iGEM 2025 — Team cjuh-jlu-china

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{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
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![](https://static.igem.wiki/teams/5542/notebook/notebook-cover.webp)

![](https://static.igem.wiki/teams/5542/home/home/label/notebook-label.webp)

* [Wet Lab
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#notebook-wetlab)

  [December
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wetlab-dec)
  [January
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wetlab-jan)
  [February
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wetlab-feb)
  [March
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wetlab-mar)
  [April
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wetlab-apr)
  [May
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wetlab-may)
  [June
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wetlab-jun)
  [July
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wetlab-jul)
  [August
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wetlab-aug)
  [September
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wetlab-sep)
* [Dry Lab
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#notebook-model)

  [December
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-model-dec)
  [January
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-model-jan)
  [February
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-model-feb)
  [March
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-model-mar)
  [April
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-model-apr)
  [May
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-model-may)
  [June
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-model-jun)
  [July
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-model-jul)
  [August
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-model-aug)
  [September
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-model-sep)
* [HP & Edu
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#notebook-hpedu)

  [December
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-hpedu-dec)
  [January
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-hpedu-jan)
  [February
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-hpedu-feb)
  [March
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-hpedu-mar)
  [April
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-hpedu-apr)
  [May
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-hpedu-may)
  [June
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-hpedu-jun)
  [July
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-hpedu-jul)
  [August
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-hpedu-aug)
  [September
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-hpedu-sep)
* [Wiki
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#notebook-wiki)

  [December
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wiki-dec)
  [January
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wiki-jan)
  [February
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wiki-feb)
  [March
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wiki-mar)
  [April
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wiki-apr)
  [May
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wiki-may)
  [June
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wiki-jun)
  [July
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wiki-jul)
  [August
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wiki-aug)
  [September
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-wiki-sep)
* [Presentation
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#notebook-pre)

  [December
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-pre-dec)
  [January
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-pre-jan)
  [February
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-pre-feb)
  [March
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-pre-mar)
  [April
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-pre-apr)
  [May
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-pre-may)

  [July
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-pre-jul)
  [August
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-pre-aug)
  [September
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#notebook-pre-sep)

![](https://static.igem.wiki/teams/5542/img/reel-lower.webp)
![](https://static.igem.wiki/teams/5542/img/pen-bg.webp)

Wet Lab
![](https://static.igem.wiki/teams/5542/img/h1-background.webp)
![](https://static.igem.wiki/teams/5542/img/h1-leave-left.webp)
![](https://static.igem.wiki/teams/5542/img/h1-leave-right.webp)

December

Experiment team member recruitment

*2024.12.14-2024.12.22*

---

Upon the completion of our recruitment process, we are proud to introduce our group members:
Wet Lab Group: Yuetong Ge, Qiyu Tan, Yueting Guo, Ziyan Qin, Qiuyan Wang, Mingcong Li, Hepeng Wang.

January

The team's first general meeting

*2025.1.4*

---

At our first general meeting, all members met each other for the first time. Led by our PI and team leader, we introduced ourselves and clarified the upcoming tasks.

![](https://static.igem.wiki/teams/5542/notebook/exp/dec-1a.webp)

Figure 1: Meeting on 1.4, we got to know each other and confirmed what to do next.

Sharing of Gold Medal Cases in Previous Years

*2025.1.4-2025.1.27*

---

We had 3 meetings online in total. Each member of the experiment group share one gold medal project which they thought was a example of a good iGEM project,and analysed the advantages and highlights of the project. In this process, we’d come to learn what is a good iGEM project.

February

Brainstorms of the project

*2025.2.9-2025.2.14*

---

Since we decided to develop a breast cancer detecting project, we began searching for available methods that could make our project possible and then looking for more papers on breast cancer to improve our understanding of the disease.

Collaborative interviews

*2025.2.15-2025.2.22*

---

During this period, we conducted extensive research with the HP team to understand the needs and perspectives of both the general public and medical professionals regarding breast cancer detection. This led us to design a breast cancer recurrence monitoring system capable of maintaining long-term functionality in vivo while providing detectable readouts ex vivo.

Background research

*2025.2.15-2025.2.22*

---

We found studies in the literature on the related properties of cancer-associated adipocytes (CAAs), which suggested that adipocytes have a function in response to changes in their surrounding breast cancer and may serve as a chassis for detection systems. We began an extensive literature search to understand the relationship between CAA and breast cancer. We also collect different sensing technology that can may be compatible with our objectives and communicate with model group to try to find the targets.

Collaborative interviews

*2025.2.23-2025.2.28*

---

To investigate whether adipocytes could serve as our engineering chassis cells, we collaborated with the HP group to conduct interviews with surgeons. These interviews focused on the application of autologous fat grafting technology in post-mastectomy reconstruction and breast augmentation surgeries. The positive feedback from our collaborative interviews with the HP group, which explored the clinical use of adipocytes in procedures like breast reconstruction, confirmed their suitability. This confirmation led us to finally select adipocytes as our engineering chassis cells and proceed with the investigation into biomarkers and output signals.

Background research

*2025.2.23-2025.2.28*

---

Our investigation into Cancer-Associated Adipocytes (CAAs) revealed that the transformation from normal adipocytes to CAAs involves the specific upregulation of multiple genes. During our survey of reporter genes, we identified two potential methods for ex vivo signal output: Gaussia Luciferase (Gluc) and melanin. Based on these findings, we began searching for a detection tool capable of sensing the upregulation of endogenous mammalian genes and featuring an editable signal output module.

March

Collaborative interviews

*2025.3.1-2025.3.10*

---

Following discussions with the HP group, we finalized Gluc as the output method for our detection system and, acting on interviewees' recommendations, initiated extensive literature research to preliminarily identify target genes that are specifically upregulated in response to factors secreted by breast cancer cells.

During this phase, we have selected four candidate target genes: PLOD2 (upregulated by PAI-1), LIF (upregulated by CXCLs), Fam3c (upregulated by TGF-β), and IL-6 (upregulated by IL-1β).

Find suitable detection system

*2025.3.1-2025.3.10*

---

Through literature research, we have identified three detection techniques suitable for our project.We selected RADAR (adenosine deaminase based programmable RNA sensor technology), CASP sensor (RNA sensing technology based on CASP high sensitivity to detect single point mutations) and PAS aptamer (aptamer based detection technology for small molecule proteins) to achieve the detection of high-expressed genes in CAAs.

Find the best group of target gene

*2025.3.11-2025.3.30*

---

Collaborative interviews with the HP group highlighted accuracy as the most critical factor in diagnostic projects. Consequently, we decided to adopt the RADAR technology-capable of simultaneous multi-gene detection-as our core methodology to achieve precise breast cancer detection. Building on this foundation, we partnered with the Modeling team to conduct screening analyses of different gene combinations, ultimately identifying PLOD2 and LIF as our target genes for detection.

Design of experiments at the stage of proof of concept

*2025.3.11-2025.3.30*

---

Upon finalizing the RADAR system as the core of our project, we conducted a thorough literature review and, based on the references, completed the experimental design for the proof-of-concept phase along with the construction of plasmid maps. Accordingly, we have compiled a comprehensive list of required experimental materials.

Finalized the project

*2025.3.11-2025.3.30*

---

We have reviewed our overall progress at this stage and improved the general introduction about the project and clarified the background, solution and application scenarios of the project.

April

Materials purchase

*2025.4.1-2025.4.20*

---

Under the guidance of our PI, we have already completed the plan of the proof-of-concept experiment. With the help of our instructor, we purchase the respective meterials in our proof-of-concept experiment plan.

Design the further experiment plan

*2025.4.1-2025.4.20*

---

Different students in the wet lab group were responsible for different parts. Yuetong Ge and Hepeng Wang carried out the design of RADAR. Qiyu Tan organized all of the parts we were going to use. Qiuyan Wang carried out the design of cell viability detection. Ziyan Qin finished the design of Gluc production as the output part. Mingcong Li and Yueting Guo carried out the design of verifying the target genes of adipocytes up-regulated by breast cancer secreted cytokines.

![](https://static.igem.wiki/teams/5542/notebook/exp/apr-fig1.webp)

Figure 1: Team members shared their ideas on the group meeting

Materials purchase

*2025.4.20-2025.4.30*

---

We listed and purchased the required experimental materials according to the designed experimental plan.

Laboratory Safety Training

*2025.4.20-2025.4.30*

---

All members of experiment group traind together on laboratory safety.

May

## Plasmid preparation

### 2025.05.12 - 2025.05.17

Event 1: Transformation: plasmid 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9→DH5α

Event 1: Transformation: plasmid 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9→DH5α

Plasmids

For ease of recording, we numbered our plasmids according to the following table.

![Result 2](https://static.igem.wiki/teams/5542/notebook/exp/may-week1-0.webp)

This week we transformed plasmids 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 into DH5α.

Procedures

1. Prepare LB liquid medium according to the protocol: **LB liquid medium**
2. Dilute the Ampicillin (Amp) to the final concentration of 100mg/ml
3. Pour LB agar plates with 0.1mg/mL Amp
4. Dissolve 4μg plasmids in 20μL ddH2O
5. The transformation process follows protocol: **Bacterial Transformation**.
   We used 3μL of plasmid.
6. Spread 150 μL competent cells without transformation on the plates (Amp+) as negative control.
7. Pick single colonies from plates and culture in 5ml LB liquid medium with shaking at 220rpm for 6h.
8. Store the transformed DH5α in a system of 200 μL glycerin and 800 μL bacteria solution.

Results

![Result 1](https://static.igem.wiki/teams/5542/notebook/exp/may-week1-1.webp)

![Result 2](https://static.igem.wiki/teams/5542/notebook/exp/may-week1-2.webp)

![Result 3](https://static.igem.wiki/teams/5542/notebook/exp/may-week1-3.webp)

Discussions

* Untransformed DH5α couldn’t grow on the plates with Amp, which proves the effectiveness of antibiotic.
* Transformed bacteria grew well.
* Transformation is considered successful.

Event 2: Isolation: plasmid 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9

Event 2: Isolation: plasmid 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9

Plasmids

This week we isolated plasmids 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 3.1.

Procedures

1. Expand culture: Remove 1ml transformed DH5α solution into 200ml LB liquid medium with Amp, and shake at 220 rpm for 7h.
2. Isolate plasmids by Vigorous Maxiprep Kit following the protocol: **Vigorous Plasmid Maxiprep**.
3. Test the plasmid concentration by nano100.

Results

![Result 3](https://static.igem.wiki/teams/5542/notebook/exp/may-week1-4.webp)

Discussions

* The concentration of plasmid 1.1 is higher then expected, we plan to perform electrophoresis to verify if the plasmids we extracted are correct.

### 2025.05.19 - 2025.05.24

Event 1: Digestion and agarose gel electrophoresis: plasmid 1.3, 1.4, 1.6, 1.7, 1.8, 1.9, 1.1, 1.2, 1.5

Event 1: Digestion and agarose gel electrophoresis: plasmid 1.3, 1.4, 1.6, 1.7, 1.8, 1.9, 1.1, 1.2, 1.5

We digested plasmid 1.1-1.9 and performed agarose gel electrophoresis twice to verify that the plasmids were successfully transformed and isolated.

Round 1: Tested plasmid 1.3, 1.4, 1.6, 1.7, 1.8, 1.9

Round 2: Tested plasmid 1.1, 1.2, 1.5

In Round 1 we noticed some of the plasmids' concentration were lower than we expected, so we configured 2 systems in Round 2 to ensure the concentration of our plasmids.

Procedures

1. We perform digestion according to the protocol: **Restriction Digest of Plasmid DNA**. The digestion systems are listed below: (the arrows in the "Plasmid Concentration" column indicate dilution, for example, 8200→600 means dilute the plasmids from 8200ng/μL to 600ng/μL)

![Result 2](https://static.igem.wiki/teams/5542/notebook/exp/plasmid/519524-1-1.avif)

Table1: Digestion system of Round 1 (plasmid 1.3, 1.4, 1.6, 1.7, 1.8, 1.9)

![Result 2](https://static.igem.wiki/teams/5542/notebook/exp/519524-1-2.webp)

Table2: Digestion system of Round 2 (plasmid 1.1, 1.2, 1.5)

2. We perform agarose gel electrophoresis following the protocol: **Agarose gel electrophoresis (DNA)**

   In Round 1 the concentration of agarose is 0.83%. In Round 2 the concentration of agarose is 1%.

   4μL 6x loading buffer was added into every 20μL sample.

   Run the gel at 135V for 40min.

Results

![Result 1](https://static.igem.wiki/teams/5542/notebook/exp/may-week2-8.webp)

![Result 1](https://static.igem.wiki/teams/5542/notebook/exp/may-week2-6-2.webp)

Figure 1: Profile of Round 1 (DNA mass 600ng)

![Result 2](https://static.igem.wiki/teams/5542/notebook/exp/may-week2-9.webp)

![Result 2](https://static.igem.wiki/teams/5542/notebook/exp/may-week2-7-2.webp)

Figure 2: Profile of Round 2 (DNA mass 600ng)

Discussions

* Round 1:
   The results of round 1 are basically correct. There could be something wrong with the gel, making the marker not straight enough.
* Round 2:
   The bands of p1.1 are correct and the concentration seems to be right.

  There wasn’t any band of p1.2 on the gel, so we plan to extract p1.2 one more time.

  Some of the bands of p1.5 are correct, but there were indeterminate bands,so p1.5 needs to be further verified.

Event 2: Transformation: plasmid 3.1→DH5α

Event 2: Transformation: plasmid 3.1→DH5α

This week we transformed plasmids 3.1 into DH5α.

Procedures

1. Procedures were the same as described in week1

Results

![Result 3](https://static.igem.wiki/teams/5542/notebook/exp/may-2-10-1009.avif)

Discussions

* Untransformed DH5α couldn’t grow on the plates (Amp+), which proves the effectiveness of ampicillin.
* Transformed bacteria grew well.

Event 3: Isolation: plasmid 1.2, 1.5, 3.1

Event 3: Isolation: plasmid 1.2, 1.5, 3.1

This week we isolated plasmid 1.2, 1.5, 3.1 in DH5α.

Procedures

1. Procedures were the same as described in week1

Results

![Result 3](https://static.igem.wiki/teams/5542/notebook/exp/plasmid/519524-1-3.avif)

Discussions

* All the plasmids seem to be extracted successfully and need further verification.

June

### 2025.06.02

Event 1: Digestion and agarose gel electrophoresis: plasmid 1.2, 1.5, 3.1

Event 1: Digestion and agarose gel electrophoresis: plasmid 1.2, 1.5, 3.1

We performed agarose gel electrophoresis to confirm that the previous operations on plasmid 1.2, 1.5, 3.1 were successful.

Procedures

1. We perform digestion according to the protocol: Restriction digestion. The restriction digestion systems are as follows:

(The arrows in the "Plasmid Concentration" column indicate dilution, for example, 8200→600 means dilute the plasmids from 8200ng/μL to 600ng/μL)

![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/plasmid/6-1.avif)

Table1: Digestion system of plasmid 1.2, 1.5, 3.1

2. We perform agarose gel electrophoresis following the protocol: **Agarose gel electrophoresis**.

   The concentration of the agarose gel is 1%.

   4μL 6x loading buffer is added in 20μL sample.

   Run the gel at 135V for 45min.

Results

![Electrophoresis result](https://static.igem.wiki/teams/5542/notebook/exp/plasmid/6-1r.avif)

Figure 2: Profile of digestion (DNA mass 600ng)

![Electrophoresis result](https://static.igem.wiki/teams/5542/notebook/exp/plasmid/6-1rr.avif)

Discussions

* The bands of plasmid 1.2 are correct, available to perform subsequent experiments.
* Plasmid 1.5, 3.1 have unspecific bands, which indicates that these two plasmids may contain impurities.

July

## Plasmid preparation

### 2025.07.01-2025.07.07

Event 1: Digestion and agarose gel electrophoresis: plasmid 1.1, 1.3, 1.6, 1.9

Digestion and agarose gel electrophoresis: plasmid 1.1, 1.3, 1.6, 1.9

We digested the plasmids and performed agarose gel electrophoresis in 3 rounds, respectfully on 05/July, 06/July and 07/July.

**- Round 1: Tested plasmid 1.1, 1.6**

**- Round 2: Tested plasmid 1.1, 1.3, 1.6, 1.9**

**- Round 3: Tested plasmid 1.1, 1.3, 1.6, 1.9**

Procedures

1. We perform digestion according to the protocol: Restriction Digest. The digestion systems are listed below: (the arrows in the "Plasmid Concentration" column indicate dilution, for example, 8200→600 means dilute the plasmids from 8200ng/μL to 600ng/μL)

   ![](https://static.igem.wiki/teams/5542/notebook/exp/jul-week1-1.webp)

   Table1: Digestion system of Round 1(plasmid 1.1, 1.6)

   ![](https://static.igem.wiki/teams/5542/notebook/exp/jul-week1-2.webp)

   Table2: Digestion system of Round 2(plasmid 1.1, 1.3, 1.6, 1.9)

   ![](https://static.igem.wiki/teams/5542/notebook/exp/jul-week1-3-2.webp)

   Table3: Digestion system of Round 3(plasmid 1.1, 1.3, 1.6, 1.9)

   (1) Digest under 37°C for 2h.

   (2) Each systems contain 600ng plasmids.

   (3) All systems are supplemented with ddH2O to 20μL.

Results

![Result 3](https://static.igem.wiki/teams/5542/notebook/exp/july-week1-1.webp)

![Result 3](https://static.igem.wiki/teams/5542/notebook/exp/july-week1-3.webp)

![Result 3](https://static.igem.wiki/teams/5542/notebook/exp/july-week1-5.webp)

![](https://static.igem.wiki/teams/5542/notebook/exp/jul-week1-2-2.webp)

Figure 1: Profile of round 1

![](https://static.igem.wiki/teams/5542/notebook/exp/jul-week1-4-2.webp)

Figure 2: Profile of round 2

![](https://static.igem.wiki/teams/5542/notebook/exp/jul-week1-6-2.webp)

Figure 3: Profile of round 3

Discussions

* **Round 1:**

  We suspect that the loss of plasmid during the digestion may be the reason why bands 1.1, 1.6 are not visible.
* **Round 2:**
  The electrophoresis results for samples 1.3, 1.6, and 1.9 are clear and correct. For sample 1.1, we suspect that concentration of 1.1 is too low.

### 2025.07.08-2025.07.14

Event 1: Isolation: plasmid 1.2, 1.4, 1.8, 1.9

Event 1: Isolation: plasmid 1.2, 1.4, 1.8, 1.9

We cultured bacteria and used maxiprep kit to extract more.

Procedures

1. Added 900μL bacteria solution stored with glycerin to 200mL LB liquid medium (Amp+) and 100μL to 5mL LB liquid medium (Amp+), shake both at 220rpm for 8h. After centrifugation at 8000rpm for 5 min, we harvested the bacteria precipitate.
2. Isolate plasmids by Vigorous Maxiprep Kit following the protocol: **Vigorous Plasmid Maxiprep**.
3. Test the plasmid concentration by nano100.

Results

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/july-week2-1.webp)

Event 2: Repeat isolation: plasmid 1.2, 1.4

Event 2: Repeat isolation: plasmid 1.2, 1.4

We extracted plasmid 1.2, 1.4 for a second time.

Procedures

1. Add 900μL stored bacteria solution to 200mL LB (Amp+) and 100ul bacteria solution to 5mL LB (Amp+), then shake at 220rpm, 37°C overnight.
2. Centrifuge at 8000rpm for 5 min.
3. Isolate plasmids by Vigorous Maxiprep Kit following the protocol: **Vigorous Plasmid Maxiprep**.
4. Test the plasmid concentration by nano100.

Results

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/july-week2-2.webp)

Event 3: Digestion and agarose gel electrophoresis: plasmid 1.1, 3.1, 1.8, 1.9, 1.4

Event 3: Digestion and agarose gel electrophoresis: plasmid 1.1, 3.1, 1.8, 1.9, 1.4

We digested the plasmids and performed agarose gel electrophoresis twice.

**- Round 1: Tested plasmid 1.1, 3.1, 1.8, 1.9**

**- Round 2: Tested plasmid 1.4**

Procedures

1. We perform digestion according to the protocol: **Restriction Digestion**. The digestion systems are listed below:

   ![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/jul-2-3-3.avif)

   Table1: Digestion system of Round 1 (plasmid 1.1, 3.1, 1.8, 1.9)

   ![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/jul-week2-4-2.webp)

   Table2: Digestion system of Round 2 (plasmid 1.4)

   Digest 600ng plasmids in 20μL system at room temperature overnight.
2. We perform agarose gel electrophoresis following the protocol: **Agarose Gel Electrophoresis**.

   Concentration of the gel is 1%.

   Run the gel at 135V for 40min.

Results

![Result 1](https://static.igem.wiki/teams/5542/notebook/exp/july-week2-5.webp)

Figure 1: Profile of Round 1 (DNA mass 600ng)

![Result 2](https://static.igem.wiki/teams/5542/notebook/exp/july-week2-7.webp)

Figure 2: Profile of Round 2 (DNA mass 600ng)

![Result 1](https://static.igem.wiki/teams/5542/notebook/exp/jul-week2-6-2.webp)

![Result 2](https://static.igem.wiki/teams/5542/notebook/exp/jul-week2-8-2.webp)

### 2025.07.15-2025.07.21

Event 1: Transformation: plasmid 1.2→DH5α

Event 1: Transformation: plasmid 1.2→DH5α

This week we transformed plasmid 1.2 into DH5α.

Procedures

1. Add 600ng plasmids (3μL) to 50μL competent cells.
2. The transformation process follows protocol: **Bacterial Transformation**.
3. After transformation, spread 150μL bacteria solution on one plate (Amp+) and 200μL on another.
4. Spread 200μL competent cells without transformation as a control to test the effectiveness of Ampicillin.

Event 2: Isolation: plasmid 1.2

Event 2: Isolation: plasmid 1.2

We extracted plasmid 1.2 for the third time.

Procedures

1. We picked 4 colonies (2 from each plates) and shake in 5mL LB liquid medium at 220rpm for 6.5h, then stored the bacteria solution with glycerin.
2. We add 1mL bacteria solution from each tube (from 200μL plates) to 200mL LB liquid medium and shake at 220rpm overnight.
3. Isolate plasmids by Vigorous Maxiprep Kit following the protocol: **Vigorous Plasmid Maxiprep**.
4. Test the plasmid concentration by nano100.

Results

* The concentration of plasmid 1.2 is 335ng/μL.

Event 3: Digestion and agarose gel electrophoresis: plasmid 1.2

Event 3: Digestion and agarose gel electrophoresis: plasmid 1.2

We digested the plasmid 1.2 and performed agarose gel electrophoresis.

Procedures

1. Digest the plasmids at room temperature overnight according to the following system:

   ![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/jul-week3-1-2.webp)

   Table1: Digestion system of plasmid 1.2
2. We perform digestion according to the protocol: **Restriction Digestion**.
3. We perform agarose gel electrophoresis following the protocol: **Agarose Gel Electrophoresis**.

   Concentration of the gel is 1%.

   Run the gel at 135V for 40min.

Results

![Result](https://static.igem.wiki/teams/5542/notebook/exp/july-week3-2.webp)

Figure 1: Profile of digestion (DNA mass 600ng)

![Result](https://static.igem.wiki/teams/5542/notebook/exp/jul-week3-3-2.webp)

### 2025.07.22-2025.07.28

Event 1: Transformation: plasmid 1.10,1.11→DH5α

Event 1: Transformation: plasmid 1.10,1.11→DH5α

This week we transformed plasmid 1.10,1.11 into DH5α

Procedures

1. Add 600ng plasmids (3μL) to 50μL competent cells.
2. The transformation process follows protocol: **Bacterial Transformation**.
3. After transformation, spread 150μL bacteria solution on one plate (Amp+) and 200μL on another.
4. Spread 200μL competent cells without transformation as a control to test the effectiveness of Ampicillin.

Event 2: Isolation: plasmid 1.10, 1.11

Event 2: Isolation: plasmid 1.10, 1.11

We extracted plasmid 1.10,1.11 for the first time.

Procedures

1. We picked 4 colonies (2 from each plates) and shake in 5mL LB liquid medium at 220rpm for 6.5h, then stored the bacteria solution with glycerin.
2. We add 1mL bacteria solution from each tube (from 200μL plates) to 200mL LB liquid medium and shake at 220rpm overnight.
3. Isolate plasmids by Vigorous Maxiprep Kit following the protocol: **Vigorous Plasmid Maxiprep**.
4. Test the plasmid concentration by nano100.

