumatin protein capable of binding to the human sweet receptor protein T1R2.

The absorbance (OD value) was measured at a wavelength of 450 nm using a microplate reader. After obtaining the data, the OD values of the sample wells were subtracted by those of the blank wells. Subsequently, the OD values of the experimental groups were normalized by dividing by the average OD value of the positive control group. The resulting values were plotted as a scatter graph, and the mean values of each dataset were compared(Figure 21).

![](https://static.igem.wiki/teams/5160/rtrtrt.png)

**Figure 21. Comparative analysis of the binding effects of proteins in fruits of 35S-Thaumatin and E8-Thaumatin with T1R2.**

The chart indicates that the expression levels of 35S-Thaumatin are comparable to those of E8-Thaumatin. Given the limited sample size we utilized, it is not feasible to conclusively determine which promoter confers a superior expression profile. Nevertheless, based on the analysis of the 11 samples, we can deduce that there is no substantial difference in the expression of sweet proteins under the control of the 35S and E8 promoters.

## Electronic Tongue Detection

We subjected the control group of tomatoes (non-infected wild-type Micro-Tom tomatoes), transgenic tomatoes with the 35S promoter, and transgenic tomatoes with the E8 promoter to electronic tongue detection. Subsequently, the taste characteristics detected by the electronic tongue were input into the Support Vector Regression (SVR) model for analysis.
Based on the predictive results of the model, we can clearly see that there is a significant gap between the wild type and the experimental group. Moreover, compared to the wild type, the data from the experimental group is larger. This indicates that the sweet protein has been successfully expressed in the genetically modified tomatoes and has shown sweetness. At the same time, to obtain more accurate data, we carried out further calculations. From the calculation results, we know that, the wild-type's predicted result was 0.0109, indicating that the wild type of tomato samples is equivalent to a 0 ppm standard Thaumatin tomato solution, further confirming the usability of our model. The transgenic tomatoes with the 35S promoter resulted in 21.6013, meaning that the Thaumatin contained within is equivalent to a 21.6013 ppm standard Thaumatin solution. Similarly, it can be concluded that the Thaumatin in tomatoes with the E8 promoter is equivalent to a 21.6040 ppm standard Thaumatin solution.

![](https://static.igem.wiki/teams/5160/wet-lab/result/svrr.webp)

**Figure 22. SVR result. Under SVR model analysis, the recombinant Thaumatin is equivalent to what concentration of naturally sweet Thaumatin with the same sweetness level.**

Following the method established by Grant E. DuBois and D. Eric Walters, we calculated the Thaumatin concentration-response curve. It was determined that the sweetness level of Thaumatin in transgenic tomatoes with the 35S promoter is 8.65, and similarly, the sweetness level of Thaumatin in transgenic tomatoes with the E8 promoter is also 8.65.

Ultimately, based on the sucrose concentration-sweetness level calculation, the Thaumatin in transgenic tomatoes is equivalent to a standard sucrose solution with a concentration of 8.65%.

In summary, from the aforementioned experiments, we have measured the sweetness of Thaumatin in transgenic tomatoes. Thaumatin with a concentration of 11.0591 mg/L in transgenic tomatoes containing the E8 promoter is equivalent in sweetness to a standard sucrose solution with a concentration of 8.65%. This sets a standard for our future product manufacturing, enabling us to provide more precise consumption guidance to users.

However, in practical operation, we found that the electronic tongue also has some limitations, such as the detection accuracy for certain substances is not high, leading to inaccurate results. Therefore, we communicated with experts and scholars on this issue and wrote a letter of suggestions for improving the electronic tongue to the engineers, hoping to promote the vigorous development of this industry. After all, as a forward-looking human sensory assessment system, the electronic tongue has gradually replaced traditional human sensory assessments, bringing more possibilities.

# **Vacuole Localization**

After confirming the expression level and functionality of the sweet protein, we aim to enhance the product further. Concerned about the susceptibility of sweet proteins to degradation, we opted to use SPS-NTPP to guide the aggregation of sweet proteins in vacuoles for targeted storage[11][13]. To verify that the addition of SPS-NTPP can direct Thaumatin to vacuoles, we first wanted to replicate the targeting effect of this peptide in our lab, ensuring we can proceed to the next phase of experiments. The most convenient method is to fuse SPS-NTPP with Thaumatin and attach an EGFP protein at the C-terminus; the fluorescence emitted by EGFP will indicate the location of Thaumatin. By comparing the fluorescence distribution of both, we can test whether SPS-NTPP can serve the vacuolar targeting role in this project.

## Verification of the intrinsic action of the positioning peptide.

Based on the above concept, we constructed two plasmids, pGD\_SPS-NTPP-Thaumatin-EGFP and pGD\_Thaumatin-EGFP and introduced them separately into *Agrobacterium* GV3101 (Figure 23). Considering the time cost and our immediate objectives (to test SPS-NTPP), we used tobacco (*Nicotiana benthamiana*) for transient infection. Then, confocal observation was used to compare the distribution and aggregation of fluorescence foci.