Results

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/plasmid/july-week4-1.avif)

Event 3: Digestion and agarose gel electrophoresis: plasmid 1.10, 1.11

Event 3: Digestion and agarose gel electrophoresis: plasmid 1.10, 1.11

We digested the plasmids 1.10,1.11 and performed agarose gel electrophoresis.

Procedures

1. Digest the plasmids at room temperature overnight according to the follow system:

   ![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/july-week4-2.webp)

Results

![Result](https://static.igem.wiki/teams/5542/notebook/exp/july-week4-3.webp)

Figure 1: Profile of digestion (DNA mass 600 ng)

![Result](https://static.igem.wiki/teams/5542/notebook/exp/jul-week4-4-2.webp)

## Proof of concept in HEK293T

### 2025.07.16-2025.07.21

Event 1: Cell revival of HEK293T

Event 1: Cell revival of HEK293T

We revived HEK293T cells for future transfection experiments.

Procedures

1. We prepared complete DMEM medium following protocol: **Complete DMEM Medium.**

   (1)Remove 50 mL from a 500 mL bottle of DMEM medium and store it separately as serum-free medium.

   (2)Filter 50 mL FBS and 5 mL penicillin-streptomycin into the remaining 450 mL DMEM medium using a 0.22 μm filter membrane.

   (3)Mix well and store at 4°C.
2. Retrieve HEK293T cells from liquid nitrogen storage.
3. Thaw cells rapidly in a 37°C water bath, then immediately transfer to 5 mL complete DMEM medium and mix gently.
4. Centrifuge at 1000 rpm, 4°C for 5 minutes, then discard the supernatant.
5. Resuspend the pellet in 5 mL PBS using a pipette, then centrifuge again at 1000 rpm, 4°C for 5 minutes.
6. Discard the supernatant and resuspend cells in 1 mL complete DMEM medium.
7. Transfer the suspension into a 10 cm culture dish containing 9 mL complete DMEM and incubate overnight at 37°C with 5% CO2.

Event 2: Cell passage of HEK293T

Event 2: Cell passage of HEK293T

We passaged HEK293T cells for future transfection experiments.

Procedures

1. Take out the cells from incubator after culturing for 1 day since revival, aspirate the old culture medium, add 2 mL of PBS, and gently wash the cells.
2. Discard the supernatant, add 1 mL of PBS and 1 mL of trypsin to the culture dish, and digest at room temperature for 1 minute.
3. Discard the supernatant, return the old medium aspirated in step 1 to the culture dish, and resuspend the cells.
4. Evenly transfer the cell suspension into one 15 mL tube, and centrifuge at 1000 rpm, 4°C for 5 minutes.
5. Discard the supernatant, resuspend the cells in 5 mL of PBS, and centrifuge at 1000 rpm, 4°C for 5 minutes.
6. Discard the supernatant, resuspend the cells in 1 mL of complete DMEM medium per tube, and evenly distribute the resulting cell suspension into three culture dishes pre-filled with 9 mL of complete DMEM medium.
7. Incubate at 37°C with 5% CO2.

Event 3: Cell seeding of HEK293T

Event 3: Cell seeding of HEK293T

We seed HEK293T cells in 24-well plates for subsequent transfection.

Procedures

1. Take out the culture dishes containing cells passaged at 1:3 ratio yesterday. Aspirate spent medium, wash with 2 mL PBS, then add 1 mL PBS and 1 mL trypsin for 1-minute digestion at 37°C.
2. Adding back the original conditioned medium. Gently dissociate cells by pipetting.
3. Transfer the cell suspension from all dishes to 15 mL tubes. Centrifuge at 1000xg, 4°C for 5 minutes.
4. Aspirate supernatant, resuspend pellets in 5 mL PBS per tube, then centrifuge again at 1000xg, 4°C for 5 minutes.
5. After final supernatant removal, resuspend each cell pellet in 1 mL complete DMEM medium.
6. Take 200μl from one cell suspension tube and dilute in 24 mL complete DMEM. Dispense 500 μl aliquots into each well of two 24-well plates (total 48 wells).
7. Incubate at 37°C with 5% CO2.
8. We passage 1 dish of cells and preserve the residual cells in freezing medium (90% FBS + 10% DMSO).

Event 4: Transfection: system1(plasmid 1.1, 1.2, 1.3, 1.8, 1.9)→HEK293T

Event 4: Transfection: system1(plasmid 1.1, 1.2, 1.3, 1.8, 1.9)→HEK293T

We transfected system1 (p1.1,1.2,1.3,1.8,1.9) to HEK293T cells and performed florescence imaging of EGFP and mCherry in transfected cells to explore the effectiveness of the RADAR system.

Procedures

1. We transfect plasmids into HEK293T cells as follows, Set up three replicate wells for each group:

   ![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/july-week3-1.webp)

   Table 1. List of transfection system
2. Transfection procedures follow protocol: **Cell transfection.**
3. Perform fluorescence imaging of EGFP and mCherry in post-transfectHEK293T cells.

Results

* All groups transfected with Sensor 1/Sensor 1-UGG showed strong red fluorescence, while the Sensor 1-UGG group also showed intense green fluorescence, confirming high transfection efficiency and proper functionality of mCherry and EGFP. However, transfection with Trigger 1 failed to induce EGFP expression in Sensor 1. We hypothesize that this may be due to endogenous TetR gene expression in the HEK293T cells, which could act on the TetO sequence preceding the Triggers and suppress Trigger expression. Alternatively, function of Sensor 1 might be weaker than anticipated, resulting in EGFP expression levels below the detection limit of fluorescence microscopy. Consequently, we plan to proceed with Sensor 2 and include tetracycline induction during transfection to determine the exact cause of the experimental failure.

### 2025.07.22-2025.07.28

Event 1: Cell passage of HEK293T

Event 1: Cell passage of HEK293T

We passaged HEK293T cells for future transfection experiments.

Procedures

1. We routinely passaged HEK293T cells at a 1:3 ratio every other day to maintain cell viability for subsequent experiments.

Event 2: Cell seeding of HEK293T

Event 2: Cell seeding of HEK293T

We seed HEK293T cells in 24-well plates for subsequent transfection.

Procedures

1. Procedures were the same as the seeding process of **July Week3**

   We seed one 24-well plate using 200μL cell resuspension.

Event 3: Transfection: system2(plasmid 1.1, 1.4, 1.5, 1.8, 1.9)→HEK293T

Event 3: Transfection: system2(plasmid 1.1, 1.4, 1.5, 1.8, 1.9)→HEK293T

We transfected system2 (p1.1,1.4,1.5,1.8,1.9) to HEK293T cells and performed florescence imaging of EGFP and mCherry in transfected cells to explore the effectiveness of the RADAR system.

Procedures

1. We transfected plasmids into HEK293T cells according to the following systems below, and all treatments are performed in three replicate wells:

   ![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/july-week4-1.webp)

   Table 1. List of transfection system
2. Prior to transfection, we replaced the old culture medium with fresh medium containing tetracycline.
3. After transfection, we replace the medium on the following day. The concentration of tetracycline remains the same.
4. Take photographs under 10× magnification in three different light conditions: mCherry excitation light, EGFP excitation light and brightfield (daylight).

Results

* The transfection results were similar to the previous experiment, as we still didn't observed Trigger-induced EGFP expression. Literature research revealed that this issue might be attributed to the high degradation rate of tetracycline. Consequently, we re-ordered doxycycline, which is more suitable for inducing the TetO system, and simultaneously performed amplification of Trigger plasmids (1.10, 1.11) lacking the TetO element.

### 2025.07.29-2025.08.04

Event 1: Cell passage of HEK293T

Event 1: Cell passage of HEK293T

We passaged HEK293T cells for future transfection experiments.

Procedures

1. We routinely passaged HEK293T cells at a 1:3 ratio every other day to maintain cell viability for subsequent experiments.

Event 2: Cell seeding: HEK293T

Event 2: Cell seeding: HEK293T

We seed HEK293T cells in two 24-well plates for subsequent transfection.

Procedures

1. Procedures were the same as the seeding process in **Week1**.
   For 24 well plate, we add 100μL cell suspension to 6mL complete DMEM medium and add 500ul diluted cell suspension to each well,8 wells in total.
2. We use the rest cells for passaging.

Event 3: Transfection: plasmid 3.1→HEK293T

Event 3: Transfection: plasmid 3.1→HEK293T

We transfected HEK293T cells with plasmid 3.1 to express the Gaussia luciferase (Gluc).

Procedures

1. We transfect HEK293T cells with plasmid 3.1 as follows:

   ![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/july-week5-1.webp)
2. Transfection procedures follow protocol: **Cell transfection**.
   (1)On the following 2 days, we collect the conditioned medium and replace with the fresh medium each day. The conditioned medium was collected in 1.5mL EP tube and centrifuge at 1000rpm for 5 minutes to get the supernatant.
   (2)The suprenatant was then stored at -80°C for further detection.

Event 4: Culture medium collection and Gluc detection: plasmid 3.1→ HEK293T

Event 4: Culture medium collection and Gluc detection: plasmid 3.1→ HEK293T

Procedures

1. Prepared the Gaussia-Lumi™ luciferase assay working solution by mixing the substrate with buffer at a 1:100 ratio.
2. Add 100 μL of collected supernatant and 100 μL working solution to each well of the 96-well plate.
3. Measure the chemiluminescence immediately using a microplate reader.
4. Also, we use chemiluminescence imaging analysis system and cellphone to capture the luminescence images.

Results

![Result](https://static.igem.wiki/teams/5542/notebook/exp/july-week5-2.webp)

Figure 1: Luminescence of culture supernatant from HEK293T cells collected at 24h, 48h and 72h after transfection.

![Result](https://static.igem.wiki/teams/5542/results/results-comparison-of-sustained-luminescence.webp)

Figure 2: Analysis of sustained luminescence over 25 minutes between kinetics of luminescence production from Gluc.

August

## Plasmid preparation

### 2025.08.01-2025.08.07

Event 1: Isolation: plasmid 1.6,1.7,1.10,1.11

Event 1: Isolation: plasmid 1.6,1.7,1.10,1.11

We extracted plasmid 1.6,1.7 for the first time and plasmind 1.10,1.11 for a second time.

Procedures

1. We picked 4 colonies (2 from each plates) and shake in 5mL LB liquid medium at 220rpm for 6.5h, then stored the bacteria solution with glycerin.
2. We add 1mL bacteria solution from each tube (from 200μL plates) to 200mL LB liquid medium and shake at 220rpm overnight.
3. Isolate plasmids by Vigorous Maxiprep Kit following the protocol: **Vigorous Plasmid Maxiprep**.
4. Test the plasmid concentration by nano100.

The concentration of plasmid maxiprep are as follows:

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/plasmid/aug-week1-1.avif)

Event 2: Digestion and agarose gel electrophoresis: plasmid 1.6,1.7,1.10,1.11

Event 2: Digestion and agarose gel electrophoresis: plasmid 1.6,1.7,1.10,1.11

We digested the plasmids and performed agarose gel electrophoresis in 2 rounds.
Round 1: Tested plasmid 1.6,1.11
Round 2: Tested plasmid 1.6,1.7,1.10

Procedures

1. We digest the plasmids at room temperature overnight according to the follow system:

   ![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/8-1-2-1-1.webp)

   ![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/8-1-2-2-1.webp)

Results

![Result](https://static.igem.wiki/teams/5542/notebook/exp/8-1-2-1-r.avif)

Figure 1: Profile of round1 (DNA mass 600 ng)

![Result](https://static.igem.wiki/teams/5542/notebook/exp/8-1-2-2-r.avif)

Figure 2: Profile of round2 (DNA mass 600 ng)

![Result](https://static.igem.wiki/teams/5542/notebook/exp/8-1-2-1-2.webp)

![Result](https://static.igem.wiki/teams/5542/notebook/exp/8-1-2-2-2.webp)

### 2025.08.08-2025.08.14

Event 1: Digestion and agarose gel electrophoresis: plasmid 1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,1.10,1.11,3.1

Event 1: Digestion and agarose gel electrophoresis: plasmid 1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,1.10,1.11,3.1

We digested the plasmids and performed agarose gel electrophoresis in 3 rounds.
Round 1: Tested plasmid 1.1,1.2,1.3,1.4
Round 2: Tested plasmid 1.3,1.4,1.5,1.6,1.7,1.8,1.9,1.10
Round 3: Tested plasmid 1.6,1.7,1.8,1.9,1.11,3.1

Procedures

1. We digest the plasmids at room temperature 1.5h according to the follow system:

   ![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/8-2-1-1-1.webp)

   ![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/8-2-1-2-1.webp)

   ![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/8-2-1-3-1.webp)

Results

![Result](https://static.igem.wiki/teams/5542/notebook/exp/8-2-1-1-r.avif)

Figure 1: Profile of round1 (DNA mass 600 ng)

![Result](https://static.igem.wiki/teams/5542/notebook/exp/8-2-1-2-r1.avif)

Figure 2.1: Profile of round2 (DNA mass 600 ng)

![Result](https://static.igem.wiki/teams/5542/notebook/exp/8-2-1-2-r2.avif)

Figure 2.2: Profile of round2 (DNA mass 600 ng)

![Result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week2-event1-round3-1.webp)

Figure 3.1: Profile of round3 (DNA mass 600 ng)

![Result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week2-event1-round3-2.webp)

Figure 3.2: Profile of round3 (DNA mass 600 ng)

![Result](https://static.igem.wiki/teams/5542/notebook/exp/8-2-1-1-2.webp)

![Result](https://static.igem.wiki/teams/5542/notebook/exp/8-2-1-2-2.webp)

![Result](https://static.igem.wiki/teams/5542/notebook/exp/8-2-1-3-2.webp)

Event 2: Transformation: plasmid 5.1→DH5α

Event 2: Transformation: plasmid 5.1→DH5α

This week we transformed plasmid 5.1 into DH5α

Procedures

1. Add 600ng plasmids (3μL) to 50μL competent cells.
2. The transformation process follows protocol: **Bacterial Transformation**.
3. After transformation, spread 150μL bacteria solution on one plate (Amp+) and 200μL on another.
4. Spread 200μL competent cells without transformation as a control to test the effectiveness of Ampicillin.

Event 3: Isolation: plasmid 5.1

Event 3: Isolation: plasmid 5.1

We extracted plasmid 5.1 for the first time.

Procedures

1. We picked 2 colonies ( from 150μL plate) and shake in 5mL LB liquid medium at 220rpm for 8h, then stored the bacteria solution with glycerin for 2 tubes.
2. We add 1mL bacteria solution from each tube (from 200μL plates) to 200mL LB liquid medium and shake at 220rpm overnight.
3. Isolate plasmids by Vigorous Maxiprep Kit following the protocol: Vigorous Plasmid Maxiprep.
4. Test the plasmid concentration by nano100.

Results

![](https://static.igem.wiki/teams/5542/notebook/exp/plasmid/aug-meow.avif)

### 2025.08.15-2025.08.21

Event 1: Transformation: plasmid 5.2,5.3,5.4→DH5α

Event 1: Transformation: plasmid 5.2,5.3,5.4→DH5α

This week we transformed plasmid 5.2,5.3,5.4 into DH5α

Procedures

1. Add 600ng plasmids (3μL) to 50μL competent cells.
2. The transformation process follows protocol: **Bacterial Transformation**.
3. After transformation, spread 150μL bacteria solution on one plate (Amp+) and 200μL on another.
4. Spread 200μL competent cells without transformation as a control to test the effectiveness of Ampicillin.

Event 2: Isolation: plasmid 1.10,1.11

Event 2: Isolation: plasmid 1.10,1.11

We extracted plasmind 1.10,1.11 for a third time.

Procedures

1. We add stored 1mL bacteria solution to 200mL LB liquid medium and shake at 220rpm overnight.
2. Isolate plasmids by Vigorous Maxiprep Kit following the protocol: **Vigorous Plasmid Maxiprep**.
3. Test the plasmid concentration by nano100.

The concentration of plasmid maxiprep are as follows:

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/aug-plasmid-add.avif)

Event 3: Digestion and agarose gel electrophoresis: plasmid 1.6,1.7,1.10,1.11,3.1,5.1

Event 3: Digestion and agarose gel electrophoresis: plasmid 1.6,1.7,1.10,1.11,3.1,5.1

We digested the plasmids and performed agarose gel electrophoresis twice.
Round 1: Tested plasmid 5.1,3.1
Round 2: Tested plasmid1.10,1.11,5.1
Round 3: Tested plasmid 1.6,1.7,1.10

Procedures

1. We digest the plasmids at room temperature overnight according to the follow system:

   ![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/8-3-2-1-1.webp)

   ![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/8-3-2-2-1.webp)

   ![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/8-3-2-3-1-6666.avif)

Results

![Result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week3-event2-round1.webp)

Figure 1: Profile of round1 (DNA mass 600 ng)

![Result](https://static.igem.wiki/teams/5542/notebook/exp/8-3-2-2-r.avif)

Figure 2: Profile of round2 (DNA mass 600 ng)

![Result](https://static.igem.wiki/teams/5542/notebook/exp/8-3-2-3-r-6666.avif)

Figure 3: Profile of round3 (DNA mass 600 ng)

![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/8-3-2-1-2.webp)

![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/8-3-2-2-2.webp)

![Digestion system table](https://static.igem.wiki/teams/5542/notebook/exp/8-3-2-3-2-6666.avif)

### 2025.08.22-2025.08.28

Event 1: Isolation: 5.2, 5.3, 5.4

Event 1: Isolation: 5.2, 5.3, 5.4

Procedures

1. We picked 4 colonies (2 from each plates) and shake in 5mL LB liquid medium at 220rpm for 6.5h, then stored the bacteria solution with glycerin.
2. We add 1mL bacteria solution from each tube (from 200μL plates) to 200mL LB liquid medium and shake at 220rpm overnight.
3. Isolate plasmids by Vigorous Maxiprep Kit following the protocol: **Vigorous Plasmid Maxiprep**.
4. Test the plasmid concentration by nano100.

Results

The concentration of plasmid maxiprep are as follows:

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/8-w4-concentration.webp)

Event 2: Digestion and agarose gel electrophoresis: plasmid 5.2, 5.3, 5.4

Event 2: Digestion and agarose gel electrophoresis: plasmid 5.2, 5.3, 5.4

We digest the plasmids at room temperature overnight according to the follow system:

Procedures

1. We digest the plasmids at room temperature overnight according to the follow system:

   ![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/8-5-2-1-1.webp)

Results

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/mmm.avif)

Figure 1: Profile of digestion (DNA mass 600 ng)

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/8-5-2-1-2.webp)

## Proof of concept in HEK293T

### 2025.08.05-2025.08.11

Event 1: Cell passage of HEK293T

Event 1: Cell passage of HEK293T

We passaged HEK293T cells for future transfection experiments.

Procedures

1. We routinely passaged HEK293T cells at a 1:3 ratio every other day to maintain cell viability for subsequent experiments.

Event 2: Cell seeding of HEK293T

Event 2: Cell seeding of HEK293T

We seed HEK293T cells in two 24-well plates for subsequent transfection.

Procedures

1. Procedures were the same as the seeding process of **July Week3**.
   We seed two 24-well plate using 200 μL cell suspension.

Event 3: Transfection: System1 (plasmid 1.1,1.2,1.3,1.8,1.9,1.10,1.11) and system2 (plasmid 1.1,1.4,1.5,1.8,1.9,1.10,1.11) →HEK293T

Event 3: Transfection: System1 (plasmid 1.1,1.2,1.3,1.8,1.9,1.10,1.11) and system2 (plasmid 1.1,1.4,1.5,1.8,1.9,1.10,1.11) →HEK293T

We transfected system1 and system2 to HEK293T cells and performed florescence imaging of EGFP and mCherry in transfected cells to explore the effectiveness of the RADAR system.

Procedures

1. We transfect plasmids to HEK293T cells according to the following system
2. For each system, 3 groups were set:
   (1) Two groups received plasmids lacking the Tet operator (p1.10 and p1.11), with one group induced using 100 ng/mL Doxycycline hyclate and the other serving as an uninduced control.
   (2) One group received plasmids containing the Tet operator (p1.8 and p1.9) and was induced with 100 ng/mL Doxycycline hyclate.
3. The 24-well plate for system1:

   ![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week1-1.webp)

   Table 1. List of transfection system
4. The 24-well plate for system2:

   ![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week1-2.webp)

   Table 2. List of transfection system

Results

As anticipated, EGFP expression was successfully triggered by their corresponding Triggers across all systems and treatment groups. This outcome not only validates the functionality of our RADAR system but also confirms the inducing effect of doxycycline. However, we observed that the untreated control groups (without doxycycline) showed the strongest signal, potentially due to cytotoxic effects caused by DMSO – the solvent used for doxycycline preparation – as illustrated below.

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week1-3.webp)

Figure 1: Images of HEK293T 48h after transfection.

Event 4: Cell seeding of HEK293T

Event 4: Cell seeding of HEK293T

We seed HEK293T cells in two 24-well plates for subsequent transfection.

Procedures

1. Procedures were the same as the seeding process of **July Week3**.

   We seed 15 wells of one 24-well plate using 125 μL cell suspension.

Event 5: Transfection: AND system→HEK293T

Event 5: Transfection: AND system→HEK293T

We transfected AND system to HEK293T cells and performed florescence imaging of EGFP and mCherry in transfected cells to explore the effectiveness of the RADAR system.

 Round 1: p1.1, p1.6, p1.7, p1.8, p1.9

 Round 2: p1.1, p1.6, p1.7, p1.10, p1.11

Procedures

1. We transfect plasmids to HEK293T cells as follows
2. Round1

   ![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week1-4.webp)

   Table 1. List of transfection system
3. Round2

   ![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week1-5.webp)

   Table 2. List of transfection system

Results

In Round 1, We found that when both Triggers were co-transfected, the induced EGFP expression was not significant. Combined with the results of Event 3, we speculate that this may be due to the TetO sequence still exerting a certain inhibitory effect on Trigger expression. Therefore, we decided to replace the plasmids with p1.10 and p1.11 and repeat the experiment.

In Round 2, We observed that the EGFP expression induced by co-transfection with both Triggers was not significant. To rule out operational factors, we have decided to repeat the experiment.

### 2025.08.12-2025.08.18

Event 1: Cell passage of HEK293T

Event 1: Cell passage of HEK293T

We passaged HEK293T cells for future transfection experiments.

Procedures

1. We routinely passaged HEK293T cells at a 1:3 ratio every other day to maintain cell viability for subsequent experiments.

Event 2: Cell seeding of HEK293T

Event 2: Cell seeding of HEK293T

We seed HEK293T cells in one 24-well plate for subsequent transfection.

Procedures

1. Procedures were the same as the seeding process of **July Week3**.

   We seed one 24-well plate using 200 μL cell suspension.

Event 3: Transfection of HEK293T

Event 3: Transfection of HEK293T

We stransfect the two systems as follows to verify the efficiency of RADAR system.

Procedures

1. We transfect plasmids to HEK293T cells in 6 wells according to the following system, each group for 2 wells:

   ![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week2-1.webp)

   Table 1. List of transfection system
2. We transfect plasmids to HEK293T cells in 10 wells according to the following system, each group for 2 wells:

   ![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week2-2.webp)

   Table 2. List of transfection system

Results

For the transfection of Sensor 1-Gluc and other system, We observed that the luminescence intensity of the culture supernatant from cells co-transfected with Sensor 1-Gluc and Trigger 1 was significantly higher than that from cells transfected with Sensor 1 alone or with Sensor 1 and Trigger 2. This demonstrates that Gluc can function effectively as an output gene in the RADAR system.

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week2-3.webp)

Figure 1: Luminescence of HEK293T cells transfected with Sensor 1-Gluc and other plasmids.

For the transfection of AND system, we found that co-transfection with both Trigger 1 and Trigger 2 can significantly induced EGFP expression, whereas individual Trigger transfection or the no-Trigger control group showed barely detectable EGFP. This result demonstrates that the system can simultaneously and specifically detect both Triggers.

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week2-4.webp)

Figure 2:Images of HEK293T 48h after transfection.

Event 4: Cell seeding of HEK293T

Event 4: Cell seeding of HEK293T

We seed HEK293T cells in one 24-well plate for subsequent transfection.

Procedures

1. Procedures were the same as the seeding process of **July Week3**.

   We seed one 24-well plate using 200 μL cell suspension.

### 2025.08.19-2025.08.25

Event 1: Cell passage of HEK293T

Event 1: Cell passage of HEK293T

We passaged HEK293T cells for future transfection experiments.

Procedures

1. We routinely passaged HEK293T cells at a 1:3 ratio every other day to maintain cell viability for subsequent experiments.

### 2025.08.26-2025.09.03

Event 1: Cell passage of HEK293T

Event 1: Cell passage of HEK293T

We passaged HEK293T cells for future transfection experiments.

Procedures

1. We routinely passaged HEK293T cells at a 1:3 ratio every other day to maintain cell viability for subsequent experiments.

Event 2: Cell seeding of HEK293T

Event 2: Cell seeding of HEK293T

We seed HEK293T cells in one 24-well plate for subsequent transfection.

Procedures

1. Procedures were the same as the seeding process of **July Week3**.

   We seed two 24-well plates for transfection, one plate used 200μL cell suspension for 24 wells seeding, and one plate used 67μL for 8 wells seeding.

Event 3: Transfection of HEK293T

Event 3: Transfection of HEK293T

We transfected plasmids to the cells to verify the efficiency of RADAR system

Procedures

1. We transfect plasmids to HEK293T cells in 8 wells according to the following system, each group for 2 wells:

   ![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week4-1.webp)

   Table 1. List of transfection system
2. We transfect plasmids to HEK293T cells in 10 wells according to the following system, each group for 2 wells:

   ![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week4-2.webp)

   Table 2. List of transfection system
3. We transfect plasmids to HEK293T cells in 6 wells of another plate according to the following system, each group for 2 wells:

   ![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week4-3.webp)

   Table 3. List of transfection system
4. We collect the supernatant of conditioned medium at 12,24,36,48,60,72 hours post transfection

Results

The transfection results for Sensor 1-Gluc and its related plasmids were consistent with previous findings. Notably, the supernatant from cells transfected with the Sensor 1(PC)-Gluc plasmid (p5.2) exhibited exceptionally high Gluc expression, confirming the functionality of both the RADAR system and the positive control. Furthermore, the RADAR AND-gate sensor with Gluc as the output performed effectively, demonstrating high luminescence levels only upon co-transfection with both Trigger 1 and Trigger 2.

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week4-4.webp)

Figure 1:Luminescence of conditioned medium of HEK293T cells after transfection, Sensor 1+2-Gluc is a RADAR sensor with Gluc as the output, created by connecting Sensor 1 and Sensor 2 in tandem.

We also collected culture supernatants at various time points from cells co-transfected with the AND Sensor-Gluc, Trigger 1, Trigger 2, and ADAR to investigate the kinetics of the RADAR system, the results are as follows:

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/aug-week4-5.webp)

Figure 2: Luminescence of conditioned medium from transfected HEK293T cells that were collected at indicated times.

## Validation in chassis-adipocytes

### 2025.08.05-2025.08.11

Event 1: Cell passaging: human preadipocytes

Event 1: Cell passaging: human preadipocytes

We passage the human preadipocytes to get more cells for experiments.

Procedures

1. The procedure of human preadipocytes passaging is similar to the HEK293T, the only difference was that the cells were maintained in 8 mL of specialized medium within a T25 flask.

### 2025.08.12-2025.08.18

Event 1: Medium changing: human preadipocytes

Event 1: Medium changing: human preadipocytes

We change the medium of human preadipocytes to keep the viability.

Procedures

1. We monitor cell morphology and medium color daily and perform medium changes accordingly to maintain cell viability.

Event 2: Cell seeding of human preadipocytes

Event 2: Cell seeding of human preadipocytes

We seed the cells into well plates for further adipogenic differentiation.

Procedures

1. We seed the preadipocytes in 10 wells of two 6-well plates and 26 wells of two 24-well plates using cells in three T25 flask.
2. We passage the rest cells into two T25 flasks.

Event 3: Adipogenic Differentiation

Event 3: Adipogenic Differentiation

To obtain adipocytes for experiments, we subjected the preadipocytes to adipogenic differentiation.

Procedures

1. We change the medium in the two 24-well plates to adipogenic differentiation
   equipment to induce the preadipocytes to differentiate into adipocytes.
2. From then on, we replaced the old adipogenic induction medium with fresh medium every 2 days and maintained this regimen for over 14 days to ensure continuous induction.

Event 4: Cell seeding of human preadipocytes

Event 4: Cell seeding of human preadipocytes

We seed the cells into well plates for further adipogenic differentiation.

Procedures

1. We seed the preadipocytes in 10 wells of one 12-well plate using cells in one T25 flask.
2. We passage the rest cells into two T25 flasks.

### 2025.08.19-2025.08.25

Event 1: Medium changing: human preadipocytes

Event 1: Medium changing: human preadipocytes

We change the medium of human preadipocytes to keep the viability.

Procedures

1. We monitor cell morphology and medium color daily and perform medium changes accordingly to maintain cell viability.

Event 2: Adipogenic Differentiation

Event 2: Adipogenic Differentiation

To obtain adipocytes for experiments, we subjected the preadipocytes to adipogenic differentiation.

Procedures

1. We change the medium in the two 6-well plates (10 wells in total) and one 12-well plates (10 wells in total) to adipogenic differentiation equipment to induce the preadipocytes to differentiate into adipocytes.
2. From then on, we replaced the old adipogenic induction medium with fresh medium every 2 days and maintained this regimen for over 14 days to ensure continuous induction.

Event 3: Cell seeding of human preadipocytes

Event 3: Cell seeding of human preadipocytes

We seed the cells into well plates for further adipogenic differentiation.

Procedures

1. We seed the preadipocytes in 3 wells of one 6-well plate using cells in one T25 flask.
2. We passage the rest cells into two T25 flasks.

Event 4: Cell revival: human preadipocytes

Event 4: Cell revival: human preadipocytes

We revival the new human preadipocytes got from the company.

Procedures

1. We revival all of the cells in one T25 flask.
2. Add 3ml fresh medium into the flask per 24h for 3 days.

Event 5: Adipogenic Differentiation

Event 5: Adipogenic Differentiation

To obtain adipocytes for experiments, we subjected the preadipocytes to adipogenic differentiation.

Procedures

1. We change the medium in the one 6-well plates (3 wells in total) and to adipogenic differentiation equipment to induce the preadipocytes to differentiate into adipocytes.
2. From then on, we replaced the old adipogenic induction medium with fresh medium every 2 days and maintained this regimen for over 14 days to ensure continuous induction.

Event 6: Cell seeding of human preadipocytes

Event 6: Cell seeding of human preadipocytes

We seed the cells into well plates for further adipogenic differentiation.

Procedures

1. We seed the preadipocytes in 23 wells of one 24-well plate using cells in two T25 flasks.

### 2025.08.26-2025.09.01

Event1: Cell passaging: human preadipocytes

Event1: Cell passaging: human preadipocytes

We passage the human preadipocytes to get more cells for experiments.

Procedures

1. We have passaged the newly resuscitated human preadipocytes into two T25 flasks.

Event2: Adipogenic Differentiation

Event2: Adipogenic Differentiation

To obtain adipocytes for experiments, we subjected the preadipocytes to adipogenic differentiation.

Procedures

1. We change the medium in the one 24-well plate (23 wells in total) and to adipogenic differentiation equipment to induce the preadipocytes to differentiate into adipocytes.
2. From then on, we replaced the old adipogenic induction medium with fresh medium every 2 days and maintained this regimen for over 14 days to ensure continuous induction.

Event3: Cell seeding of human preadipocytes

Event3: Cell seeding of human preadipocytes

We seed the cells into well plates for further adipogenic differentiation.

Procedures

1. We seed the preadipocytes in 20 wells of one 24-well plate and 2 wells of one 12-well plate using cells in one T25 flask.
2. The rest cells are passaged in one T25 flask.

Event4: Adipogenic Differentiation

Event4: Adipogenic Differentiation

To obtain adipocytes for experiments, we subjected the preadipocytes to adipogenic differentiation.

Procedures

1. We change the medium in the one 24-well plate (20 wells in total) and 12-well plate (2 wells in total) to adipogenic differentiation equipment to induce the preadipocytes to differentiate into adipocytes.
2. From then on, we replaced the old adipogenic induction medium with fresh medium every 2 days and maintained this regimen for over 14 days to ensure continuous induction.

September

### 2025.09.02-2025.09.08

Event 1: Oil red O staining

Event 1: Oil red O staining

We assessed the extent of adipocyte differentiation by Oil Red O staining.

Procedures

1. We use the cells in the two 6-well plates for Oil Red O staining.
2. Prepare the Oil Red O working solution by mixing the Oil Red O stock solution with distilled water at a 3:2 ratio and thoroughly vortexing the mixture. Filter the solution through filter paper and collect the filtrate, which is the ready-to-use Oil Red O working solution.
3. After completing the induction of differentiation, carefully aspirate the cell culture supernatant and wash the cells 1-2 times with 1×PBS. Add an appropriate amount of cell fixative and incubate at room temperature for 30 minutes for fixation.
4. Upon completion of cell fixation, carefully aspirate the fixative solution and wash the cells twice with 1×PBS.
5. Slowly add Oil Red O staining solution along the well wall, using 1mL per well, and incubate for 30 minutes at room temperature.
6. After staining, remove the staining solution and rinse with PBS to eliminate non-specific staining.

Results

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/sep-week1-1.webp)

Figure 1: Differentiated Adipocytes

Event 2: rAAV infection

Event 2: rAAV infection

We used rAAV to deliver Sensor 1-Gluc, Trigger 1, and ADAR to validate the functionality of both the RADAR system and rAAV.

Procedures

1. Thaw viral stock on ice.
2. repare infection mix.
3. Add infection mix to cells, mix well.
4. Incubate (37°C, 5% CO2) for 24h and change the medium.
5. Using microscope to observe the mcherry.
6. Round 1

   We infected the cells in the 24-well plates by rAAV carrying Sensor 1-Gluc first to explore the optimal viral dosage.

   We try to use 10μL, 15μL and 25μL virus per well to infect the cells.
7. Round 2
   We infected the cells in the 24-well plates by 15μL rAAV carrying Sensor 1-Gluc and Trigger 1, with 5μL rAAV carrying ADAR, Using cells infected only with rAAV carrying Sensor 1-Gluc and ADAR as the control group, and non-infected cells as the blank group.

Results

Round 1
Through observing expression of mcherry using the fluorescence microscope, we found that using 15μL can already achieved satisfactory infection efficiency, we decided to use 15 μL for all subsequent infections.

Round 2
Three days post-infection, the cell culture supernatant was collected and mixed with substrate for chemiluminescence detection. The results showed that co-infection with Trigger 1 induced a significant increase in luminescence from Sensor 1-Gluc, demonstrating that rAAV can effectively deliver the RADAR system and that the RADAR system functions robustly within adipocytes.

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/sep-week1-2.webp)

Figure 1:Luminescence of the conditioned medium from adipocytes infected by rAAVs carrying Sensor 1 and Trigger 1

Event 3: Cytokine treatment

Event 3: Cytokine treatment

We treated the cells with cytokines to validate the upregulation of the target gene in adipocytes.

Procedures

1. Adipocytes in 12-well plates were treated with PAI-1, CXCL3, CXCL8, and a combination of PAI-1 and CXCL8, respectively, using untreated cells as a control.
2. We use cytokines treat the cells for 48 hours.

Event 4: qPCR

Event 4: qPCR

We quantified the expression of specific genes by qPCR.

Procedures

RNA extraction

1. Remove the supernatant and wash the cells twice with PBS.
2. Lyse the cells by adding a total of 500μL of Transzol up per group. Collect the cell lysate.
3. Add infection mix to cells, mix well.
4. Centrifuge the lysate at 12,000xg for 10 minutes at 4°C. After centrifugation, transfer the supernatant to a new tube.
5. Add 100μL of chloroform per tube, vortex for 15 seconds, and then shake for 30 seconds until the mixture turns a uniform pink color.
6. Incubate the mixture at room temperature for 5 minutes. And centrifuge the samples at 12,000xg for 15 minutes at 4°C.
7. Carefully transfer the upper to a fresh tube.Precipitate the RNA by adding 250μL of isopropyl alcohol pertube. Incubate at room temperature for 10 minutes.
8. Centrifuge at 12,000xg for 10 minutes at 4°C. Carefully discard the supernatant.
9. Wash the RNA pellet by adding 500ul of 75% ethanol Vortex briefly to dislodge the pellet.
10. Centrifuge at 7,500xg for 5 minutes at 4°C. Carefully aspirate the ethanol.
11. Air-dry the pellet for approximately 10 minutes and dissolve the purified RNA in an appropriate volume of nuclease-free water. Measure the concentration.

qPCR

1. Prepare the reverse transcription mixture
2. Use ddH2O to dilute 1000ng RNA extraction to 10μL
3. Adding the mixture and the RNA to the Thermal Cycler. Set the system at 50°C for 5min and 80°C for 0.05min. Waiting for the system temperature until it decrease to 4°C.
4. Adding 140μL ddH2O to the tube. And remove the reverse transcription product to the 96 well PCR plate (4μL each)
5. Adding 5μL SYBR and 1μL primer, then seal the plate with a membrane.
6. Centrifuge at 2250 rpm for 3 minutes. Put the plate into the Real-Time PCR Instrument.

Results

Treatment with either PAI-1 or CXCL8 specifically upregulated their respective target genes (PLOD2 and LIF), with CXCL8 inducing a more pronounced upregulation than CXCL3. Furthermore, co-treatment with PAI-1 and CXCL8 simultaneously enhanced the expression of both genes.

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/sep-week1-3.webp)

Figure 1: Expression of PLOD2 and LIF in adipocytes treated with PAI-1 and CXCL8.

Event 5: Cell seeding of human preadipocytes

Event 5: Cell seeding of human preadipocytes

We seed the cells into well plates for further adipogenic differentiation.

Procedures

1. We seed the preadipocytes in 6 wells of one 6-well plate using cells in 1.5 T25 flask.
2. The rest cells are passaged in one T25 flask.

Event 6: Oil Red O staining

Event 6: Oil Red O staining

We stained undifferentiated adipocytes to supplement the control groups.

Procedures

1. We use the cells in the 6-well plate for Oil Red O staining
2. Prepare the Oil Red O working solution by mixing the Oil Red O stock solution with distilled water at a 3:2 ratio and thoroughly vortexing the mixture. Filter the solution through filter paper and collect the filtrate, which is the ready-to-use Oil Red O working solution.
3. After completing the induction of differentiation, carefully aspirate the cell culture supernatant and wash the cells 1-2 times with 1×PBS. Add an appropriate amount of cell fixative and incubate at room temperature for 30 minutes for fixation.
4. Upon completion of cell fixation, carefully aspirate the fixative solution and wash the cells twice with 1×PBS.
5. Slowly add Oil Red O staining solution along the well wall, using 1mL per well, and incubate for 30 minutes at room temperature.
6. After staining, remove the staining solution and rinse with PBS to eliminate non-specific staining.

Results

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/sep-week1-3-333333.avif)

Figure 1: Undifferentiated human preadipocytes,with or without oil red o staining.

### 2025.09.09-2025.09.15

Event 1: Cell revival:MDA-MB-231

Event 1: Cell revival:MDA-MB-231

We revived the human breast cancer cell line MDA-MB-231 to obtain conditioned medium.

Procedures

1. The procedure follows the one in July Week3.
2. We maintained MDA-MB-231 cells in DMEM supplemented with 10% FBS.

Event 2: rAAV infection

Event 2: rAAV infection

We used rAAV to deliver the PLOD2 sensor,LIF sensor and ADAR to validate the functionality of our RADAR system.

Procedures

1. We infected the cells in the 24-well plates (23 wells in total) by 15μL rAAV carrying PLOD2 sensor or LIF sensor, with 5μL rAAV carrying ADAR mixed.
2. We infected each group with 6 wells.

Event 3: Cytokine treatment and luminescence detection

Event 3: Cytokine treatment and luminescence detection

We treated the cells with cytokines to up-regulate the target gene in adipocytes.

Procedures

1. 48 hours post transfection, we treat the cells with PAI-1 and CXCL8.
2. After cytokines treatment, we collect the supernatant of conditioned medium and detect the luminescence.

Results

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/sep-week2-1.webp)

Figure 1:Luminescence of conditioned medium supernatant from infected adipocytes, with or without cytokine treatment.

Event 4: Conditioned medium collection

Event 4: Conditioned medium collection

We seed the MDA-MB-231 cells into 6-well plate to collect the conditioned medium for further experiments.

Procedures

1. We seed 6 wells of a 6-well plate with MDA-MB-231.
2. day after seeding,we exchange the medium to 0.2% FBS DMEM medium and cultured for another 24h.
3. Then, we collect the conditioned medium and filter using a 0.22 µm filter.
4. The conditioned medium are stored at -80°C for further experiments.

### 2025.09.16-2025.09.20

Event 1: rAAV infection

Event 1: rAAV infection

We used rAAV to deliver the PLOD2-LIF sensor and ADAR to validate the functionality of our RADAR system.

Procedures

1. We infected the cells in the 24-well plates (12 wells in total) by 15μL rAAV carrying PLOD2-LIF sensor, with 5μL rAAV carrying ADAR mixed.
2. We infected 6 wells with above rAAVs.

Event 2: Cytokine treatment and luminescence detection

Event 2: Cytokine treatment and luminescence detection

We treated the cells with cytokines to up-regulate the target gene in adipocytes.

Procedures

1. 48 hours post transfection, we treat the cells with PAI-1 and CXCL8.
2. After cytokines treatment, we collect the supernatant of conditioned medium and detect the luminescence.

Results

We observed a significant increase in luminescence intensity in the post-infection culture supernatant following co-treatment with both cytokines, an effect not elicited by individual cytokine treatment or in the absence of cytokines. This confirms that our designed PLOD2-LIF sensor exhibits excellent detection performance and specificity.

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/sep-week2-2.webp)

Figure 1: Luminescence of conditioned medium supernatant from adipocytes infected by rAAV carry PLOD2-LIF sensor, with or without cytokines treatment.

Event 3: Conditioned medium treatment and luminescence detection

Event 3: Conditioned medium treatment and luminescence detection

We treated the cells with conditioned medium of MDA-MB-231 to detect the efficiency for our engineered adipocytes to detect breast cancer.

Procedures

1. 48 hours post transfection, we treat the cells with MDA-MB-231 CM,with 0.2%FBS DMEM treatment as a control.
2. Then, we collect the supernatant of conditioned medium and detect the luminescence.

Results

We observed that MDA-MB-231 conditioned medium (CM) induced a significant increase in luminescence intensity, indicating that our detection system functions effectively.

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/sep-week2-3.webp)

Figure 1: Luminescence of conditioned medium supernatant from adipocytes infected by rAAV carrying PLOD2-LIF sensor and ADAR, with or without CM treatment.

Event 4: Measurement of cell viability by CCK-8 assay

Event 4: Measurement of cell viability by CCK-8 assay

We assessed the impact of rAAV infection on cell viability using the CCK-8 assay.

Procedures

1. Thaw CCK-8 solution and protect from light (wrap in foil).
2. Add 50μL CCK-8 reagent per well.
3. Return plate to CO2 incubator for 2h.
4. Collect the supernatant, transferred to a 96-well plate, and its absorbance was measured at 450nm using a microplate reader.

Results

We observed no significant change in the viability of adipocytes following infection with rAAV carrying the PLOD2-LIF sensor and ADAR.

![Plasmid concentration result](https://static.igem.wiki/teams/5542/notebook/exp/sep-week2-4.webp)

Figure 1:The viability of infected and uninfected adipocytes revealed by CCK-8 assay.

Dry Lab
![](https://static.igem.wiki/teams/5542/img/h1-background.webp)

December

Model group member recruitment

*2024.11.24-2024.12.22*

---

After the team recruitment, our model group was set up by three outstanding
undergraduate students from Jilin University, Hanqiu Yang from the School
of Computer Science and Technology, Wenyuan Deng from the School of Mathematics and Chenguang Shi from the Third Bethune Clinical Medical College.

CJUH-JLU-China First Full Group Meeting

*2024.12.23-2025.01.04*

---

During the group meeting, members of the Model group met offline for the first time and made their choices regarding the research directions during the brainstorming phase: Hanqiu Yang chose the research direction of large sequence models, while Wenyuan Deng opted for the direction of biomolecular dynamics simulation,and Chenguang Shi chose bioinformatics analysis as his research direction. Moving forward, the members will engage in learning and replication in their respective fields of interest, gradually accumulating experience.

January

The First Online Meeting of the Model Group (The First Brainstorm)

*2025.01.05-2025.01.19*

---

At the first meeting of the Model group, Hanqiu Yang reported on his learning progress, which covered knowledge such as fully connected neural networks in deep learning. He also researched the application direction of large sequence models in synthetic biology and proposed the application of AI in the Wiki group. Wenyuan Deng shared her learning experience with MATLAB and simbiology, analyzed and replicated the case of the 2023 AFCM - Egypt Best Model nominee, pointed out the advantages and disadvantages of the case as well as the direction for improvement, and meanwhile, stated the existing problems and the subsequent plan. Chenguang Shi shared the application of programming software such as R and Python in bioinformatics analysis.Consultant Renxiu Song and the former leader of the Model group, Haoyang Liu, shared their Model competition experience and put forward corresponding suggestions for improvement.

![](https://static.igem.wiki/teams/5542/notebook/model/jan-1a.webp)

Figure 1:Screenshot of the PPT for the First Online Group Meeting

Preliminary preparation and the exploration of principles of models

*2024.12.23-2025.01.26*

---

During the initial phase of the Model group's activities, members established foundational
research directions through brainstorming and collaborative discussions. Domains of large
sequence models and biomolecular dynamics simulation through tools like MATLAB and SimBiology
are studied by team members. Our work also includes the case studies of best model nominees
such as the 2023 AFCM - Egypt and 2021 Vilnius.
Subsequently, we learnt the mathematical principles of Transformer architecture, reproducing
self-attention mechanisms and explored applications of large-scale models in gene sequence analysis.
Furthermore, we also advanced our proficiency in non-compartmental analysis and parameter fitting using SimBiology's Model Analyzer.

![](https://static.igem.wiki/teams/5542/notebook/model/jan-2a.webp)

Figure 2: Screenshot of the PPT for the Second Online Group Meeting.

February

Data search

*2025.02.17-2025.02.23*

---

This week, we officially began the search work in the database with the EXP team. We systematically learned the use of databases such as GEO, TCGA, GTEx, and ArrayExpress. After comprehensive consideration, we ultimately chose GEO, TCGA, and GTEx as our available databases.

![](https://static.igem.wiki/teams/5542/notebook/model/feb-1a.webp)

Figure 1:GEO, TCGA, GTEx Databases

Determine the data source

*2025.02.24-2025.03.03*

---

After analysis, we found that the GEO database data has issues such as small scale, which is difficult to meet our work needs. Therefore, we ultimately decided to search for the required data from TCGA and GTEx.

![](https://static.igem.wiki/teams/5542/notebook/model/feb-2a.webp)

Figure 2: Analysis Conducted on GEO

March

Through literature research, we have identified several genes which exhibit abnormal expression in adipocytes after being affected by breast cancer cells.

*2025.03.04-2025.03.10*

---

Through literature research, we have identified several genes which exhibit abnormal expression in adipocytes after being affected by breast cancer cells.

![](https://static.igem.wiki/teams/5542/notebook/model/mar-1a.webp)

Figure 1:Relevant Literature

Determination of Gene Combination for Monitoring

*2025.03.11-2025.03.28*

---

Utilizing an MLP algorithm incorporating the Attention mechanism-better suited to capture complex non-linear relationships in high-dimensional sparse data-we performed feature extraction on the data matrix. Based on data from TCGA and GTEx, we evaluated the predictive performance of different gene combinations and ultimately determined the gene combination for subsequent monitoring.

![](https://static.igem.wiki/teams/5542/notebook/model/mar-2a.webp)

Fighure2: ROC Curve of the Top 5 Gene Combinations.

April

Preliminary Work and Exploration on the Construction of Ethical Risk Assessment Model

*2025.03.29-2025.04.06*

---

This week with the HP team, we developed a Synthetic Biology Ethical Risk Assessment Model. Current research remains qualitative (philosophy/social science) with no standardized risk grading or mathematical models. Proposed solutions (ethical matrices, AI evaluation, structural equation modeling) all face challenges like subjective quantification. Next week focuses on feasibility analysis and preliminary modeling.