![](https://static.igem.wiki/teams/5160/wet-lab/result/vacuole-1.jpg)

**Figure 23. Colonies grown successfully on selective agar medium.**

The results showed that green fluorescence appeared within the contours of the vacuoles, preliminarily proving that SPS-NTPP has the function of directing protein transport to the vacuoles. However, after the localization work was completed, whether SPS-NTPP can be cleaved and whether thaumatin has correctly folded, although the sweetness experiment has confirmed that our sweet protein can exert a certain degree of sweetness function, further WB and other molecular level experiments are still needed for verification(Figure 24).

![](https://static.igem.wiki/teams/5160/wet-lab/result/laser4x2tobacco2.png)

**Figure 24. Confocal microscopy results image of tobacco.
Red: ChlorophyII autofluorescence (to indicate cytoplasmic location); Green: EGFP**

## Application on Tomatoes

However, we also know that our ultimate goal is to allow the sweet protein in transgenic tomatoes to be localized in the vacuoles of fruit cells for storage to increase the expression of sweet protein. In order to further verify the feasibility of SPS-NTPP localization peptides in tomato fruits, and considering the complexity of transgenic plant culture, after team discussion, we decided to conduct a preliminary leaf infiltration experiment in tomatoes to obtain a general understanding of the SPS-NTPP guidance for sweet protein localization. For more details, please see the [**Outline**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27outline%27%29%20%7D%7D) webpage.

[ ]

References

[1] Singh, S. M., & Panda, A. K. (2005). Solubilization and refolding of bacterial inclusion body proteins. Journal of bioscience and bioengineering, 99(4), 303-310.
[2] Refolding the sweet-tasting protein Thaumatin II from insoluble inclusion bodies synthesised in Escherichia coli.Food Chemistry Volume 71, Issue 1, October 2000.
[3] Joseph JA, Akkermans S, Nimmegeers P, Van Impe JFM. Bioproduction of the Recombinant Sweet Protein Thaumatin: Current State of the Art and Perspectives. Front Microbiol. 2019 Apr 8;10:695. doi: 10.3389/fmicb.2019.00695. PMID: 31024485; PMCID: PMC6463758.
[4] Transgenic Plants as Producers of Supersweet Protein Thaumatin II, Living reference work entry, 19 August 2016.
[5] Hirai T, Kim YW, Kato K, Hiwasa-Tanase K, Ezura H. Uniform accumulation of recombinant miraculin protein in transgenic tomato fruit using a fruit-ripening-specific E8 promoter. Transgenic Res. 2011 Dec;20(6):1285-92. doi: 10.1007/s11248-011-9495-9. Epub 2011 Feb 27. PMID: 21359850.
[6] Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113-30. doi: 10.1016/0022-2836(86)90385-2. PMID: 3537305.
[7]An E8 promoter-HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin, Original Paper, Published:11 January 2013.
[8] Ohta K, Masuda T, Tani F, Kitabatake N. The cysteine-rich domain of human T1R3 is necessary for the interaction between human T1R2-T1R3 sweet receptors and a sweet-tasting protein, Thaumatin. Biochem Biophys Res Commun. 2011 Mar 18;406(3):435-8. doi: 10.1016/j.bbrc.2011.02.063. Epub 2011 Feb 15. PMID: 21329673.
[9]Jill Deikman, Randy Kline, Robert L. Fischer, Organization of Ripening and Ethylene Regulatory Regions in a Fruit-Specific Promoter from Tomato (Lycopersicon esculentum) , Plant Physiology, Volume 100, Issue 4, December 1992.
[10] Lamia Azzi, Cynthia Deluche, Frédéric Gévaudant, Nathalie Frangne, Frédéric Delmas, Michel Hernould, Christian Chevalier, Fruit growth-related genes in tomato, Journal of Experimental Botany, Volume 66, Issue 4, February 2015.
[11] Marin Viegas VS, Ocampo CG, Petruccelli S. Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields. Bioengineered. 2017;8(3):203-211. doi:10.1080/21655979.2016.1222994
[12] Quinet M, Angosto T, Yuste-Lisbona FJ, Blanchard-Gros R, Bigot S, Martinez J-P and Lutts S (2019) Tomato Fruit Development and Metabolism. Front. Plant Sci. 10:1554.
[13] Jha S, Agarwal S, Sanyal I, Jain GK, Amla DV. Differential subcellular targeting of recombinant human α₁-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants. Plant Sci. 2012;196:53-66. doi:10.1016/j.plantsci.2012.07.004
[14] Jill Deikman, Randy Kline, Robert L. Fischer, Organization of Ripening and Ethylene Regulatory Regions in a Fruit-Specific Promoter from Tomato (Lycopersicon esculentum) , Plant Physiology, Volume 100, Issue 4, December 1992.

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