![](https://static.igem.wiki/teams/5542/notebook/model/apr-1a.webp)

Figure 1:Exploring modeling approaches for ethical risk assessment in collaboration with the HP.

Case Analysis and Model Exploration on the Construction of Ethical Risk Assessment Model

*2025.04.07-2025.04.15*

---

This week, we made further progress on our synthetic biology ethical risk assessment mode. we adopted a hybrid approach combining mathematical modeling with Likert-scale questionnaires covering biosafety and social equity dimensions. Our next steps involve applying structural equation modeling to analyze latent variables through path coefficients for quantitative validation.

![](https://static.igem.wiki/teams/5542/notebook/model/apr-2a.webp)

Figure 2: Screenshot of the PPT for Online Group Meeting

Structural Design Optimization and Model Exploration

*2025.04.16-2025.04.24*

---

we finalized the adoption of a Structural Equation Modeling (SEM) + Bayesian Network hybrid approach. After deepening our theoretical understanding of SEM, we established a dual-track strategy combining mathematical modeling with questionnaire surveys. This involves: 1) collecting data through customized questionnaires to analyze key influencing factors and their path coefficients, 2) developing quantitative assessment models, and 3) exploring Bayesian Network-SEM integration with proposed linear/Sigmoid mapping solutions for weight-to-probability conversion. Collaborating with the HP team, we refined ethical assessment dimensions in the questionnaire.

![](https://static.igem.wiki/teams/5542/notebook/model/apr-3a.webp)

Figure 3: Screenshot of the PPT for Online Group Meeting.

May

Building and Expanding the Model through Preliminary Questionnaire Collection and Data Validation

*2025.04.25-2025.05.07*

---

In our seventh team meeting, we finalized a comprehensive modeling framework integrating multi-dimensional variables through Structural Equation Modeling (SEM) and Bayesian Networks to evaluate both the potential impact and occurrence probability of ethical risks. Building on established evaluation dimensions and indicators from literature, we've successfully designed and distributed questionnaires, collecting 322 diverse samples. For SEM implementation, we've completed data simulation tests yielding path diagrams and reliability indices, while for Bayesian Networks, we've established fundamental principles including weight-to-probability conversion methods and risk assessment procedures using Bayesian inference.

![](https://static.igem.wiki/teams/5542/notebook/model/may-1a.webp)

Figure 1: Exchanging perspectives with the HP on
ethical risk assessment model construction

Transitioning to Bayesian Networks

*2025.05.08-2025.05.16*

---

In the ethical risk assessment model, we transitioned from structural equation modeling to Bayesian networks. This involved converting path coefficients to form a topological structure, discretizing continuous variables, and applying softmax normalization to build a conditional probability table. We also began learning to use Netica software.

![](https://static.igem.wiki/teams/5542/notebook/model/may-2a.webp)

Figure 2: the Photos of Group Meeting.

Bayesian Network Application

*2025.05.17-2025.05.24*

---

We have established an ethical risk assessment system and developed a generalized synthetic biology risk assessment model based on structural equation and Bayesian network, achieving risk quantification.

![](https://static.igem.wiki/teams/5542/notebook/model/may-3a.webp)

Figure 3: the Photos of Group Meeting and Bayesian Network.

June

Case Validation of the Ethical Risk Assessment Model

*2025.05.25-2025.06.02*

---

We designed a universal questionnaire within our ethical risk assessment model and used two case studies-"synthetic reconstruction of the smallpox virus" and "producing the antimalarial drug artemisinin(青蒿素) with synthetic yeast"-to test the model's validity. Through this process, we successfully generated the corresponding Bayesian network diagrams, which verified that the model is sound and reasonable.

![](https://static.igem.wiki/teams/5542/notebook/model/jun-1a.webp)

Figure 1: the results of Bayesian network.

Model Framework Refinement and Project Self-Assessment

*2025.06.03-2025.06.11*

---

We further refined the overall framework of the ethical risk assessment model, creating its flowchart and drafting the relevant documentation. Concurrently, we conducted an ethical risk assessment on our own project using the model, ultimately concluding that our project is categorized as low-risk.

![](https://static.igem.wiki/teams/5542/notebook/model/jun-2a.webp)

Figure 2: Framework and the result of Bayesian network.

Ethical Assessment System Documentation Refinement

*2025.06.12.-2025.06.22*

---

This week, we finalized the name "BEAM" (Bio-ethical Assessment Model) for our ethical assessment model, intending to illuminate ethical evaluation in synthetic biology. Subsequently, we revised and discussed the documentation outline.

![](https://static.igem.wiki/teams/5542/notebook/model/jun-3a.webp)

Figure 3: The Photos of BEAM Framework and Logo.

Design of sensor-RNA Sequences

*2025.06.23-2025.06.30*

---

Upon obtaining the key gene combination, we designed multiple sensor-RNA sequences for plasmid construction based on their expression profiles. We employed the iPKnot++ algorithm, capable of predicting long-chain RNA structures including pseudoknots, to simulate RNA secondary structures. We quantitatively evaluated the functionality of the RNA sequences using two metrics: the unpaired probability in the sensor region and the exposed length near editing sites. Ultimately, we determined the sensor-RNA sequences for plasmid construction.

![](https://static.igem.wiki/teams/5542/notebook/model/jun-4a.webp)

Figure 4: Reference of iPknot++ and simulation of RNA secondary structure.

July

Preliminary Construction of the RADAR System Dynamics Model

*2025.07.01.-2025.07.07*

---

This week, we established the preliminary dynamic model of the RADAR system based on the previously designed RNA sensors. By abstracting the RNA editing and downstream reporter expression process, we obtained the overall reaction framework and formulated an equation system for subsequent validation.

![](https://static.igem.wiki/teams/5542/notebook/model/jul-1a.webp)

Figure 1: RADAR System Response Process.

RADAR System Simulation

*2025.07.08.-2025.07.14*

---

We noticed that the data scale of the GEO database was insufficient for our needs. After screening, we identified TCGA (The Cancer Genome Atlas) and GTEx (Genotype-Tissue Expression) as the primary data sources.

![](https://static.igem.wiki/teams/5542/notebook/model/jul-2a.webp)

Figure 2: Simulation Curves of RADAR.

Preliminary Construction of GLUC Renal Filtration Membrane Model

*2025.07.15.-2025.07.21*

---

This week, we focused on the dynamic process of GLUC in the glomerular filtration barrier. By modeling the structural features and diffusion mechanisms, we demonstrated how GLUC molecules pass through the filtration barrier and obtained the concentration change trends in body fluids.

![](https://static.igem.wiki/teams/5542/notebook/model/jul-3a.webp)

Figure 3: Schematic of GLUC Filtration Model

Dry Lab Documentation Refinement

*2025.07.22.-2025.07.28*

---

This week,We have not only validated the RADAR response pattern at the cellular level but also demonstrated the feasibility of GLUC as a detectable output signal. Our model is named DISCERN, signifying "Dynamic Integration of Simulation for Cancer Evaluation and Recognition," while also embodying the meaning of "discernment and recognition."

![](https://static.igem.wiki/teams/5542/notebook/model/jul-4a.webp)

Figure 4: The Photo of Discern Logo.

August

Backend Algorithm Implementation

*2025.07.29 - 2025.08.08*

---

This week, we focused on refining the core algorithm logic, completing the code implementation for data processing and modeling, and conducting preliminary performance tests.

![](https://static.igem.wiki/teams/5542/notebook/model/aug-1a.webp)

Figure 1: Code Snippet.

Frontend Development and Functionality Debugging

*2025.08.09 - 2025.08.19*

---

We established the frontend framework and designed the overall webpage structure, achieving seamless integration with the backend. Page interaction optimization and visual design adjustments were implemented to ensure clarity, aesthetics, and functionality. Initial functional testing was initiated to identify and resolve issues in frontend-backend interaction.

![](https://static.igem.wiki/teams/5542/notebook/model/aug-2a.webp)

Figure 2: Webpage Design Optimization.

Bug Fixes and Software Documentation

*2025.08.20 - 2025.08.31*

---

This week, we systematically addressed bugs identified during testing and optimized the code to enhance overall stability and efficiency. Concurrently, we drafted the accompanying Software webpage documentation.

September

Final Refinement and Conclusion

*2025.09.01 - 2025.09.07*

---

We conducted a final round of testing and functionality verification. Files were organized, documentation formats were finalized, and the submission version was prepared.

HP & Edu
![](https://static.igem.wiki/teams/5542/img/h1-background.webp)

December

HP & Education group member recruitment

*2024.12.4-2024.12.7*

---

After the recruitment process, 7 undergraduates from Jilin University have joined the HP & Education group of the 2025 CJUH-JLU-China iGEM team.

January

Braille reading materials production

*2025.1.7-2025.1.9*

---

We successfully transformed and printed the 2024 original team-created picture book "Invisible Bacteria"
into a Braille version through the Comet-Light Hanbilingual Translation Platform. This provided unique
biological science popularization educational content for visually impaired children.

![](https://static.igem.wiki/teams/5542/notebook/edu/jan-1a.webp)

Figure 1: Braille reading materials.

Synthetic biology Winter Camp

*2025.1.9-2025.1.12*

---

The "Explore the code of life and program a journey into synthetic biology" synthetic biology fun
camp was held in the Song Zhiping Gymnasium of Jilin University, attracting teenagers curious about
life sciences to participate.

![](https://static.igem.wiki/teams/5542/notebook/edu/jan-2a.webp)

Figure 2: Synthetic Biology Fun Camp in winter.

Return-to-school promotion activities

*2025.1.16-2025.1.21*

---

As the winter vacation arrives, our team members carried out several lectures back at their high schools which shaped and cultivated them, with the aim of inspiring curiosity and enthusiasm in synthetic biology and scientific research among the young talents.

![Gene Component Auction lecture](https://static.igem.wiki/teams/5542/notebook/edu/jan-3a.webp)

Figure 3: Lecture on the theme of "Synthetic Future, Innovators Say" by Wenxian Gao.

Figure 3: Lecture on the theme of "Synthetic Future, Innovators Say" by Wenxian Gao.

![Planting the Seeds of Synthetic Biology lecture](https://static.igem.wiki/teams/5542/notebook/edu/jan-3b.webp)

Figure 4: Promotional speech of iGEM by Jingzhe Yu.

Figure 4: Promotional speech of iGEM by Jingzhe Yu.

![Biologyand The Art of Synthesis lecture](https://static.igem.wiki/teams/5542/notebook/edu/jan-3c.webp)

Figure 5: Online promotional speech of iGEM by Jiayu Cong.

Figure 5: Online promotional speech of iGEM by Jiayu Cong.

February

Listening to the Community

*2025.2.9-2.11*

---

We explored public health needs in several communities in Changchun, including Qunying, Huxi, and Nanling Baiyi. Surveys and interviews revealed strong concern about breast cancer, especially around early detection and current diagnostic limitations. Patients expressed the need for better awareness, less invasive screening, and more accessible technologies. These insights guided our project toward developing improved, patient-centered breast cancer diagnostics.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/feb-1a.webp)

Figure 1: Public interviews in three communities.

Understanding Public Health Needs in Yanbian

*2025.2.12-2.14*

---

After learning about breast cancer challenges in the Yanbian Korean Autonomous Prefecture, we visited the local CDC for more information. They shared issues like limited medical resources and gaps in disease monitoring. Although screening programs exist, effective follow-up for breast cancer remains a major challenge. Current methods may miss early recurrence or cause unnecessary anxiety. This inspired us to develop a technology for long-term in vivo monitoring with easy at-home readouts, aiming to make follow-up care more accessible and reliable.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/feb-2a.webp)

Figure 2: Exchange activity with Yanbian State CDC and residents.

Strengthening Our Idea at the MedTech Summit 2025

*2025.2.15-2.17*

---

To find suitable technologies for our in vivo monitoring with in vitro readout system, we attended the MedTech Summit 2025. The conference focused on medical innovation and patient-centered care. We discovered that combining in vitro diagnostics (IVD) with implantable sensors and external readers offers a faster, easier, and less invasive way to monitor breast cancer. One key idea we learned was "real-time early warning", which uses continuous monitoring and smart analysis to catch problems early. This inspired us to include real-time features in our design to improve early detection, reduce patient burden, and support long-term follow-up care.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/feb-3a.webp)

Figure 3: Attend the MedTech Summit 2025 online.

Interview with Dr. Guang Sun, Breast Surgery Department

*2025.2.17-2.21*

---

We interviewed Dr. Guang Sun from Jilin University to get clinical feedback on our project. He explained that breast cancer detection in China has improved with imaging, but many areas still have limited screening and low patient compliance. He pointed out two main problems: the high chance of cancer returning, especially in aggressive types, and weak long-term follow-up systems. Dr. Sun agreed that the focus is moving from early detection to preventing recurrence and improving long-term care, which supports our project’s goals.

![](https://static.igem.wiki/teams/5542/ihp/ihp-inspiration4-guangsun1.webp)

Figure 4: Interview with Dr. Guang Sun, Breast Surgery Department.

Interview with Dr. Wan Wang, Breast Surgeon

*2025.2.22-2.24*

---

Noticing the rise in breast reconstruction surgeries, we explored integrating a diagnostic system with reconstruction to aid both monitoring and recovery. After consulting Dr. Wan Wang from Jilin University, we learned that autologous fat grafting is widely used and feasible for implanting engineered adipocytes during surgery. Although breast-conserving surgery rates are relatively low and immediate reconstruction even lower, this approach offers a promising integrated diagnosis and treatment strategy for patients undergoing reconstruction.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/feb-5a.webp)

Figure 5: Interview with Dr. Wan Wang, Breast Surgeon.

Interview with Dr. Lianbo Zhang, Plastic Surgeon

*2025.2.25-2.27*

---

To expand our project’s impact beyond breast cancer patients undergoing reconstruction, we explored the use of autologous fat grafting in cosmetic breast augmentation. Guided by insights from plastic surgeon Dr. Lianbo Zhang, we learned that fat grafting is both clinically safe and increasingly popular due to its natural results and minimal invasiveness. Based on this, we decided to broaden our target group to include women choosing cosmetic augmentation, using fat grafting as a platform to extend the application of our diagnostic technology.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/feb-6a.webp)

Figure 6: Interview with Dr. Lianbo Zhang, Plastic Surgeon.

Interview with Dr. Bin Yang, Breast Surgeon

*2025.2.28-3.1*

---

While evaluating output options for our system, we compared melanin and Gaussia luciferase (Gluc) in terms of user convenience and psychological impact. Through an interview with Dr. Bin Yang, we learned that visible melanin marks could negatively affect body image, while Gluc offers a non-invasive and technically feasible luminescent readout. Based on this, we chose Gluc as our reporter gene and confirmed its sensitivity and practicality through lab tests. Its ability to produce detectable signals in body fluids like urine supports its potential for real-world, patient-friendly monitoring.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/feb-7a.webp)

Figure 7: Interview with Dr. Bin Yang, Breast Surgeon.

Knowledge sharing program at at Huxi Community

*2025.2.1-2.4*

---

At the Huxi Community, Changchun, we held a discussion-focused outreach, bringing together thirteen community members to explore the applications of synthetic biology and ethical considerations.

![](https://static.igem.wiki/teams/5542/notebook/edu/feb-1a.webp)

Figure 1: Community knowledge sharing program.

Education wiki refinement

*2025.2.12-2025.2.15*

---

Our team discussed how to improve the layout and content presentation of our Education wiki. Ideas include embedding scroll-triggered animations, hand-drawn comics, real-time online graffiti walls and many more.

![](https://static.igem.wiki/teams/5542/notebook/edu/feb-2a.webp)

Figure 2: Edu wiki improvement solution.

March

Interview with Professor Walter Bodmer, Fellow of the Royal Society

*2025.3.5-3.11*

---

We began by exploring public databases to identify genes highly expressed in cancer-associated adipocytes (CAAs), but the available datasets were limited in size and lacked strong statistical power. After consulting Professor Walter Bodmer, we learned that observed gene expression might reflect general inflammation rather than breast cancer specificity.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/mar-1a.webp)

Figure 1: Interview with Professor Walter Bodmer, Fellow of the Royal Society.

Interview with Project Manager at DIRUI

*2025.3.23-3.29*

---

To select suitable cancer biomarkers, we consulted DIRUI Industrial Co., Ltd., a company experienced in tumor marker detection. Their experts emphasized the importance of accuracy and shared strategies to optimize screening and reduce false positives. Inspired by this, we adopted a combined biomarker approach and identified four candidate gene pairs from literature.

![](https://static.igem.wiki/teams/5542/ihp/ihp-optimization1-dirui3.webp)

Figure 2: Interview with Project Manager at DIRUI.

Interview with Research Scientist Hongyu Wu

*2025.3.12-3.16*

---

We consulted Dr. Wu, an expert in molecular biology, to assess the feasibility of using adenovirus for gene delivery in adipocytes. She confirmed adenovirus's high infection efficiency but noted its short-lived expression, potential immune response, and reduced effectiveness in adipose tissue. Based on her advice, we switched to adeno-associated virus (AAV) for its lower immunogenicity and better stability in non-dividing cells.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/mar-3a.webp)

Figure 3: Interview with research scientist Hongyu Wu.

1st WeChat press release

*2025.3.6*

---

We published the first WeChat official account post displaying the 2025 CJUH-JLU-China team recruitment activity.

![](https://static.igem.wiki/teams/5542/notebook/edu/mar-1a.webp)

Figure 1: 1st WeChat press release.

2nd WeChat press release

*2025.3.13*

---

We published the second WeChat official account post titled "Synthesizing the Future: Voice of Innovators", kicking off our iGEM promotion series.

![](https://static.igem.wiki/teams/5542/notebook/edu/mar-2a.webp)

Figure 2: 2nd WeChat press release.

"Super Little Inventor" education activity

*2025.3.14*

---

We carried out the "Super Little Inventor" educational activity in Jilin University Affiliated Primary School, aiming to inspire children's interest in exploring the world of biology by designing "super little creatures".

![](https://static.igem.wiki/teams/5542/notebook/edu/mar-3a.webp)

Figure 3: "Super Little Inventor" education activity.

3rd WeChat press release

*2025.3.20*

---

We published the third WeChat official account post, with the theme of "the Innovative Blueprint of Synthetic Biology and Cell Factories".

![](https://static.igem.wiki/teams/5542/notebook/edu/mar-4a.webp)

Figure 4: 3rd WeChat press release.

Debate on Ethics of IP in Synthetic Biology

*2025.3.23*

---

To spark deeper reflection on syn-bio ethics and innovation, we hosted a debate centered the ethical implications of IP openness in synthetic biology, and invited the debate team from JLU School of Business and Management as our counterpart.

![](https://static.igem.wiki/teams/5542/notebook/edu/mar-5a.webp)

Figure 5: Intellectual Property Debate Competition.

April

Interview with Professor Yunsheng Dong

*2025.4.2-4.15*

---

To overcome the challenge of balancing subjective judgment and objective criteria in constructing our ethical risk assessment framework, we sought guidance from Prof. Dong, a specialist in philosophy and sociology. He emphasized the importance of scenario-specific ethical assessments, as ethical concerns vary by application. Following his advice, we refined our framework. This made our framework more targeted, practical, and adaptable.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/apr-1a.webp)

Figure 1: Interview with Professor Yunsheng Dong.

Interview with Professor Wenbin Chen

*2025.4.16-4.24*

---

While developing our ethical risk assessment framework, we initially built a multi-dimensional system and used Structural Equation Modeling (SEM) to determine the weight of each factor. However, we faced challenges in quantitatively assessing uncertain risks based on these dimensions. To address this, we consulted Prof. Wenbin Chen, who advised us to explore probabilistic relationships among factors. Following his guidance, we introduced a Bayesian network, which enabled dynamic risk inference and improved adaptability. .

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/apr-2a.webp)

Figure 2: Interview with Professor Wenbin Chen.

Survey on Ethical Perceptions of Synthetic Biology

*2025.4.25-4.26*

---

After analyzing ethical issues across different scenarios, we recognized that expert perspectives alone were insufficient for a comprehensive evaluation. To incorporate public perception, we designed a detailed questionnaire covering key ethical dimensions and attitudes toward emerging synthetic biology applications. The collected data became the foundation for our model, offering both insights into public concerns and quantitative input for model training. This step helped bridge expert analysis with societal expectations, ensuring our framework remains grounded and socially relevant.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/apr-3a.webp)

Figure 3: The Questionnaire on Ethical Concepts in Synthetic Biology.

Attending the International Academic Exchange Conference on Plastic Surgery, "China-Korea Plastic Surgery Academic Salon"

*2025.4.27-4.28*

---

We attended the China-Korea Plastic Surgery Academic Salon to gain insight into the needs and business models of plastic surgery departments in public hospitals. Dr. Lianbo Zhang’s presentation provided key insights into clinical evaluation standards in public hospitals, emphasizing practicality, evidence, and protocol alignment. This guided us to pursue research collaborations and clinical validation. Additionally, Dr. Haiyan Qin’s talk helped us identify a technical pathway to integrate our project into medical aesthetics.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/apr-4a.webp)

Figure 4: Attending the China-Korea Plastic Surgery Academic Salon.

Interview with Dr. Zhao, president of the private medical aesthetics clinic Jingdian Medical Beauty

*2025.4.3*

---

To better understand the needs of private medical aesthetics clinics, we interviewed Dr. Zhao, who emphasized that fat cell survival is crucial for successful breast augmentation and patient satisfaction. She pointed out the industry's challenge of unstable fat cell survival, which could hinder the adoption of ABCS. In response, we identified improving the survival rate of engineered fat cells during transplantation as a key research focus, aiming to optimize methods tailored.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/apr-5a.webp)

Figure 5: Interview with Dr. Zhao.

"Fuzzy Sticks" education activity

*2025.4.3*

---

We held the "Fuzzy Sticks" education activity at Changchun Jilong Middle School. Through creative hands-on practice, we led students into the fascinating world of synthetic biology and helped them experience the unique charm of science and innovation.

![](https://static.igem.wiki/teams/5542/notebook/edu/apr-1a.webp)

Figure 1: "Fuzzy Sticks" education activity.

1st post on REDnote

*2025.4.4*

---

We established the REDnote account and published our first post,
briefly introducing our team and our past highlight moments.

![](https://static.igem.wiki/teams/5542/notebook/edu/apr-2a.webp)

Figure 2: 1st post on REDnote.

4th WeChat press release

*2025.4.5*

---

We released the fourth WeChat official account post, displaying our visit to the Changchun International
Medical and Health Industry Expo and our exploration of the public's understanding on medical and health.

![](https://static.igem.wiki/teams/5542/notebook/edu/apr-3a.webp)

Figure 3: 4th WeChat press release.

The "Cell Kingdom on Kites" education activity

*2025.4.11*

---

We conducted the Cell Kingdom on Kites education activity at Jilin University
Affiliated Primary School. We introduced the kids to a variety of cells and
organelles, explained how they work, and let them illustrate their favorite
cells on kites, aiming to guide children in exploring microscopic world mysteries
and feel the unique charm of the collision between science and art.

![](https://static.igem.wiki/teams/5542/notebook/edu/apr-4a.webp)

Figure 4: The "Cell Kingdom on Kites" education activity.

5th WeChat press release

*2025.4.12*

---

We released the fifth WeChat official account post, centering the past and the present of synthetic biology.

![](https://static.igem.wiki/teams/5542/notebook/edu/apr-5a.webp)

Figure 5: 5th WeChat press release.

The "Letter Exchange Across the Nation" activity

*2025.4.13*

---

Through the platform of Jilin University, we reached out to students at multiple
universities nationwide through open letters, sharing our team's iGEM journey
last year to spark their interest in iGEM and synthetic biology.

![](https://static.igem.wiki/teams/5542/notebook/edu/apr-6a.webp)

Figure 6: the "Letter Exchange Across the Nation" activity.

2nd post on REDnote

*2025.4.13*

---

We released the second post on REDnote, introducing the cycle and award settings of the iGEM competition.

![](https://static.igem.wiki/teams/5542/notebook/edu/apr-7a.webp)

Figure 7: 2nd post on REDnote.

6th WeChat press release and 3rd post on REDnote

*2025.5.1-2025.5.25*

---

We released the sixth WeChat official account post and the third post on REDnote, introducing the whole process of the Apricot Blossom Festival (an annual tradition here in JLU) in detail.

![Gene Component Auction lecture](https://static.igem.wiki/teams/5542/notebook/edu/apr-8a.webp)

Figure 8: 6th WeChat press release.

![Planting the Seeds of Synthetic Biology lecture](https://static.igem.wiki/teams/5542/notebook/edu/apr-9a.webp)

Figure 9: 3rd post on REDnote.

The "Spring in Pinwheels, Nature in Slides" activity

*2025.4.20*

---

On the occasion of the 10th apricot blossom festival of Jilin University, we held the "Spring in Pinwheels, Nature in Slides" activity at the Nanling campus, Jilin University. Faculty and students actively participated, experiencing the integration of science and spring’s beauty.

![](https://static.igem.wiki/teams/5542/notebook/edu/apr-10a.webp)

Figure 10: Pictures at the activity site.

The "Cell Chess" game

*2025.4.24*

---

We developed the educational game "Cell Chess" and engaged international students
in the chess play, enabling them to discover biology’s wonders.

![](https://static.igem.wiki/teams/5542/notebook/edu/apr-11a.webp)

Figure 11: the "Cell Chess" game with international students.

7th WeChat press release

*2025.4.26*

---

We released the seventh WeChat official account post, reviewing the memorable
moments in the Apricot Blossom Festival, from DIY windmills to a magical world
under a microscope, we have achieved a vivid interaction between spring and science.

![](https://static.igem.wiki/teams/5542/notebook/edu/apr-12a.webp)

Figure 12: 7th WeChat press release.

Synthetic Biology in Seven Days-Advanced Edition - Chapter 1 Video

*2025.4.27*

---

We recorded a video explaining the first chapter of the learning material, focusing on the concept of iGEM, its development history, and the award settings.

![](https://static.igem.wiki/teams/5542/notebook/edu/apr-13a-1007.webp)

Figure 13: Chapter 1 video cover.

4th post on REDnote

*2025.4.27*

---

We released the fourth post on REDnote, presenting the wonderful moments of
the Apricot Blossom Festival event.

![](https://static.igem.wiki/teams/5542/notebook/edu/apr-14a.webp)

Figure 14: 4th post on REDnote.

May

Attending the Symposium on Clinical Research and Overseas Expansion of China’s New Drug R&D in the New Environment

*2025.5.17-6.4*

---

As the ABCS project approached commercialization, we sought to bridge the gap between research, clinical application, and market readiness by attending a symposium on clinical research and new drug development. There, we developed a multi-dimensional strategy involving small-scale clinical trials to improve system stability, a tiered market approach targeting both hospitals and consumer channels, and plans to co-develop supporting equipment.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/may-1a.webp)

Figure 1: Participating Online in the Symposium.

8th WeChat press release

*2025.5.1*

---

We released the eighth WeChat official account post, centering the introduction to some of the iGEM villages.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-1a.webp)

Figure 1: 8th WeChat press release.

The "Science Carnival on Snakes and Ladders" education activity

*2025.5.8*

---

We conducted a science popularization activity for students of Dehui Experimental
Primary School, introducing human cells, cell organelles, and Escherichia
coli. Then, we designed the Snakes and Ladders Q&A activity, combining
education with entertainment to arouse students' curiosity about exploring
biological mysteries.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-2a.webp)

Figure 2: The "Science Carnival on Snakes and Ladders" education activity.

9th WeChat press release

*2025.5.10*

---

We released the ninth WeChat official account post, introducing the whole
process of the "Yinyin Journey" grassland music festival in detail and
the gameplay of snakes and ladders.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-3a.webp)

Figure3: 9th WeChat press release.

The first session of the SUSTech-SynBio Community Exchange

*2025.5.10*

---

We participated in the first session of the SUSTech-SynBio Community Exchange
as the invited guest, sharing this year's project design and the Human
Practices experience in 2024.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-4a.webp)

Figure 4: The first session of the SUSTech-SynBio Community Exchange.

5th post on REDnote

*2025.5.11*

---

We released the fifth post on REDnote, documenting the wonderful moments
of the Apricot Blossom Festival (an annual tradition here in JLU) in the
form of a video.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-5a.webp)

Figure 5: 5th post on REDnote.

The "Bio-Melody" - Science Outreach at the Grassland Music Festival

*2025.5.11*

---

On the occasion of the "Yinyin Journey" grassland music festival at Jilin University, we held a variety of scientific activities at the Xinmin Campus of Jilin University such as SynBio&Smile smiley face board to immerse participants in a joyful atmosphere of science and music fusion.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-6a.webp)

Figure 6: Photos at the activity site.

The 2nd Northeast Regional iGEMer Exchange Conference

*2025.5.11*

---

We participated in the second Northeast Regional iGEMer Exchange Conference, sharing this year's
projects and listening to the insightful views of other teams on experimental
techniques, project design and other aspects.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-7a.webp)

Figure 7: The 2nd Northeast Regional iGEMer Exchange Conference.

The "Life Code Designer" education activity

*2025.5.12*

---

We guided high school students from Dehui Experimental High School to briefly understand what synthetic
biology is and its fascinating inventions. Using common daily-life phenomenon
as a gateway to introduce synthetic biology, we encouraged students to unleash
their imagination and create simple synthetic biology designs.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-8a.webp)

Figure 8: The "Life Code Designer" education activity.

Educational activity at Dehui Special Education School

*2025.5.14*

---

Using daily-life phenomenon as the starting point, we introduced bacteria to children at Dehui Special Education School. We guided them to collaboratively create bacteria models with plasticine. Despite many facing self-care challenges, they cooperated warmly, sharing genuine smiles. These children, like angels with folded wings, may find in biology an interest that fuels their journey ahead, inspiring them to persevere.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-9a.webp)

Figure 9: Educational activity at Dehui Special Education School.

Exchange conference with Fudan University iGEM team

*2025.5.16*

---

We exchanged with Fudan University iGEM team, reaching a consensus on cooperation.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-10a.webp)

Figure 10: Exchange conference with Fudan University iGEM team.

6th post on REDnote

*2025.5.17*

---

We released the sixth post on REDnote, reviewing the gains from the 2nd Northeast
Regional iGEMer Exchange Conference.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-11a.webp)

Figure 11: 6th post on REDnote.

"Plasmid Construction, Campus Orienteering" Challenge

*2025.5.18*

---

We developed the "Plasmid Construction, Campus Orienteering" to promote synthetic biology by orienteering while collecting plasmid fragments at different sites and eventually completing a plasmid at the finishing line.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-12a.webp)

Figure 12: "Plasmid Construction, Campus Orienteering" Challenge.

10th WeChat press release

*2025.5.18*

---

We released the tenth WeChat official account post as a supplement to the eighth post, centering the introduction to the rest iGEM villages.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-13a.webp)

Figure 13: 10th WeChat press release.

11th WeChat press release

*2025.5.24*

---

We released the eleventh WeChat official account post, reviewing
the moments at the second Northeast Regional iGEMer Exchange Conference.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-14a.webp)

Figure 14: 11th WeChat press release.

7th post on REDnote

*2025.5.26*

---

We released the seventh post on REDnote, reviewing our highlights at the grassland music festival.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-15a.webp)

Figure 15: 7th post on REDnote.

Synthetic Biology in Seven Days-Advanced Edition - Chapter 2 Video

*2025.5.28*

---

We recorded a video explaining the second chapter of the learning material, focusing on the award criteria of the iGEM competition and conducted case interpretation on some special awards.

![](https://static.igem.wiki/teams/5542/notebook/edu/may-16a.webp)

Figure 16: Chapter 2 video cover.

June

ABCS Ethical Risk Assessment

*2025.6.5-6.13*

---

We conducted a survey to assess how different stakeholders perceive the ethical risks and benefits of the ABCS system. This targeted survey aligned directly with BEAM indicators, transforming the model from a theoretical framework into a practical assessment tool. The results revealed differing concerns among experts, patients, and the public, allowing us to adjust risk weights and develop the ABCS Ethical Risk Assessment.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/jun-1a.webp)

Figure 1: The ABCS Ethical Risk Assessment Questionnaire.

Interview with Director Jun Wei of the Ethics Committee

*2025.6.14-6.23*

---

We recognized the importance of breast cancer patients’ firsthand insights but faced challenges related to handling sensitive personal data during interviews. To ensure ethical compliance and protect patient rights, we consulted Wei Jun from the Ethics Committee. He advised us to standardize interview procedures and improve the informed consent form for clarity and transparency. Following his guidance, we revised the consent document to clearly explain the interview purpose, data use, and privacy protections in patient-friendly language.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/jun-2a.webp)

Figure 2: Interview with Director Jun Wei and the Informed Consent Form.

Interview with Professor Yujiao Jia

*2025.6.24-6.27*

---

We encountered challenges in respecting patients’ subjectivity and fostering equal dialogue. To address this, we consulted Prof. Yujiao Jia, an expert in philosophy and social sciences. She recommended using a narrative cognitive theory-based life history interview method, involving multi-layered questioning to capture patients’ experiences fully. Prof. Jia also emphasized the importance of suspending the interviewer’s authority, promoting humility, and engaging in open, jargon-free conversations to truly honor patients as narrators of their own lives.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/jun-3a.webp)

Figure 3: Interview with Professor Yujiao Jia.

Interview with Dr. Zijun Zhou, a specialist in breast surgery

*2025.6.28-6.29*

---

As we prepared for in-depth interviews with breast cancer patients, we recognized that technical readiness alone was insufficient due to the patients’ complex psychological states. To address this, we consulted experienced breast surgeon Dr. Zhou who shared insights on patients’ common psychological challenges-identity confusion, anxiety about recurrence, and social withdrawal-and offered communication strategies emphasizing empathy and active listening.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/jun-4a.webp)

Figure 4: Interview with Dr. Zijun Zhou.

Interview with the AI Simulated Patient

*2025.6.30-7.7*

---

Before actually interviewing patients, we were still concerned that our approach might not be mature enough and could unintentionally cause secondary emotional harm. So, we decided to use Doubao AI to help us assess whether our interview style met the requirements suggested by the experts we consulted.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/jun-5a.webp)

Figure 5: Interview with the AI Simulated Patient.

The second iBridge exchange meeting

*2025.6.1*

---

We participated in the second iBridge exchange meeting, presenting this year's projects and sharing the competition experiences with other teams.

![](https://static.igem.wiki/teams/5542/notebook/edu/jun-1a.webp)

Figure 1: Screenshot of the online meeting.

Virtual laboratory-1: Preparation of competent cells

*2025.6.3*

---

We have completed the virtual experiment of competent cells preparation, and completed the interface layout design of the virtual laboratory.

![](https://static.igem.wiki/teams/5542/notebook/edu/jun-2a.webp)

Figure 2: Virtual laboratory-1: Preparation of competent cells.

8th post on REDnote

*2025.6.10*

---

We released the eighth post on REDnote, describing the detailed work content of each group in our iGEM team.

![](https://static.igem.wiki/teams/5542/notebook/edu/jun-3a.webp)

Figure 3: 8th post on REDnote.

Virtual laboratory-2: Bacterial transformation

*2025.6.12*

---

We completed the virtual experiment of bacterial transformation, and carried out the function test and performance optimization.

![](https://static.igem.wiki/teams/5542/notebook/edu/jun-4a.webp)

Figure 4: Virtual laboratory-2: Bacterial transformation.

12th WeChat press release

*2025.6.14*

---

We released the twelfth WeChat official account post, reviewing our highlights at the grassland music festival.

![](https://static.igem.wiki/teams/5542/notebook/edu/jun-5a.webp)

Figure 5: 12th WeChat press release.

Synthetic Biology in Seven Days-Advanced Edition - Chapter 3 Video

*2025.6.20*

---

We recorded this video to explain the third chapter of the learning material, focusing on the concept and key points of synthetic biology.

![](https://static.igem.wiki/teams/5542/notebook/edu/jun-6a.webp)

Figure 6: Chapter 3 video cover.

13th WeChat press release

*2025.6.28*

---

We released the thirteenth WeChat official account post, centering the CRISPR gene editing technology.

![](https://static.igem.wiki/teams/5542/notebook/edu/jun-7a.webp)

Figure 7: 13th WeChat press release.

Virtual laboratory-3: DNA digestion and ligation

*2025.6.29*

---

We have completed the process decomposition, animation production and algorithm implementation of DNA digestion and ligation experiment, aiming to establish a virtual experiment platform and integrate popular science game elements.

![](https://static.igem.wiki/teams/5542/notebook/edu/jun-8a.webp)

Figure 8: Virtual laboratory-3: DNA digestion and ligation.

July

Interview with Professor Ling Ren from the School of Business

*2025.7.8-7.9*

---

We faced challenges in defining our marketing strategy. To address this, we consulted Prof. Ren Ling from the School of Business, who advised us to adopt a phased approach-starting with ToB technical services before expanding to ToC products. Guided by her insights, we formulated a market segmentation strategy.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/jul-1a.webp)

Figure 1: Interview with Professor Ling Ren and the Business Canvas for ABCS

Interview with Mr. Xue

*2025.7.10-7.12*

---

We realized the challenge lay in effectively communicating our technical value to investors. To address this, we interviewed Mr. Xue, an expert in biotech investment and entrepreneurship. He emphasized the importance of storytelling-translating technical strengths into accessible language, linking them to market needs, and showcasing both commercial potential and social impact. Based on his advice, we developed the "ABCS Brand Strategy House" to clearly articulate our value proposition and enhance investor appeal.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/jul-2a.webp)

Figure 2: Interview with Mr. Xue and the "ABCS Brand Strategy House"

Interview with the Human Resources Manager of Guangzhou Wonfo Biotechnology Co., Ltd

*2025.7.13-2025.7.14*

---

To better understand how to build a talent system aligned with commercialization goals, we consulted executives from Wondfo, a company experienced in medical innovation. They emphasized the importance of interdisciplinary talent and shared strategies like cross-functional collaboration, departmental rotation, and integrated workshops. Inspired by their insights, we created a tailored talent development plan for the ABCS project.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/jul-3a.webp)

Figure 3: Interview with the HR of Wondfo Biotechnology Co., Ltd.

Interview with Professor Haoran Yu, Department of Mathematics

*2025.7.15-7.16*

---

To evaluate Gluc’s renal clearance efficiency, we planned to simulate its transport across the glomerular filtration barrier but faced two data gaps: no literature on Gluc’s filtration rate and no basement membrane pore size distribution data, so we consulted Prof. Haoran Yu. He suggested using the normal distribution of stochastic biological structures and the barrier’s known physical parameters to reasonably assume the basement membrane’s pore size, and further recommended coupling macro renal clearance data with micro structural modeling.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/jul-4a.webp)

Figure 4: Interview with Professor Haoran Yu, Department of Mathematics

Interview with Senior Researcher Yin Rui

*2025.7.17-7.31*

---

Via literature review, we noted GFB is traditionally 3-layered. After confirming the first two layers’ parameters, we faced unresolvable discrepancies in the third layer’s reported parameters. We then interviewed Senior Researcher Rui Yin, who validated our exploration and explained the discrepancy: conventional “third layer” is often ambiguously defined, but should be split into two sub-layers. He advised investigating their specific parameters separately.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/jul-5a.webp)

Figure 5: Interview with Senior Researcher Yin Rui.

9th post on REDnote

*2025.7.4*

---

We participated in the SUSTech-SynBio Community Exchange Meeting as special guests, sharing the experience of achieving last year’s excellent results.

![](https://static.igem.wiki/teams/5542/notebook/edu/jul-1a.webp)

Figure 1: 9th post on REDnote.

14th WeChat press release

*2025.7.5*

---

We released the 14th WeChat official account post, centering on the technology of TALENs.

![](https://static.igem.wiki/teams/5542/notebook/edu/jul-2a.webp)

Figure 2: 14th WeChat press release.

Teachers’ Reference Book: All-ages Synthetic Biology, Course 1-5

*2025.7.5-2025.7.29*

---

We designed courses 1-5 of our teaching material Teachers’ Reference Book: All-ages Synthetic Biology for teachers, which is divided into three levels: primary school, junior high school and senior high school. The content revolves around the activities: Fuzzy Sticks, Cell Kingdom on Kites, Auction of Genetic Components, Super Little Inventor, and Life Code Designer.

![](https://static.igem.wiki/teams/5542/notebook/edu/jul-3a.webp)

Figure 3: A page from the book.

Synthetic Biology in Seven Days-Advanced Edition - Chapter 4-6 Video

*2025.7.6-2025.7.10*

---

We recorded chapter 4-6 of the learning material Synthetic Biology in Seven Days-Advanced Edition, respectively focusing on experimental technology, including both basic and specific; design modeling, including foundations, theory, and practices; ethical safety, including its position,issues and guiding principles.

![](https://static.igem.wiki/teams/5542/notebook/edu/jul-4a.webp)

Figure 4: Chapter 4 video cover.

The framework of Education wiki page

*2025.7.8*

---

We established the framework of Education page, classified all educational activities and gained an overall concept.

![](https://static.igem.wiki/teams/5542/notebook/edu/jul-5a.webp)

Figure 5: The education framework.

Dongjian’an School Activity in Shanxi Province

*2025.7.13*

---

We held the "Syn-bio Snakes & Ladders and Live Lab Tour" activity in Shanxi Dongjian'an School for the third time,allowing students to easily understand synthetic biology and glimpse authentic experimental settings and expand vision beyond the classroom.

![](https://static.igem.wiki/teams/5542/notebook/edu/jul-6a.webp)

Figure 6: Dongjian’an School Activity in Shanxi Province.

"Auction of Genetic Components" activity

*2025.7.14*

---

We held the "Auction of Genetic Components" activity in Shandong Licun Middle School, using analogy to explain synthetic biology and genetic engineering principles. Also,this activity linked three nearby schools, bridging cutting-edge technology and rural education.

![](https://static.igem.wiki/teams/5542/notebook/edu/jul-7a.webp)

Figure 7: The "Auction of Genetic Components" activity

4th experiment of the virtual laboratory-PCR

*2025.7.16*

---

We completed the fourth virtual lab experiment-PCR, introducing recombinant plasmids into competent cells using heat-shock method.

![](https://static.igem.wiki/teams/5542/notebook/edu/jul-8a.webp)

Figure 8: Virtual lab experiment-PCR

15th WeChat press release

*2025.7.21*

---

We released the fifteenth WeChat official account post, paying tribute to the contributions made by female scientist Ning Yan to the world.

![](https://static.igem.wiki/teams/5542/notebook/edu/jul-9a.webp)

Figure 9: 15th WeChat press release

5th experiment of the virtual laboratory-Cell thawing

*2025.7.22*

---

We completed the fifth virtual lab experiment-cell thawing, offering experience of the complete process of thawing frozen cells.

![](https://static.igem.wiki/teams/5542/notebook/edu/jul-10a.webp)

Figure 10: Virtual lab experiment-Cell thawing

Synthetic Biology in Seven Days-Intermediate Edition - Chapter 1-3 Video

*2025.7.24-2025.7.29*

---

We recorded chapters 1-3 of the Synthetic Biology in Seven Days-Intermediate Edition, focusing on the concept of iGEM, the award criteria and case interpretation, and basic theories. The difference from the advanced version is that we adjusted the knowledge content and presentation form.

![](https://static.igem.wiki/teams/5542/notebook/edu/jul-11a.webp)

Figure 11: Chapter 1 video cover

6th experiment of the virtual laboratory-Cell passage

*2025.7.28*

---

We completed the 6th virtual lab experiment-Cell passage, introducing the procedure of cell passaging.

![](https://static.igem.wiki/teams/5542/notebook/edu/jul-12a.webp)

Figure 12: Virtual lab experiment-Cell passage

August

Interview with Lawyer Junling Bai

*2025.8.1-8.20*

---

Recognizing the legal risks across the ABCS project’s R&D, clinical, and commercialization stages, we consulted Lawyer Bai for professional guidance. She outlined key legal considerations, including biosecurity and gene technology compliance in R&D, clinical trial approvals and device regulations in application, and IP protection and advertising laws in commercialization. Following her advice, we began strengthening our legal risk management system to ensure compliance.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/aug-1a.webp)

Figure 1: Interview with Lawyer Junling Bai.

Interview with Professor Jinshan Zhang from the School of Innovation and Entrepreneurship

*2025.8.21-8.26*

---

To explore how to transform ABCS into a real-world startup, we interviewed Prof. Zhang, an expert in innovation and entrepreneurship. He emphasized shifting from technical to entrepreneurial thinking and introduced the "entrepreneurial capability matrix", highlighting essential areas like market awareness, fundraising, and strategic planning.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/aug-2a.webp)

Figure 2: Interview with Professor Jinshan Zhang.

Obtaining the hospital ethics approval document

*2025.8.27-8.28*

---

We first submitted an application to the ethics committee of China-Japan Union Hospital of Jilin University. The ethics committee conducted a strict review of the application, and we finally successfully obtained the hospital's ethical review approval, laying a legal and compliant foundation for the offline interview activities.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/aug-3a.webp)

Figure 3: Ethics Approval Document.

Completion of the iGEM Entrepreneurship Practice Simulation handbook

*2025.8.29-9.9*

---

To help our team familiarize with entrepreneurial processes and boost decision-making capabilities via collaboration, we developed our own "iGEM Entrepreneurship Practice Simulation", which will let team members grasp the full entrepreneurial process and enhance capabilities through comprehensive sandbox-based simulations.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/aug-4a.webp)

Figure 4: The iGEM Entrepreneurship Practice Simulation Handbook.

Teachers’ Reference Book: All-ages Synthetic Biology, Courses 6-7

*2025.8.2*

---

We designed course 6-7 of Teachers’ Reference Book: All-ages Synthetic Biology for teachers. The content revolves respectively around the "Bacterial Adventure-Our Friends "Activity and popularization for autistic children.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-1a.webp)

Figure 1: A page from the book.

10th post on REDnote

*2025.8.3*

---

We participated in the iBridge Exchange Meeting, sharing the experience of achieving excellent results last year and introducing our 2025 project.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-2a.webp)

Figure 2: 10th post on REDnote.

Synthetic Biology in Seven Days-Intermediate Edition - Chapter 4-6 Videos

*2025.8.5-8.11*

---

We recorded videos explaining the 4-6 chapters of the Synthetic Biology in Seven Days-Intermediate Edition, respectively focusing on the design and analysis methods of synthetic biology, explaining practical information; the importance of modeling in the iGEM competition and its biological foundations; ethical challenges and principles.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-3a.webp)

Figure 3: Chapter 4 video cover.

Attending CCiC (Conference of China iGEMer Community)

*2025.8.6-2025.8.8*

---

We went to Beijing to attend CCiC, conducting presentations and displaying our project posters, and reached a consensus for cooperation through continuous communication with other teams. During this period, we also attended the functional nucleic acid seminar organized by PekingHSC iGEM team, and ultimately we reached a consensus to jointly produce a functional nucleic acid white paper.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-4a.webp)

Figure 4: CCiC on-site pictures.

1st podcast

*2025.8.9*

---

We released the first podcast series, discussing the fermentation function of yeast and the factors that influence its functionality.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-5a.webp)

Figure 5: 1st podcast series.

7th experiment of the virtual laboratory-Cell freezing

*2025.8.10*

---

We designed the seventh virtual lab experiment-Cell freezing, introducing the basics principles and techniques of long-term cell storage.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-6a.webp)

Figure 6: Virtual lab experiment-Cell freezing.

Exchange meetings with international iGEM teams

*2025.8.14-8.19*

---

We met online with 3 international iGEM teams-iGEM Lund, iGEM NYUAD, and iGEM McGill-during this period to exchange our projects and promote our education collaboration.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-7a.webp)

Figure 7: Screenshots from the exchange meetings.

16th WeChat press release

*2025.8.14*

---

We released the sixteenth WeChat official account post, recording the entire process we had at CCiC and sharing our gains.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-8a.webp)

Figure 8: 16th WeChat post.

11th post on REDnote

*2025.8.15*

---

We released the eleventh post on REDnote, documenting the entire process over our three days at CCiC.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-9a.webp)

Figure 9: 11th post on REDnote.

2nd podcast

*2025.8.15*

---

We released the second podcast, which tells the story of beneficial bacteria cleaning the sewers, demonstrating that bacteria can also be helpful.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-10a.webp)

Figure 10: 2nd podcast.

Synthetic Biology in Seven Days- Basic Edition - Chapter 1-2 Videos

*2025.8.17*

---

We recorded a video explaining the first chapter of the Synthetic Biology in Seven Days-Basic Edition, introducing the core concepts of the iGEM competition by using a friendly opening, strong interactivity, and a combination of rich visuals and explanations to enhance perception.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-11a.webp)

Figure 11: Chapter 1 video cover.

12th post on REDnote

*2025.8.20*

---

We released the twelfth post on REDnote, documenting the work we did at CCiC, including team communication, cooperation achievements, and so on.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-12a.webp)

Figure 12: 12th post on REDnote.

3rd podcast

*2025.8.20*

---

We released the third podcast, telling a story of lactic acid bacteria and other bacteria producing lactic acid to fend off harmful bacteria.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-13a.webp)

Figure 13: 3rd podcast.

4th podcast

*2025.8.22*

---

We released the fourth podcast, telling the story of photosynthetic bacteria converting weak light into energy and releasing oxygen through photosynthesis.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-14a.webp)

Figure 14: 4th podcast.

Synthetic Biology in Seven Days- Basic Edition - Chapter 3-6 Videos

*2025.8.29-9.9*

---

We recorded chapter 3-6 videos of the Synthetic Biology in Seven Days-Basic Edition, focusing on these topics:- Introducing iGEM in the form of physical analogies and simple interactive games.
- Introducing the basic knowledge of synbio experiments in a simple pictorial way.
- Simple hands-on biology experiments in a simulated scenario.
- introducing the rules of experiments in iGEM and reviewing the content of the previous five chapters.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-15a.webp)

Figure 15: 4th podcast.

13th post on REDnote

*2025.8.25*

---

We released the thirteenth post on REDnote and created a new series-knowledge cards, revolving which direction the public is most interested in understanding synthetic biology, listing 5 options, and explaining the one with the highest votes in 3 days.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-16a.webp)

Figure 16: 13th post on REDnote.

5th podcast series

*2025.8.25*

---

We released the fifth podcast series, telling the story that acteriophages recognize and eliminate viruses.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-17a.webp)

Figure 17: 5th podcast.

6th podcast

*2025.8.28*

---

We released the sixth podcast, telling the story of how root nodule bacteria convert atmospheric nitrogen into nutrients that can be absorbed by bean sprouts.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-18a.webp)

Figure 18: 6th podcast.

14th post on REDnote

*2025.8.28*

---

We released the fourteenth post on REDnote and created a new series--knowledge cards,revolving which application the public is most interested in understanding synthetic biology, listing 5 options, and explaining the one with the highest votes after 3 days.

![](https://static.igem.wiki/teams/5542/notebook/edu/aug-19a.webp)

Figure 19: 14th post on REDnote.

September

Internal training using the handbook iGEM Entrepreneurship Practice Simulation

*2025.9.10-9.17*

---

Our team built an entrepreneurship ability matrix by using our original "iGEM Entrepreneurship Practice Simulation" handbook, preparing for future responses to actual entrepreneurship challenges.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/sep-1a.webp)

Figure 1: Conducting Internal Training.

Patient feedback word cloud

*2025.9.18-9.20*

---

We organized the feedback collected from patient interviews into a word cloud, ranking terms by frequency to make patients’ needs, concerns, and emotions more visually accessible. This helped us quickly identify recurring themes and gain a clearer, more intuitive understanding of what patients value most.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/sep-2a.webp)

Figure 2: Patient Feedback Word Cloud.

Online patient interview

*2025.9.21-9.29*

---

The ABCS project needs to extensively collect the preferences of breast cancer patients for three testing methods (test kits, test strips, and automatic testing toilets) to ensure that the product form meets the actual needs of patients. Since offline interviews had limited reach, we expanded the research online through the platforms "Mijian" and "Dances with Cancer", ensuring that patient voices were included in shaping our project.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/sep-3a.webp)

Figure 3: Online Patient Interview.

Completion of the business plan

*2025.9.30*

---

The business plan helps us deeply analyze the commercial potential of the project and determine the implementation path. It served not only as a systematic framework connecting our technology to the broader business chain, but also as a key blueprint for attracting resources and guiding practical actions.

![](https://static.igem.wiki/teams/5542/notebook/hp-edu/sep-4a.webp)

Figure 4: The Business Plan.

8th experiment of the virtual laboratory - CCK-8 assay

*2025.9.2*

---

We designed the eighth virtual lab experiment- CCK-8 assay, simulating the adding process of CCK-8 reagent and reading absorbance values.

![](https://static.igem.wiki/teams/5542/notebook/edu/sep-1a.webp)

Figure 1: Virtual lab experiment-CCK-8 assay.

15-17th post on REDnote

*2025.9.4*

---

We released our fifteenth to seventeenth REDnote posts, launching a new knowledge card series focused on community interest. The post featured a poll that offered five different iGEM "village" topics. To overcome the platform’s limitation on voting options, we released two follow-up posts on September 15th and 19th to collect votes for the remaining villages.

![](https://static.igem.wiki/teams/5542/notebook/edu/sep-2a.webp)

Figure 2: 15-17th post on REDnote.

Exchange meeting with iGEM Munich

*2025.9.8*

---

We established contact with iGEM Munich to exchange our projects and promote collaboration on education .

![](https://static.igem.wiki/teams/5542/notebook/edu/sep-3a.webp)

Figure 3: Meeting with iGEM Munich.

9th experiment of the virtual laboratory-Cell transfection

*2025.9.10*

---

We designed the ninth virtual lab experiment-Cell transfection, focusing on choosing suitable transfection reagents and plasmid DNA.

![](https://static.igem.wiki/teams/5542/notebook/edu/sep-4a.webp)

Figure 4: Virtual lab experiment-Cell transfection.

17th WeChat press release

*2025.9.13*

---

We released the seventeenth WeChat official account post, which introduced how synthetic biology uses gene-editing technology to modify biological systems, showcasing applications in areas like eco-friendly plastics and cultured meat.

![](https://static.igem.wiki/teams/5542/notebook/edu/sep-5a.webp)

Figure 5: 17th WeChat post.

10th experiment of the virtual laboratory - Cell infection

*2025.9.21-9.29*

---

We designed the tenth virtual lab experiment-Cell infection, to help learners understand virus-mediated gene expression and its applications in research.

![](https://static.igem.wiki/teams/5542/notebook/edu/sep-6a.webp)

Figure 6: Virtual lab experiment-Cell infection.

Lab daily Vlog on TikTok

*2025.9.28*

---

We posted a video on TikTok showing the daily routines of our Experiment group.

![](https://static.igem.wiki/teams/5542/notebook/edu/sep-7a.webp)

Figure 7: The Tiktok video.

Wiki
![](https://static.igem.wiki/teams/5542/img/h1-background.webp)

December

WIKI & Videos group member recruitment

*2024.12.4-2025.1.4*

---

Upon the completion of our recruitment process, we are proud to introduce our group members: Jiaqi Qu; Fuzhi Wang; Yucheng Liu; Yilong Liao.

January

1st Brainstorm and drafts on wiki design

*2025.1.4-2025.1.15*

---

Group members focused on different wiki sections according to preference and creative ideas were put forward. The initial drafts of the Team and the HP sections were prepared, while members provided comprehensive insights on web design optimization. We shared designs of visual elements for the homepage (including the Safety, Awards, and Notebook sections), a structured navigation bar and the table of contents.

![](https://static.igem.wiki/teams/5542/notebook/wiki/jan-1a-2.webp)

Figure 1: Screenshots of PPT from the First Group Meeting.

2nd Brainstorm and drafts on Wiki design

*2025.1.15-2025.1.25*

---

The Team page design was refined through the integration of illustrated member avatars and the navigation bar. Simultaneously, the HP page layout was optimized with matching components to ensure visual consistency. We proposed the first version of our table of contents and footer, and confirmed the font design.

![](https://static.igem.wiki/teams/5542/notebook/wiki/jan-2a-2.webp)

Figure 2: Screenshots of PPT from the Second Group Meeting.

February

3rd Brainstorm and drafts on Wiki design

*2025.2.6-2025.2.13*

---

Group members focused on different wiki sections, refining IHP pages while ensuring design consistency. The navigation bar, table of contents, and background color were standardized. We updated the Team page layout, added our PI and Instructors’ labels, and initiated the Notebook page. The Description page incorporated a navigation bar, a table of contents, and a footer. In the mean time we progressed on the Model section. The footer design was revised, and homepage visuals, including the HP page illustration, were created.

![](https://static.igem.wiki/teams/5542/notebook/wiki/feb-2a.webp)

Figure 1: Screenshots of PPT from the Third Group Meeting.

4th Brainstorm and drafts on Wiki design

*2025.2.14-2025.2.22*

---

Group members continued refining sections, enhancing visual elements, and improving functionality across the wiki page. Updates were made to the IHP and other key page banners, along with adjustments to the footer for a more cohesive presentation. The Team page visuals were refined, as we optimized the character presentation for HP, Education, and Presentation group sections. We added more details to the Notebook page structure, including the design of overview images as interactive effects and the AI model underwent further modifications. Also, refinements were made to the Description page and navigation bar, ensuring proper integration of key identifiers and our university logo.

![](https://static.igem.wiki/teams/5542/notebook/wiki/feb-1a-2.webp)

Figure 2: Screenshots of PPT from the forth group meeting.

March

A series of events between March 2nd, 2025 and March 15th, 2025

Apricot Blossom Festival poster

---

A beautifully designed event poster was brought to life by our talented wiki group.

![](https://static.igem.wiki/teams/5542/notebook/wiki/mar-1a.webp)

Figure 1: The apricot Blossom Festival poster.

5th brainstorm and drafts on wiki design

---

Based on the needs of the Education group, we developed an educational platform and optimized several webpage widgets. Collaboration with group members is still ongoing.

![](https://static.igem.wiki/teams/5542/notebook/wiki/mar-1c.webp)

Figure 2: The initial version of the educational platform.

6th brainstorm and drafts on wiki design

---

We created a draft Attribution page referring to last year's CJUH-JLU-China team information and optimized our self-designed education platform.

![](https://static.igem.wiki/teams/5542/notebook/wiki/mar-1d.webp)

Figure 3: Screenshots of PPT from the seventh group meeting.

Preliminary design of the team logo

*2025.3.23-2025.3.30*

---

Based on the meaning of our project, we have preliminarily designed six versions of the team logo for selection.

![](https://static.igem.wiki/teams/5542/notebook/wiki/mar-3a.webp)

Figure 6: Six versions of the team logo.

Seventh Brainstorm and drafts on Wiki design

*2025.3.23-2025.3.30*

---

We further refined the webpages we had already built, assisted the Education group in designing jigsaw puzzles and implementing the web-based virtual lab, and also held discussions with the Model group.

![](https://static.igem.wiki/teams/5542/notebook/wiki/mar-4a.webp)

Figure 7: Our new semester started at the end of February, and our

---

 brainstorming meetings have transitioned to offline discussions.

---

Here's a photo from one of our sessions!

April

The final version of the team logo

*2025.4.1-2025.4.6*

---

After several rounds of screening, we determined the final version of the team logo.

![](https://static.igem.wiki/teams/5542/notebook/wiki/apr-1a.webp)

Figure 1: Final version of the team logo.

Progress of the Wiki group

*2025.4.1-2025.4.6*

---

We have completed the operational part and animation interaction of Experiment 1 in the virtual lab, and created the video page for the educational platform.

The design of the Aeroplane Chess cards

*2025.4.1-2025.4.6*

---

Our Gene Voyage is taking off! We've finished designing and illustrating our very own Aeroplane Chess cards.

![](https://static.igem.wiki/teams/5542/notebook/wiki/apr-2a.webp)

Figure 2: The design of the Aeroplane Chess cards.

Design of the team uniform

*2025.4.1-2025.4.6*

---

We designed multiple team uniform concepts based on the team's color scheme,
logo, and website theme. After two rounds of selection and revisions,
we finalized the uniform design and began production.

![](https://static.igem.wiki/teams/5542/notebook/wiki/apr-4a.webp)

Figure 4: Final version of the team uniform.

Design of the team flag

*2025.4.21-2025.4.30*

---

We designed our team flag by incorporating our team name, logo, and the official iGEM emblem.

![](https://static.igem.wiki/teams/5542/notebook/wiki/apr-5a.webp)

Figure 3: The team flag.

May

Drafts on Model and Engineering web page header images

*2025.5.1-2025.5.25*

---

We finished the drafts of Model and Engineering web page header images.

![Gene Component Auction lecture](https://static.igem.wiki/teams/5542/notebook/wiki/may-1a.webp)

Figure 1: Model web page header image.

![Planting the Seeds of Synthetic Biology lecture](https://static.igem.wiki/teams/5542/notebook/wiki/may-1b.webp)

Figure 2: Engineering web page header image.

June

Half-body portraits of the Model group members

*2025.6.1-2025.6.8*

---

To better showcase our team members, we began to arrange half-body portraits for our team members, starting from the Model group.

![](https://static.igem.wiki/teams/5542/notebook/wiki/jun-1a.webp)

Figure 1: Half-body portraits of the Model group members.

Refinement of the Attributions web page header image

*2025.6.8-2025.6.30*

---

In order to better suit our features, Wiki members have further improved the
Attributions page header image.

![](https://static.igem.wiki/teams/5542/notebook/wiki/jun-3a.webp)

Figure 4: The refined Attributions web page header image.

July

CCiC (Conference of China iGEMer Community) poster design

*2025.7.1-2025.7.8*

---

The annual event CCiC is a great chance to exchange with national iGEM teams and gain inspiration for improving our own work. We designed a poster for the conference and planned to share our design ideas with other iGEM teams.

![](https://static.igem.wiki/teams/5542/notebook/wiki/jul-1a.webp)

Figure 1: CCIC poster design.

Logo for the ethical risk assessment model "BEAM"

*2025.7.9-2025.7.13*

---

Based on the meaning of "BEAM", the image of a lighthouse came to our mind. Therefore, the logo features a lighthouse as its main element, radiating light in all directions. Since "beam" is an ethical model, the little figures holding hands symbolize its people-centered philosophy.

![](https://static.igem.wiki/teams/5542/notebook/wiki/jul-2a.webp)

Figure 2: Logo for BEAM.

Completion of the Contribution wiki page cover image

*2025.7.14-2025.7.20*

---

Regarding the meaning of the "contribution" page in iGEM, we started with the Earth as the basic image, then expanded the idea to include tides and the universe. This process eventually led to the final version of the full image.

![](https://static.igem.wiki/teams/5542/notebook/wiki/jul-3a.webp)

Figure 3: The Contribution wiki page cover image.

The creation of 4 cover images for the front pages

*2025.7.14-2025.7.20*

---

Based on the main ideas represented by different pages, we created the drawing for the iHP, Model, Engineering, and Collaboration page.

![](https://static.igem.wiki/teams/5542/notebook/wiki/jul-4a.webp)

Figure 4: Drafts of the 4 cover images.

Completion of 4 cover images

*2025.7.14-2025.7.20*

---

Based on the main themes, we designed the illustration for the Design, Protocol, Parts, and Results page.

![](https://static.igem.wiki/teams/5542/notebook/wiki/jul-5a.webp)

Figure 5: Drafts of the 4 cover images.

August

Virtual Laboratory page design

*2025.8.1-2025.8.10*

---

In order to make the virtual laboratory pages look more attractive, their layouts have been redesigned.

![](https://static.igem.wiki/teams/5542/notebook/wiki/aug-1a.webp)

Figure 1: New Virtual Laboratory page design.

Mechanism animation graphic design

*2025.8.11-2025.8.17*

---

To enhance the clarity and appeal of our project mechanism in the promotion video, we designed a series of custom graphics to visually explain the core science.

![](https://static.igem.wiki/teams/5542/notebook/wiki/aug-2a.webp)

Figure 2: Graphics for the promotion video.

Completed the cover image of the "Description", "Education" page

*2025.8.18-2025.8.24*

---

We improved the "Description", "Education" cover image.

![Gene Component Auction lecture](https://static.igem.wiki/teams/5542/notebook/wiki/aug-3a.webp)

Figure 3: Cover image of the "Description" page.

![Planting the Seeds of Synthetic Biology lecture](https://static.igem.wiki/teams/5542/notebook/wiki/aug-3b.webp)

Figure 4: Cover image of the "Education" page.

We moved the Description page from local hosting to the official iGEM website

*2025.8.18-2025.8.23*

---

On August 18, 2025, we deployed the Description page to our locally hosted site. After five days of revisions and polishing, on August 23, 2025, we officially published it on the iGEM website.

Completed the cover image of the "Safety" page

*2025.8.25-2025.8.31*

---

Integrating the shield and the spear, which symbolize protection, we completed the cover image of "Safety".

![](https://static.igem.wiki/teams/5542/notebook/wiki/aug-4a.webp)

Figure 5: Cover image of the "Safety" page.

September

Completion of the Model page cover image

*2025.9.1-2025.9.7*

---

Based on the elements of our two models, we designed the cover image of Model page.

![](https://static.igem.wiki/teams/5542/notebook/wiki/sep-1a.webp)

Figure 1: Cover image of the Model page.

Completion of the Software page cover image

*2025.9.1-2025.9.7*

---

Integrating the unique technological elements of the software, the cover image of the software was designed.

![](https://static.igem.wiki/teams/5542/notebook/wiki/sep-2a.webp)

Figure 2: Cover image of the Software page.

Completion of the Design page cover image

*2025.9.8-2025.9.14*

---

In response to the requirements for beautification in the promotion video, some graphic refinement has been carried out.

![](https://static.igem.wiki/teams/5542/notebook/wiki/sep-3a.webp)

Figure 3: The Design page cover image.

Completion of the iHP page cover image

*2025.9.8-2025.9.14*

---

The cover image of iHP website was designed to convey a sense of connecting to the world.

![](https://static.igem.wiki/teams/5542/notebook/wiki/sep-4a.webp)

Figure 4: Cover image of the iHP page.

Completion of the Engineering page cover image

*2025.9.8-2025.9.14*

---

We carefully designed and developed the final page drawing on the scenarios we envisioned and our understanding of the Engineering page.

![](https://static.igem.wiki/teams/5542/notebook/wiki/sep-5a.webp)

Figure 5: Cover image of the Engineering page.

Completion of the cover image for the art page

*2025.9.15-2025.9.21*

---

Drawing on the scenarios we envisioned and our understanding of the Art page, we carefully designed and developed the final Art page.

![](https://static.igem.wiki/teams/5542/notebook/wiki/sep-6a.webp)

Figure 6: Cover image of the Art page.

Completion of the Results page cover image

*2025.9.15-2025.9.21*

---

Based on the connection with "results", the image of wheat ears was added, aiming to convey the idea of achieving a harvest.

![](https://static.igem.wiki/teams/5542/notebook/wiki/sep-7a.webp)

Figure 7: Cover image of the Results page.

Home Page Design

*2025.9.15-2025.9.21*

---

The homepage hero image was created by integrating our project theme and the main character design.

![](https://static.igem.wiki/teams/5542/notebook/wiki/sep-9a.webp)

Figure 8: Home Page Design.

Completion of the Virtual Laboratory

*2025.9.21*

---

After more than half a year of dedicated effort, our Wiki team and HP & Education team have successfully completed the Virtual Laboratory. It contains ten biology experiments and represents a great deal of hard work. A detailed introduction is provided on our Wiki.

Completion of the Medal page cover image

*2025.9.22-2025.9.31*

---

Based on the association with medal, we aimed to convey a golden and radiant visual aesthetic for the cover image.

![](https://static.igem.wiki/teams/5542/notebook/wiki/sep-8a.webp)

Figure 9: Cover image of the Medal page.

Design of the Loading Animation

*2025.9.22-2025.9.31*

---

Based on our unique web style, we designed a distinctive loading page.

![](https://static.igem.wiki/teams/5542/notebook/wiki/sep-10a.webp)

Figure 10: Design of the Loading Animation.

Home Subpage in Picture Book Style

*2025.9.22-2025.9.31*

---

Aiming for a unique home subpage design, we eventually selected a picture book style rich in texture.

![](https://static.igem.wiki/teams/5542/notebook/wiki/sep-11a.webp)

Figure 11: Home Subpage in Picture Book Style.

Presentation
![](https://static.igem.wiki/teams/5542/img/h1-background.webp)

December

Presentation Group Member Recruitment

*2024.12.14-2024.12.22*

---

Through team recruitment, our presentation group was set up by 4 outstanding undergraduate
students from Jilin University, Sirui Dong, Changning Fu and Hongrui Yu from the first
clinical school and Zimu Li from the School of Chemistry.

January

Preliminary Brainstorm of Promotion Video

*2024.12.14-2025.01.20*

---

During the initial phase of the Presentation group's activities, we prepared the
Material Library of project’s promotion video to make members be more familiar
with iGEM project and presentation work. Through brainstorming, discussions and
learning promotion video of past gold projects, we finally came up with 3 different
design of promotion video.

![](https://static.igem.wiki/teams/5542/notebook/pre/jan-1a.webp)

Figure 1: Screenshot of the PPT for the Promotion Video
 Online Group Meeting.

Design of Attribution page

*2025.1.20-2025.1.27*

---

According to the review of judges in 2024 jamboree, some problems were pointed out in our attribution page that we need to solve this year. Therefore, we decided to consider our attribution page earlier to avoid the mistakes. After case studies of outstanding projects, we designed the components and texts of this year’s attribution page.

February

Design and Development of Notebook Page

*2025.01.20-2025.02.22*

---

In our review of 2024 jamboree, our notebook had problems in page loading and readability.
Therefore, we start planning our notebook at the begin of this year’s project. We conducted
case studies of top teams JU-Krakow and Heidelberg, making grate updates in way of presenting
our notebook. After design is decided, we made demo web page with the help of wiki group.

![](https://static.igem.wiki/teams/5542/notebook/pre/feb-1a.webp)

Figure 1: Screenshot of the PPT for the Notebook
Online Group Meeting.

March

Event: Drafting of questions in Web Helper

*2025.03.09-2025.03.16*

---

The idea of creating an online web helper in our wiki came up by our wiki group members. Inspired by websites that built-in chatbots or virtual assistants, we recognized that we could develop our own chatbots in our wiki to answer questions relative to our project that viewers of our wiki may ask. Therefore, our project can be interpreted better and help iGEMers locating what they are curious about more quickly. We brainstormed some questions people may ask and collect them down. We will continue collecting questions and answer them while the project develops gradually.

![](https://static.igem.wiki/teams/5542/notebook/pre/mar-1a.webp)

Figure 1: Questions for the Web Helper.

Preliminary Design and Drafting of Safety Page

*2025.03.23-2025.04.05*

---

The safety page was one of the weak points in our wiki last year. In the past, we considered safety to be a limited concept that only involved wet lab safety. However, after viewing other teams’ wiki, we have realized that safety is a multifaceted concept, which not only about laboratory safety but also biosafety of projects and the social and psychological safety in HP activities etc. For this reason, we started preparing the safety page in advance, redesigning its content outline and developing the page layout.

![](https://static.igem.wiki/teams/5542/notebook/pre/mar-2a.webp)

Figure 2: Screenshot of the PPT for the Safety Page
Online Group Meeting.

Preliminary Preparation of Presentation video

*2025.02.26-2025.03.09*

---

We prepared the Material Library of project’s presentation video early this year to ensure our video attractive and suitable to project. Through brainstorming, discussions and learning presentation video of past iGEM projects, we finally came up with 3 different designs of presentation video.

Illustration of Project Mechanism Flowchart

*2025.03.23-2025.04.20*

---

We created the project mechanism flowchart with the help of BioRender. With the help of the experiment group, we drafted the initial chart and improved it through wide discussion. After 2 times of completely revised and redrawn, we illustrated the first version of project mechanism flowchart.

![](https://static.igem.wiki/teams/5542/notebook/pre/mar-3a.webp)

Figure 3: The Iterative Process of Illustrating the
Project Mechanism Flowchart.

April

Launch of Safety page

*2025.04.05-2025.04.20*

---

After a month of dedicated effort, we completed the drafting and refinement of the safety
documentation. We also visited the laboratory to photograph and organize visual materials
of the safety equipment. With the support of the Wiki group, we finalized a local demo
version of the Safety webpage.

![](https://static.igem.wiki/teams/5542/notebook/pre/apr-1a.webp)

Figure 1: Photos collected for Safety page.

Refinement of project mechanism flowchart

*2025.04.20-2025.05.18*

---

We update the project mechanism flowchart with the help of BioRender.
We revisited and restructured the modeling of tumor cell–adipocyte interactions,
with a particular focus on detailing the cytokine-mediated pathways affecting
adipocytes, to enhance the clarity and communicability of our project’s underlying
mechanism.

![](https://static.igem.wiki/teams/5542/notebook/pre/apr-2a.webp)

Figure 2: Updated project mechanism flowchart

May

Preliminary design of the promotion video

*2025.04.28-2025.05.18*

---

We have started designing the promotion video and completed the first draft of the
script, along with a preliminary plan for the visual style and content.
After discussions with Experiment group, the script was refined and some
inaccuracies in the part mentioning the project’s theoretical foundation
were corrected.

![](https://static.igem.wiki/teams/5542/notebook/pre/may-1a.webp)

Figure 1:Part of our initial design.

Drafting the Description section

*2025.05.06-2025.05.18*

---

We developed an outline and accordingly finished the first draft
of the Description section.

![](https://static.igem.wiki/teams/5542/notebook/pre/may-2a.webp)

Figure 2:The initial outline of Description.

Description restructuring and content revision

*2025.05.18-2025.06.05*

---

Our first draft felt too rigid, so we revised the content based on a restructured
framework, resulting in a more reader-friendly Description section.

![](https://static.igem.wiki/teams/5542/notebook/pre/may-3a.webp)

Figure 3: The restructured Description framework.

July

Filming the promotion video

*2025.7.10- 2025.8.1*

---

We began filming the footage of our promotion video. Unlike last year’s animation, this year we adopted live-action shooting, and involved many of our team members in the filming process.

![](https://static.igem.wiki/teams/5542/notebook/pre/jul-1a.webp)

Figure 1: CCIC poster design

August

Editing the promotion video

*2025.8.1-8.12*

---

After filming the footage in July, we edited and uploaded this year’s promotion video.

![](https://static.igem.wiki/teams/5542/notebook/pre/aug-1a.webp)

Figure 1: Cover of the promotion video.

Description page writing

*2025.8*

---

started writing the Description page. This page presents the core objectives, technical road map, and scientific significance of the experiment to the entire project.

Education page writing

*2025.8*

---

This month we co-created the Education page with the Education group, revised the text and translated it into English.

Improving the Team page

*2025.8*

---

In the writing of the iGEM Team page, we clearly presented the interdisciplinary architecture of the team, introduced the modules they were responsible for, such as experimental operations, data simulation, and promotional design, and deeply linked the team roles with project goals, refining their work content.

September

Human Practices page writing

*2025.9*

---

This month we co-created the Human Practices page with the HP group, revised the text and translated it into English.

Presentation video design and script writing

*2025.9*

---

We started planning our presentation video, and finished the draft of our script along with initial visual design.

Manuscript writing and revision of Model Page

*2025.9*

---

We finished writing the Model wiki content. By communicating with members of the Model group, we gained a good understanding of the models, and then revised and polished the manuscript and the wiki page’s image layout.

{% endblock %}
{% block addJS %}

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block addCSS %}

{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5542/engineering/engineering-cover.webp)

![](https://static.igem.wiki/teams/5542/home/home/label/engineering-label.webp)

* [Overview
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#engineering-overview)
* [Cycle1: Identification of biomarker combinations
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#engineering-cycle1)

  [Design
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle1-design)
  [Build
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle1-build)
  [Test
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle1-test)
  [Learn
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle1-learn)
* [Cycle 2: Optimal design of sensor sequences
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#engineering-cycle2)

  [Design
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle2-design)
  [Build
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle2-build)
  [Test
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle2-test)
  [Learn
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle2-learn)
* [Cycle 3: Exploration of the reaction kinetics
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#engineering-cycle3)

  [Design
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle3-design)
  [Build
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle3-build)
  [Test
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle3-test)
  [Learn
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle3-learn)
* [Cycle 4: Ethical assessment of our project with BEAM
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#engineering-cycle4)

  [Design
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle4-design)
  [Build
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle4-build)
  [Test
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle4-test)
  [Learn
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#engineering-cycle4-learn)

![](https://static.igem.wiki/teams/5542/img/reel-lower.webp)
![](https://static.igem.wiki/teams/5542/img/pen-bg.webp)

Overview
![](https://static.igem.wiki/teams/5542/img/h1-background.webp)
![](https://static.igem.wiki/teams/5542/img/h1-leave-left.webp)
![](https://static.igem.wiki/teams/5542/img/h1-leave-right.webp)
Our project, ABCS is a breast cancer surveillance system specifically designed for women who had breast cancer or undergone breast surgery. The core of our project is to develop an effective AND gate sensor sequence that can detect RNA biomarkers of cancer associated adipocytes and report an output signal that can be easily detected.
Here we show 4 engineering cycles used in our project:

Cycle 1
Identification and validation of optimal biomarker combinations

Cycle 2
Design and test of the optimal sensor sequences

Cycle 3
Exploration of the reaction kinetics of output signal

Cycle 4
Ethical assessment of our project with the Bio-Ethical Assessment Model

![](https://static.igem.wiki/teams/5542/engineering/engineering-1-1.webp)
Four engineering cycles contribute to project progress step by step

Each engineering cycle represents a crucial problem we need to solve through combination of wet lab, dry lab and integrated human practice when developing ABCS. In the stage of Design, we utilize modeling to guide our experiment. Following the design, we build our system in eligible chassis. In the stage of Test, we verify our building by experiments. Finally, we summarize our findings and reflect on design improvements to advance our project in the stage of Learn.

Cycle1: Identification of biomarker combinations
![](https://static.igem.wiki/teams/5542/img/h1-background.webp)
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Design
To establish an ideal surveillance system for breast cancer, the initial problem we need to solve is what biomarker we detect for.
Through reported research we knew that specific genes can be significantly upregulated in adipocytes as they transformed into cancer-associated adipocytes (CAAs) under the influence of tumor microenvironment of breast cancer.
Aiming to find out the optimal single gene or gene combination with high specificity for breast cancer recognition, we integrated data from public databases (TCGA, GTEx, UCI) and developed a multi-layer perceptron classification model incorporating an Attention mechanism (Attention-MLP) to make it perform better when handling high-dimensional data. Parameters in Attention-MLP were continuously optimized through binary cross-entropy loss and backpropagation, while the classification threshold determined by Youden's index. Attention-MLP shows better performance over traditional MLP across multiple UCI public datasets.
Subsequently, through literature review, we shortlisted four candidate genes that have been reported to have significantly increased expression in CAAs: PLOD2, LIF, FAM3C, and IL-6. We then evaluated the performances of all combinations of these 4 genes using Attention-MLP Model. The results were assessed based on ROC curves and the AUC metric.
As illustrated in Figure.2, the PLOD2 and LIF gene combination achieved the highest AUC value, indicating the best predictive performance and thus was selected as the optimal gene combination to target in our engineered adipocytes.

![](https://static.igem.wiki/teams/5542/model/model-bcs-top5-combinations-roc.webp)
ROC Curve of Gene Combinations

Build
Following the analysis of optimal gene combination by Attention-MLP model, we proceeded to validate the best gene combination with wet-lab experiments. The aim was to confirm that the expression of PLOD2 and LIF in human mature adipocytes is significantly and synergistically upregulated within a specific tumor microenvironment.
First, human preadipocytes were successfully differentiated into mature adipocytes using a commercial adipogenic induction kit.
Based on literature review we knew that PLOD2 and LIF expressions can be upregulated by PAI-1 and CXCLs that are secreted from breast cancer cells respectively, so the differentiated adipocytes were treated by PAI-1 and CXCL8. Subsequently, qPCR analysis was performed to measure the expression levels of PLOD2 and LIF, with GAPDH used as an internal reference.
Test
We treated differentiated human adipocytes with PAI-1 and CXCL8 and subsequently measured the expression levels of PLOD2 and LIF via qPCR.

![](https://static.igem.wiki/teams/5542/engineering/engineering-3.webp)
Expression levels of target genes after cytokines treatment

The qPCR result revealed that, compared to untreated cells, PAI-1 treatment significantly upregulated PLOD2 expression, and CXCL8 treatment significantly upregulated LIF expression (Figure 3).
Learn
From the results of our test, we can confirm that the selected gene combination, PLOD2 and LIF, undergoes upregulation in the treatment of cytokines that are secreted from breast cancer cells, indicating that PLOD2 and LIF are optimal biomarker combination in adipocytes to detect breast cancer.
The validated result of PLOD2+LIF in wet experiments also demonstrated that Attention-MLP Model is effective for biomarker identification. However, due to the limitation in time we haven’t finished the verification of other gene combinations, which is required to sufficiently validate the accuracy Attention-MLP model.

Cycle 2: Optimal design of sensor sequences
![](https://static.igem.wiki/teams/5542/img/h1-background.webp)
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Design
To design RADAR sensor sequences targeting PLOD2 and LIF with high binding specificity, we used a variety of bioinformatical tools. The function of RNA sensor not only depends on its sequence but also highly depends on its secondary structure after folding. In our project, the secondary structure of sensors will directly influence the biomarker recognition and dsRNA formation between sensor sequences and their targets, which ultimately determine the efficiency of ADAR-mediated editing. Multiple studies demonstrate that the binding efficiency of the sensor region to its target RNA is positively correlated with the degree of exposure of the sensor region, i.e. the more exposed the binding region, the more readily binding occurs.
Therefore, we employed the iPknot++ algorithm to simulate the secondary structures of candidate sensor sequences. By calculating the unpaired probability and the exposure length of the editing site, we selected sensor regions with high exposure and favorable editing site accessibility as candidate sequence variants.
Specifically, we first identified candidate sensor sequences for each gene, then defined the "unpaired probability" as the ratio of unpaired bases to total bases in the sensor region, and the "exposed length of the editing site" as the longest consecutive unpaired bases near the editing site. These two metrics were used to quantitatively evaluate the candidate sequences.
Finally, we simulated the secondary structures of several constructed candidate RNA sequences (as listed in the table below) and screened Seq C as the functionally optimal sensor sequence, based on both unpaired probability and exposed length.
In Figure 4 we show simulated conformation and predicted results.
For detailed computational prediction procedures, please refer to the [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) page.

![](https://static.igem.wiki/teams/5542/engineering/engineering-4.webp)
Results of Seq C functional structure assessment

Build
Based on the optimal PLOD2&LIF sensor sequence (Seq C) designed by our model group, we constructed the PLOD2&LIF–Gluc sensor sequence and integrated it into the RADAR system to validate its effectiveness.
Mature adipocytes derived from induced differentiation were infected with rAAV9 carrying the PLOD2&LIF–Gluc sensor sequence. Two days post-infection, the cells were subjected to different treatment conditions: no cytokine treatment, individual treatments with PAI-1 or CXCLs, and combined treatment with both cytokines.
To better simulate the in vivo interaction between breast cancer cells and adipocytes, we also treated the engineered adipocytes with conditioned medium from MDA-MB-231 cells. The effectiveness of the PLOD2&LIF–Gluc system were assessed by measuring luminescence in the culture supernatant.
Test
In the cytokine treatment experiment involving engineered adipocytes, we first assessed viral infection efficiency by measuring mCherry expression via fluorescence microscopy imaging post-infection. After collecting the supernatant, luminescence was measured following the addition of the substrate coelenterazine.
The results revealed a significant increase in luminescence intensity in the sample from cells treated with both cytokines, compared to the untreated control and samples from cells treated with a single cytokine, demonstrating that Gluc reporter expression is induced only when both PLOD2 and LIF are upregulated simultaneously (Figure 5). Most importantly, this result confirms that the PLOD2&LIF sensor can specifically respond to the simultaneous presence of both cytokines and induced the express of its downstream output gene Gluc.

![](https://static.igem.wiki/teams/5542/engineering/engineering-5.webp)
Luminescence detection of conditioned medium of infected adipocytes, with or without cytokines treatment.

Considering that cytokine treatment alone cannot fully replicate the complex situation within the breast cancer tumor microenvironment, we further treated adipocytes with the conditioned medium from MDA-MB-231 human breast cancer cells. After 24 hours of culture, the conditioned medium was collected and luminescence was measured following the addition of the substrate of Gluc, coelenterazine.

![](https://static.igem.wiki/teams/5542/engineering/engineering-6.webp)
Luminescence situation of conditioned medium from adipocytes treated with the supernatant of the conditioned medium from MDA-MB-231 or cultured alone.

The results showed a significant increase in luminescence intensity in the supernatant of engineered adipocytes treated with the conditioned medium from MDA-MB-231, demonstrating that the adipocytes equipped with the PLOD2&LIF-Gluc sensor can effectively respond to the presence of breast cancer and induce the express of Gluc signal for detection purposes.
Learn
In cycle 2, the effectiveness of the PLOD2&LIF sensor sequence designed by our model group has been confirmed, which confirms that RADAR detection element with optimal sensor sequences works in intracellular environment of adipocyte.
In addition, the results show that the iPknot++ algorithm exhibited strengths in predicting RNA secondary structures, which might be a valuable algorithmic tool for other projects that also use RADAR system.

Cycle 3: Exploration of the reaction kinetics
![](https://static.igem.wiki/teams/5542/img/h1-background.webp)
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Design
To enable patients to conveniently assess their breast health status by ABCS, we needed to design a clearly visible output detection method without specialized equipment or complicated procedures. Our choice fell upon Gaussia Luciferase (Gluc).
Gluc is a novel luciferase that can be secreted from cells, metabolized by the kidneys, and excreted in urine, thereby has the potential to translate internal breast tissue changes into a detectable signal outside the body. When Gluc reacts with its substrate coelenterazine, it generates luminescence signal that is detectable in dark conditions.
Reaction kinetics of Gluc production in the RADAR system are essential for determining detection time and method. Therefore, we conducted dynamics modeling. We first analyzed the reaction mechanism of the RADAR system. The interaction between the sensor RNA and its target trigger RNA induces a response, which recruit ADAR to convert the stop codon UAG upstream of the Gluc reporter gene into UIG, thereby initiating its translation. Building upon the interaction and catalytic processes of the RADAR system, we developed kinetic equations accounting for four key processes: transcription and basal degradation, translation and degradation, complex formation, and catalysis and reporter production. Based on biological and mathematical principles, we converted these kinetic equations into a set of ordinary differential equations (ODEs).

![](https://static.igem.wiki/teams/5542/engineering/engineering-7.webp)
Transition from chemical reactions to ordinary differential equations

Parameter values were determined through literature research, and numerical solutions were obtained using Python's solve\_ivp function with the LSODA method. This modeling allowed us to predict the temporal concentration changes of Gluc within the RADAR system, as illustrated in the figure below.

![](https://static.igem.wiki/teams/5542/engineering/engineering-8.webp)
Gluc expression curves in dual input RADAR system

Kinetic modeling results demonstrate that the Gluc concentration reaches its peak of 1.91×10⁵ molecules (equivalent to 316.76 nM) at 35.75 hours, surpassing the visualization threshold (150 nM) at 14.13 hours. The concentration reaches half of its maximum value at 14.63 hours. These data indicate that the dual-input RADAR system requires approximately 14 hours to generate a visible output signal upon detection of target RNAs.
The modeling result confirms the possibility of using Gluc as the output signal, as it demonstrates that Gluc can be expressed in the RADAR system and accumulates visually detectable concentrations. We plan to further validate through wet-lab experiments.
Build
To verify the result of Gluc expression dynamics modeling in dual-input RADAR system, we constructed plasmid carrying an established AND gate sensor sequence and Gluc, transfected together with plasmids carrying trigger RNAs.

![](https://static.igem.wiki/teams/5542/engineering/engineering-9.webp)
Plasmid map of Sensor 1+2-Gluc

Test
The constructed plasmids were transfected into HEK-293T cells. Conditioned medium was collected from the transfected HEK-293T cells at 12, 24, 36, 48, 60, and 72-hour post-treatment. Immediately after collection, the substrate coelenterazine was added, and luminescence signals were measured using an ALLSHENG Feyond-A300 microplate reader. The corresponding results are shown in Figure 10. Results indicate that the conditioned medium showed an upward trend and reached the peak at 60 hours post-cytokine treatment in the luminescence intensity. After 60 hours, the luminescence intensity began to descent.

![](https://static.igem.wiki/teams/5542/engineering/engineering-10.webp)
Time-course curve of Gluc reaction fluorescence intensity

Learn
By monitoring Gluc luminescence intensity at multiple time points, we characterized its expression and secretion dynamics: a rising phase between 12 and 60 hours, peak secretion at 60 hours, followed by a subsequent decline. Compared to the reaction kinetics predicted by our modeling, the expression of Gluc is slower in wet lab experiments. We consider this phenomenon might be caused by two main reasons. The first reason is that in our modeling the initial plasmid value was set as the amount of plasmid already in cells, while in actual experiment, plasmids require time to enter the cells. The second reason is that in our modeling we assumed maximum plasmid transfection efficiency as the calculation parameter, but the actual transfection efficiency in experiments is usually lower. Nevertheless, the curves exhibit similar trends, demonstrating that the experimental result fits the reaction kinetics of Gluc simulated by our model in general.
In short words, the modeling of Gluc reaction kinetics could provide valuable information and guidance for detection of Gluc in the subsequent application of our ABCS project.

Cycle 4: Ethical assessment of our project with BEAM
![](https://static.igem.wiki/teams/5542/img/h1-background.webp)
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Design
During the development of our ABCS project, we focus on not only technical optimization but also real-world applicability. Due to the application of autologous adipocyte transplantation in ABCS, we need to ensure that our project is ethically sound and socially acceptable. This requires careful ethical review and attention to broader societal feedback.
Traditional ethical validation relies on interviews with patients, stakeholders, and experts. However, insights obtained from interviews are often difficult to interpret systematically or present in an intuitive manner. To address this gap, we sought to transform dispersed ethical considerations into a structured, data-driven evaluation framework, which led to the Bio-Ethical Assessment Model (BEAM).
In BEAM, we identified five key ethical assessment dimensions through literature review and expert consultation. We plan to employ BEAM as a supporting tool to achieve a systematic and quantitative approach to ethical evaluation.
Build
Building on five core evaluation dimensions identified in the Design phase, we further expanded and refined them to form a more comprehensive ethical evaluation dimension path structure. Subsequently, through expert interviews this path structure was revised and validated to ensure its scientific rigor and rationality.
After identifying path structure, our next task is to evaluate relationship among dimensions and quantify the path coefficients. Based on data collected from the ethical dimension evaluation questionnaire, we constructed the Structural Equation Modeling (SEM) to estimate the path coefficients between dimensions and thereby determine their relative weights within the overall ethical risk framework.
Finally, to analyze risks in complex systems and reveal interactions among multiple factors, we employed a Bayesian network to convert the weights into probabilistic relationships. To map the weights to probabilities we used functions, thereby defined conditional probability tables for each node. Bayesian network can infer and predict risk probabilities based on input dimension scores.
Test
To test the performance of BEAM, we selected several representative cases in synthetic biology—such as the synthetic reconstruction of the horsepox virus—to evaluate the model’s predictive capability.
After testing, we use BEAM to analyze our project using data collected from the public ethics assessment questionnaire conducted by our Human Practices team. The evaluation yielded a score of 32.2, corresponding to the mild risk category.

![](https://static.igem.wiki/teams/5542/engineering/engineering-11.webp)
Evaluation results of ABCS by BEAM

Learn
According to BEAM, we noticed that in our project ABCS, risk scores in genetic abnormality (41.3) and technology equity (41.5) are relatively high. Genetic abnormality refers to risk of abnormal genetic mutations and technology equity represents risk in regional technological inequity. In ABCS, autologous adipocyte engineering and transplantation are major causes of public concern in these two dimensions. This score inspires us to consider more when applying autologous adipocyte technological approach.
As a universal framework for quantifying ethics, BEAM shows how ethical concerns in synthetic biology can be turned into structured, data-driven models. Its validation proves that ethical risks can be systematically analyzed and improved. It is worth noting that, beyond the ethical issues we had already anticipated e.g. genetic abnormality, BEAM also pointed out potential risk we had not considered, e.g. the risks of ABCS on regional technological inequity.
Together, these efforts complete a full methodological cycle, turning ethical evaluation from abstract discussion into quantitative analysis and continuous improvement.

{% endblock %}
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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
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## What Should this Page Contain?

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Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block addCSS %}

{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5542/results/results-cover.webp)

![](https://static.igem.wiki/teams/5542/home/home/label/results.webp)

* [Proof of concept in HEK293T
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#results-proof)

  [Verification of RADAR system
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#results-proof-radar)
  [Verification of Gaussia Luciferase (Gluc)
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#results-proof-gluc)
  [Validation of RADAR system with Gluc
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#results-proof-radarwithgluc)
* [Validation of chassis-adipocytes
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#results-validation)

  [Induced differentiation of preadipocytes
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#results-validation-preadipocytes)
  [Validation of rAAV9 delivery
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#results-validation-raav9)
  [Test the viability of engineered adipocytes
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#results-validation-test)
* [Test of our RADAR in adipocytes
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#results-test)

  [Verification of target gene expression
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#results-test-expression)
  [Test of single input RADAR systems
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#results-test-single)
  [Test of double input RADAR systems
  ![](https://static.igem.wiki/teams/5542/img/star-dark.webp)](#results-test-double)
* [Conclusion
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#results-conclusion)
* [Discussion
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#results-discussion)
* [Reference
  ![](https://static.igem.wiki/teams/5542/img/pen.webp)](#results-ref)

![](https://static.igem.wiki/teams/5542/img/reel-lower.webp)
![](https://static.igem.wiki/teams/5542/img/pen-bg.webp)

![](https://static.igem.wiki/teams/5542/extended/results-overview-1009.avif)

Proof of concept in HEK293T
![](https://static.igem.wiki/teams/5542/img/h1-background.webp)
![](https://static.igem.wiki/teams/5542/img/h1-leave-left.webp)
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Verification of RADAR system
At this stage, our objective is to validate the effectiveness of the RADAR system reported by K. Eerik Kaseniit and explore optimal conditions and experimental setups for RADAR, thereby laying the foundation for the development of our RADAR system for breast cancer surveillance. We selected two RADAR sensors, Sensor 1 and Sensor 2, which have been documented by K. Eerik Kaseniit to perform well, for experimental validation. Subsequently, we used the AND gate sensor—— Sensor 1+2, formed by connecting Sensor 1 and Sensor 2 together, to verify its function of simultaneously detecting two distinct Triggers. At the same time, a sequence with the stop codon (UAG) replaced by a tryptophan codon (UGG) was used as a positive control to evaluate the switching efficiency of RADAR system. The positive control sensor is labeled by adding the suffix "-UGG", such as Sensor 1-UGG[1]. We transfected HEK293T cells with plasmids carrying the Sensors, the target RNAs (Trigger RNA) of the Sensors, and ADAR. After 48 hours, we assessed transfection efficiency and effectiveness of RADAR system by detecting mCherry and EGFP expression.
Amplification and verification of plasmids
Plasmids carrying Sensors (including Sensor 1, Sensor 2, Sensor 1+2 and their positive control), triggers (including Trigger 1 and Trigger 2) and hADAR1p150 were synthesized according to the sequences provided (Table 1)[1-2].

Detailed information of plasmids

| Short Name | Full Name | Length (bp) |
| --- | --- | --- |
| ADAR | pcDNA3.1-hADAR1p150 | 9028 |
| Sensor 1 | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1-EGFP | 6591 |
| Sensor 1-UGG | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1(PC)-EGFP | 6591 |
| Sensor 2 | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 2-EGFP | 6591 |
| Sensor 2-UGG | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 2(PC)-EGFP | 6591 |
| Sensor 1+2 | pcDNA3.1/Hygro(+)-PSFFV-mCherry-AND Sensor-EGFP | 6687 |
| Sensor 1+2-UGG | pcDNA3.1/Hygro(+)-PSFFV-mCherry-AND Sensor(PC)-EGFP | 6687 |
| Trigger 1 | pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 1 | 5981 |
| Trigger 2 | pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 2 | 5981 |

These plasmids were transformed into DH5α for amplification. Monoclonal colonies were cultured in LB medium, followed by plasmid Maxiprep from the bacterial cultures. The extracted plasmid was then verified by restriction digestion, and the digested plasmids were analyzed by agarose gel electrophoresis to confirm the correct size of the plasmids (Figure 1).

![](https://static.igem.wiki/teams/5542/results/results-fig1-1-1009.avif)
![](https://static.igem.wiki/teams/5542/results/results-fig1-2-1009.avif)
![](https://static.igem.wiki/teams/5542/results/results-fig1-3-1009.avif)
**A-I** Maps of plasmids;
**J-R** Plasmids digested with single/double restriction endonucleases, 1% agarose gel electrophoresis.

Single input RADAR system transfection and detection of fluorescent protein
To verify that the RADAR sensor can specifically respond to its corresponding Trigger RNA and induce the expression of the output gene (EGFP), we transfected HEK293T cells with plasmids using Lipofectamine™ 3000 according to the ratios specified in Table 2.

Transfection system of single input RADAR

| System of sensor 1 | | | System of sensor 2 | | |
| --- | --- | --- | --- | --- | --- |
| Group | Plasmid | Amount (ng) | Group | Plasmid | Amount (ng) |
| Sensor 1 | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1-EGFP | 200 | Sensor 2 | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 2-EGFP | 200 |
| Sensor 1+Trigger 1 | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1-EGFP | 200 | Sensor 2+Trigger 1 | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 2-EGFP | 200 |
| pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 1 | 250 | pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 1 | 250 |
| Sensor 1+Trigger 2 | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1-EGFP | 200 | Sensor 2+Trigger 2 | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 2-EGFP | 200 |
| pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 2 | 250 | pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 2 | 250 |
| Sensor 1+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1-EGFP | 200 | Sensor 2+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 2-EGFP | 200 |
| pcDNA3.1-hADAR1p150 | 50 | pcDNA3.1-hADAR1p150 | 50 |
| Sensor 1+Trigger 1+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1-EGFP | 200 | Sensor 2+Trigger 1+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 2-EGFP | 200 |
| pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 1 | 250 | pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 1 | 250 |
| pcDNA3.1-hADAR1p150 | 50 | pcDNA3.1-hADAR1p150 | 50 |
| Sensor 1+Trigger 2+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1-EGFP | 200 | Sensor 2+Trigger 2+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 2-EGFP | 200 |
| pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 2 | 250 | pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 2 | 250 |
| pcDNA3.1-hADAR1p150 | 50 | pcDNA3.1-hADAR1p150 | 50 |
| Sensor 1-UGG | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1(PC)-EGFP | 200 | Sensor 2-UGG | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 2(PC)-EGFP | 200 |

48 hours post transfection, fluorescence images were performed using excitation wavelengths of 488 nm (for EGFP) and 580 nm (for mCherry).

![](https://static.igem.wiki/teams/5542/results/results-hek293t-48h-after-transfection.webp)
Images of HEK293T 48h after transfection.

Fluorescence images reveal that cells transfected with either Sensor 1 or Sensor 2 exhibit strong red fluorescence, confirming high transfection efficiency. Most important, EGFP expression from sensors were only induced in the presence of their matching trigger. Notably, when ADAR was transfected, Trigger-induced EGFP expression was obviously increased, indicating that exogenous ADAR enhanced the switching effeciency of “UAG” to “UGG” of the RADAR system (Figure 2). These results demonstrate that the RADAR system can be specifically activated by matching triggers to induce the expression of output genes.
Double input RADAR system transfection and detection of fluorescent protein
Next, to further investigate whether an AND gate function can be achieved by linking the sequences of Sensor 1 and Sensor 2 together, we transfected HEK293T cells with plasmids carrying the Sensor 1+2, along with the corresponding Triggers. Since we confirmed that additional ADAR expression can enhance the efficiency of RADAR system, an additional 50 ng of the ADAR-expressing plasmid (pcDNA3.1-hADAR1p150) was included in each transfection group except Sensor 1+2-UGG, as specified in Table 3.

Transfection system of double input RADAR system verification

| Group | Plasmid | Amount (ng) |
| --- | --- | --- |
| Sensor 1+2 +ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-AND Sensor-EGFP | 200 |
| pcDNA3.1-hADAR1p150 | 50 |
| Sensor 1+2+Trigger 1+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-AND Sensor-EGFP | 200 |
| Sensor 1+2+Trigger 1+ADAR | 125 |
| pcDNA3.1-hADAR1p150 | 50 |
| Sensor 1+2+Trigger 2+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-AND Sensor-EGFP | 200 |
| pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 2 | 125 |
| pcDNA3.1-hADAR1p150 | 50 |
| Sensor 1+2+Trigger 1+Trigger 2+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-AND Sensor-EGFP | 200 |
| pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 1 | 125 |
| pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 2 | 125 |
| pcDNA3.1-hADAR1p150 | 50 |
| Sensor 1+2-UGG | pcDNA3.1/Hygro(+)-PSFFV-mCherry-AND Sensor(PC)-EGFP | 200 |

48 hours post transfection, fluorescence imagings were performed using excitation wavelengths of 488 nm (for EGFP) and 580 nm (for mCherry).

![](https://static.igem.wiki/teams/5542/results/results-hek293t-48h-after-transfection-of.webp)
Images of HEK293T 48h after transfection of Sensor 1+2 and other plasmids.

Fluorescence images reveal that EGFP expression from the Sensor 1+2 was triggered only upon co-transfection with both Trigger 1 and Trigger 2, which demonstrates that an AND gate constructed by linking two sensors together can be activated only when both triggers were present (Figure 3). Therefore, we decided to design our own RADAR sensor with AND gate function by linking two sensors together.
Verification of Gaussia Luciferase (Gluc)
Humanized Gaussia Luciferase (Gluc), a small secretory protein (19.9 kD), catalyzes the reaction of coelenterazine to coelenteramide to emit luminescence, which makes it an ideal reporter for our ABCS project[3]. At this stage, our objective is to explore and optimize the experimental setup for Gluc expression, thereby laying the groundwork for the development and testing of Gluc as a output reporter gene with RADAR system. In our experiments, we transfected HEK293T cells with plasmids carrying the humanized Gluc gene, and collect the conditioned medium at 24 h, 48 h and 72 h post transfection. Luminescence intensity was measured using a multifunctional microplate reader, and luminescence images were obtained using a fully automated chemiluminescence imaging analysis system.
Amplification and verification of plasmids
We used Gluc expressing plasmid (pCMV-Gluc-1), which carries the humanized Gluc gene (Figure 4 A). This plasmid was transformed into DH5α for amplification. Monoclonal colonies were then expanded in LB medium, followed by extraction of plasmids from the bacterial culture. The extracted plasmids were digested with restriction endonucleases and analyzed by agarose gel electrophoresis to confirm the correct plasmid size (Figure 4 B).

![](https://static.igem.wiki/teams/5542/results/results-4.webp)
**A** Map of pCMV-Gluc-1;
**B** pCMV-Gluc-1 digested with single/double restriction endonucleases, 1% agarose gel electrophoresis.

Transfection and luminescence detection
To evaluate the expression and kinetics of Gaussia Luciferase (Gluc) in HEK293T cells, we transfected HEK293T cells in 24-well plates with 500 ng of Gluc plasmid per well, including a non-transfected control group (NC). The conditioned medium were collected from the transfected HEK293T cells at 24, 48, 72 hours post transfection. Supernatants of the conditioned medium were obtained by centrifuging at 1000 rpm for 5 minutes, and subsequently analyzed using a multifunctional microplate reader after adding coelenterazine as substrate.

![](https://static.igem.wiki/teams/5542/results/results-comparison-of-luminescence.webp)
Chemiluminescence analysis of culture supernatant from HEK293T cells collected at 24h, 48h and 72h after transfection. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001.

Results showed that Gluc was successfully expressed and secreted from the transfected HEK293T cells, which remained at high levels at 24, 48 and 72h, with the peak time at 48h (Figure 5).
To visualize the luminescence produced by the Gluc-catalyzed reaction, we also performed luminescence images using both a fully automated chemiluminescence imaging analysis system and a cell phone. Similar as results shown in Figure 5, while supernatant from non-transfected control exhibited no luminescence, supernatant from cells transfected with Gluc expression plasmid displayed bright luminescence (Figure 6A). Importantly, such luminescence could also be captured by a cellphone, indicating that, in the future, the monitoring of occurence of breast cancer by our ABCS system could be easily done with a cell phone at home (Figure 6B).

![](https://static.igem.wiki/teams/5542/results/results-6.webp)
**A** Luminescence images captured by fully automated chemiluminescence imaging analysis system;
**B** Luminescence images captured by a cellphone.

Furthermore, to investigate the flash kinetic characteristics of Gluc, we analyzed these supernatants using the kinetic mode of a multifunctional microplate reader. A key finding was that all samples exhibited similar kinetic profiles—reaching maximum luminescence intensity (peak RLU) within milliseconds after substrate addition, clearly demonstrating its ultra-rapid reaction capability (Figure 7).

![](https://static.igem.wiki/teams/5542/results/results-comparison-of-sustained-luminescence.webp)
Analysis of sustained luminescence over 25 minutes between kinetics of luminescence production from Gluc.

Based on these results, we decided to collect supernatant at 48h post transfection and measure luminescence immediately after the adding of coelenterazine in subsequent experiments using Gluc as output reporter.
Validation of RADAR system with Gluc
So far, we have validated the function of the RADAR system in HEK293T cells and confirmed that Gluc can be expressed and secreted by HEK293T cells as an efficient reporter. To investigate whether Gluc can be used as an efficient output reporter for RADAR system, we constructed RADAR sensor plasmids with Gluc as output reporter and performed functional validation in HEK293T cells.
Amplification and verification of plasmids
Based on the plasmid used in the previous experiments, we constructed plasmids carrying Sensor 1-Gluc and Sensor 1-Gluc-UGG. Additionally, to validate the efficiency of the AND gate sensor using Gluc as the reporter gene in simultaneously detecting two target triggers, we constructed plasmids carrying Sensor 1+2-Gluc and Sensor 1+2-Gluc-UGG (Table 4).

Detailed information of plasmids carrying RADAR sensor with Gluc

| Short Name | Full Name | Length (bp) |
| --- | --- | --- |
| Sensor 1-Gluc | pcDNA3.1/Hyg1ro(+)-PSFFV-mCherry-Sensor 1-Gluc | 6429 |
| Sensor 1-Gluc-UGG | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1(PC)-Gluc | 6429 |
| Sensor 1+2-Gluc | pcDNA3.1/Hygro(+)-PSFFV-mCherry-AND Sensor-Gluc | 6525 |
| Sensor 1+2-Gluc-UGG | pcDNA3.1/Hygro(+)-PSFFV-mCherry-AND Sensor(PC)-Gluc | 6525 |

These plasmids were transformed into DH5α for amplification. Monoclonal colonies were cultured in LB medium, followed by extraction of plasmid DNA from the bacterial cultures. The extracted plasmids were digested with restriction enzyme and analyzed by agarose gel electrophoresis to confirm the correct plasmid size (Figure 8).

![](https://static.igem.wiki/teams/5542/results/results-8.webp)
**A-D** Maps of plasmids;
**E-H** Plasmids digested with single/double restriction endonuclease, 1% agarose gel electrophoresis.

Next, we transfected HEK293T cells according to the system outlined in Table 4.

Transfection system of single input RADAR system with Gluc as output gene

| Group | Plasmid | Amount (ng) |
| --- | --- | --- |
| Sensor 1-Gluc+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1-Gluc | 200 |
| pcDNA3.1-hADAR1p150 | 50 |
| Sensor 1-Gluc+Trigger 1+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1-Gluc | 200 |
| pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 1 | 250 |
| pcDNA3.1-hADAR1p150 | 50 |
| Sensor 1-Gluc+Trigger 2+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1-Gluc | 200 |
| pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 2 | 250 |
| pcDNA3.1-hADAR1p150 | 50 |
| Sensor 1-Gluc-UGG | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1(PC)-Gluc | 200 |

48 hours post transfection, we collected conditioned medium from transfected cells and measured chemiluminescence following the addition of coelenterazine.

![](https://static.igem.wiki/teams/5542/results/results-comparison-of-chemiluminescence.webp)
Luminescence of HEK293T cells transfected with Sensor 1-Gluc and other plasmids.

We observed that, luminescence intensity from cells transfected with Sensor 1-Gluc and Trigger 1 was significantly higher than that from cells transfected with Sensor 1-Gluc but no Trigger 1. In addition, luminescence intensity from cells transfected with Sensor 1-Gluc and Trigger 1 was significantly higher than that from cells transfected with Sensor 1-Gluc and Trigger 2, demonstrating that Sensor 1-Gluc specifically detects Trigger 1 and drives Gluc expression efficiently (Figure 9). These results validated that Gluc could serve as a effitient output reporter of RADAR system.
To further evaluate the sensitivity and specificity of an AND gate Sensor with Gluc as output reporter, we transfected HEK293T cells with plasmids as listed in Table 5, and assessed luminescence intensity for validation.

Transfection system of double input RADAR sensor with Gluc as output gene

| Group | Plasmid | Amount (ng) |
| --- | --- | --- |
| Sensor 1+2-Gluc+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1+2-Gluc | 200 |
| pcDNA3.1-hADAR1p150 | 50 |
| Sensor 1+2-Gluc+Trigger 1+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1+2-Gluc | 200 |
| pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 1 | 125 |
| pcDNA3.1-hADAR1p150 | 50 |
| Sensor 1+2-Gluc+Trigger 2+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1+2-Gluc | 200 |
| pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 2 | 125 |
| pcDNA3.1-hADAR1p150 | 50 |
| Sensor 1+2-Gluc+Trigger 1+Trigger 2+ADAR | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1+2-Gluc | 200 |
| pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 1 | 125 |
| pcDNA3.1/Hygro(+)-mTagBFP2-Trigger 2 | 125 |
| pcDNA3.1-hADAR1p150 | 50 |
| Sensor 1+2-Gluc-UGG | pcDNA3.1/Hygro(+)-PSFFV-mCherry-Sensor 1+2(PC)-Gluc | 200 |

48 hours post transfection, conditioned medium from transfected cells were collected and subjected to chemiluminescence detection after adding coelenterazine (Figure 10).

![](https://static.igem.wiki/teams/5542/results/results-chemiluminescence-analysis-of-culture-supernatant.webp)
Chemiluminescence of conditioned medium from HEK293T cells transfected with Sensor 1+2-Gluc and other plasmids.

We observed that the luminescence intensity of the conditioned medium from cells co-transfected with both Trigger 1, Trigger 2 and the Sensor 1+2 was significantly higher than that from cells transfected with only one Trigger or no Trigger, which demonstrates that the tandem AND-gate sensor with Gluc as output reporter functions effectively and specifically in detecting the presence of both Triggers (Figure 10).

![](https://static.igem.wiki/teams/5542/results/results-11.webp)
Luminescence of conditioned medium from transfected HEK293T cells that were collected at indicated times.

Furthermore, to investigate the kinetic characteristics of the Gluc expression from AND gate RADAR sensor, we collected conditioned medium at 12, 24, 36, 48, 60 and 72 hours post transfection and measured luminescence. The results showed a continuous generation of luminescence over the 72-hour period following transfection, with a peak time at 60 hours post transfection (Figure 11).

Validation of chassis-adipocytes
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Induced differentiation of preadipocytes
To obtain mature adipocytes for subsequent experiments, we chose to induce differentiation of preadipocytes. For the induction of preadipocytes, a commercial adipogenic differentiation kit was used, specific steps are described in the [Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D). To confirm successful differentiation, we performed Oil Red O staining and examined lipid droplet formation under microscopy. Following 14 days of induction, successful differentiation was observed via Oil Red O staining, thereby establishing a reliable way to generate differentiated adipocytes for subsequent experiments (Figure 12).

![](https://static.igem.wiki/teams/5542/results/results-human-preadipocytes.webp)
Induced differentiation of preadipocytes.

Validation of rAAV9 delivery
We utilized recombinant adeno-associated virus serotype 9 (rAAV9) to deliver the Sensor 1-Gluc, Trigger 1 and ADAR to investigate whether RADAR system can be successfully transduced into adipocytes and remain functional within this cell type.
Infection and detection of Gluc expression
Following adipocyte differentiation, cells were infected with rAAV carrying Sensor 1, Trigger 1, and ADAR to assess both rAAV delivery efficiency and RADAR function in adipocytes. Conditioned medium were collected two days post infection and luminescence was meatured by adding coelenterazine, thereby evaluating the expression of Gluc in differentiated adipocytes (Figure 13).

![](https://static.igem.wiki/teams/5542/results/results-detection-of-gluc.webp)
Luminescence of the conditioned medium from adipocytes infected by rAAVs carrying Sensor 1 and Trigger 1.

The result demonstrates that RADAR system can be effectively delivered and function appropriately in adipocytes.
Test the viability of infected adipocytes
The viability of adipocytes after rAAV infection is another critical aspect we must consider. We examined the viability of infected adipocytes to ensure that rAAV infection did not cause severe detrimental effects results on the viability of adipocytes.
Detection of Engineered Adipocyte Viability by CCK-8 Assay
To ensure that rAAV infection did not induce significant toxicity or necrosis in engineered adipocytes, cell viability was measured using the CCK-8 assay. In this assay, the water-soluble tetrazolium salt WST-8 is reduced by mitochondrial dehydrogenases in viable cells to an orange formazan product. The reduction rate is proportional to the number of living cells, and absorbance at 450 nm is used to quantify metabolic activity. OD450 was measured 2 hours after adding CCK-8 reagent to assess cell viability.

![](https://static.igem.wiki/teams/5542/results/results-comparison-of-viability.webp)
The viability of infected and uninfected adipocytes revealed by CCK-8 assay.

The result shows no significant difference in viability between rAAV infected and non infected adipocytes, indicating that rAAV infection might be a safe way to engineer adipocytes (Figure 14).

Test of our RADAR in adipocytes
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Following the proof-of-concept experiments of RADAR system in HEK293T cells and validation of adipocytes as the chassis, we proceeded to develop our own RADAR system to detect the emerging of cancer associated adipocytes. Since literature review and database analysis by our model group showed that PLOD2 and LIF are optimal biomarkers of cancer associated adipocytes, we decided to use them as target genes for our RADAR-based detection.
Verification of target gene expression
As literatures reported that PLOD2 and LIF expression in adipocytes could be induced by PAI-1 and CXCLs in breast tumor microenvironment[4-5], we validated these observation with experiments. To investigate the efficiency of induced expression of PLOD2 and LIF, we treated differentiated adipocytes with recombinant PAI-1 and CXCL8 proteins.
Cytokine treatment and qPCR
Differentiated adipocytes were treated with PAI-1 and CXCL8 individually or in combination. The expression levels of PLOD2 and LIF were measured via qPCR with GAPDH as internal control.

![](https://static.igem.wiki/teams/5542/results/results-expression-levels-of-target-genes-left.webp)

![](https://static.igem.wiki/teams/5542/results/results-expression-levels-of-target-genes-right.webp)

Expression of PLOD2 and LIF in adipocytes treated with PAI-1 and CXCL8.

qPCR results revealed that PAI-1 treatment significantly increased PLOD2 expression, while CXCL8 treatment significantly increased LIF expression (Figure 15).
Test of single input RADAR systems
Through analysis of RNA secondary structure with iPKnot++, we obtained the sensor sequences capable of targeting PLOD2 and LIF (details provided in the [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)). These sensors, with Gluc as the output gene, were constructed. Using rAAV, we delivered these sensors into adipocytes and treated the infected adipocytes with PAI-1 and CXCL8. The luminescence generated by Gluc were then measured to validate the function of these two RADAR sensors.
The function validation of PLOD2 sensor
We infected the differentiational adipocytes with rAAV9 carrying PLOD2 sensor and ADAR. Two days after infection, we treated the cells with PAI-1, and then collected conditioned medium to measure the luminescence by adding coelenterazine.

![](https://static.igem.wiki/teams/5542/results/results-luminescence-detection.webp)
Luminescence of conditioned medium supernatant from adipocytes infected by rAAV carrying PLOD2 sensor, with or without PAI-1 treatment.

We found that the luminescence intensity of the PAI-1-treated group was significantly higher than that of the untreated group (Figure 16). Together with the result shown in Figure 15, these results suggested that our designed PLOD2 sensor could detect PAI-1 induced expression of PLOD2 and activated the expression of output reporter gene Gluc.
The function validation of LIF sensor
We infect adipocytes with rAAV carrying LIF sensor and ADAR. We treated cells with CXCL8 and then collected conditioned medium to measure the luminescence by adding coelenterazine.

![](https://static.igem.wiki/teams/5542/results/results-17.webp)
Luminescence of conditioned medium supernatant from adipocytes infected by rAAV carrying LIF sensor, with or without CXCL8 treatment.

We found that the CXCL8-treated group exhibited significantly higher luminescence intensity compared to the untreated group (Figure 17). Together with the result shown in Figure 15, these results suggested that our designed LIF sensor could detect CXCL8-induced expression of LIF and activate the expression of output reporter gene Gluc.
The successful validation of the effectiveness of PLOD2 sensor and LIF sensor establishes a foundation for linking these two sensor sequences together to enable simultaneous detection of PLOD2 and LIF.
Test of double input RADAR system
Following the validation of PLOD2 sensor and LIF sensor, we proceeded to verify the function of our designed AND-gate sensor, i.e. PLOD2-LIF sensor. After delivering the PLOD2-LIF sensor into adipocytes using rAAV, we treated the cells with PAI-1 and CXCL8 to assess its detection capability. Subsequently, these adipocytes were treated with the conditioned medium from breast cancer cell MDA-MB-231 to further evaluate the ability of sensor to detect cytokines secreted from breast cancer cells.
The function validation of PLOD2-LIF sensor
Differentiated adipocytes were infected with rAAV9 carrying the PLOD2-LIF sensor. On day 2 post infection, cells were treated as follows: no cytokines, PAI-1 alone, CXCL8 alone, or both cytokines combined. Conditioned medium were collected for luminescence detection. Results demonstrated that the luminescence intensity was significantly higher in the conditioned medium from cells treated with both PAI-1 and CXCL8 than those from cells treated with PAI-1 only or CXCL8 only, indicating that Gluc expression from PLOD2-LIF sensor was induced only when both PLOD2 and LIF were simultaneously up-regulated. This result demonstrates that the PLOD2-LIF sensor responds to the presence of both PAI-1 and CXCL8 and activates the expression of output reporter gene Gluc.

![](https://static.igem.wiki/teams/5542/results/results-18.webp)
Luminescence of conditioned medium supernatant from adipocytes infected by rAAV carrying PLOD2-LIF sensor, with or without cytokines treatment.

To better simulate the interaction between breast cancer cells and adipocytes, conditioned medium from breast cancer cell MDA-MB-231 was used to assess the performance of the engineered adipocytes. Human breast cancer cells MDA-MB-231 were cultured in medium with 0.2% FBS for 24 hours. The conditioned medium was then collected to treat the adipocytes infected with rAAV carrying PLOD2-LIF sensor. After 48 hours of treatment, the supernatant was collected for luminescence detection.

![](https://static.igem.wiki/teams/5542/results/results-19.webp)
Luminescence of the conditioned medium from adipocytes infected by rAAV carrying PLOD2-LIF sensor, with or without treatment of conditioned medium from MDA-MB-231 cells.

Treatment with conditioned medium from MDA-MB-231 cells significantly increased the luminescence generated from conditioned medium of adipocytes with PLOD2-LIF sensor, indicating that these engineered adipocytes could respond to the cytokines secreted from breast cancer cells (Figure 19). This result suggests that these engineered adipocytes have the potential to detect breast cancer cells.

Conclusion
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We have constructed engineered adipocytes for detection of cytokines secreted from breast cancer cell. Initially, we verified in HEK293T cells the function of established RADAR systems, and the effectiveness of the RADAR sensor using Gluc as reporter. Subsequently, the RADAR system was efficiently delivered via rAAV9 into differentiated human adipocytes—confirmed by Oil Red O staining. Finally, by deploying RADAR sensors targeting PLOD2 and LIF in adipocytes, we confirmed that our designed sensors specifically responds to cytokines secreted from breast cancer cells and activates the expression of Gluc as output reporter.

Discussion
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So far, we have completed the full workflow from sensor design, adipocyte engineering to functional validation of RADAR system. Our engineered adipocytes perform effectively without significant impairment to viability, demonstrating that the ABCS has the potential of long-term monitoring of breast cancer, thereby offering a novel approach to breast cancer surveillance.
Although our current experiments are confined to the laboratory stage, we constructed a model that simulates the Glomerular Filtration Barrier (GFB) to investigate the renal metabolic characteristics of Gaussia luciferase (Gluc) in the human body (Click [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) to learn more). While the model simulation provides valuable insights for in vivo applications, issues such as the long-term safety and stability of engineered cells in real living environments still require further investigation. In addition, the sensitivity and specificity of the sensors can be further optimized—for instance, by screening more efficient ADAR isoforms or refining sensor sequence structures to reduce background noise and improve the signal-to-noise ratio.
Looking forward, we will focus on developing improved adipocytes engineering strategies to enhance the survival and functional persistence of engineered adipocytes. Concurrently, we aim to refine sensor performance through iterative design and testing, enabling high-accuracy and high-specificity detection in complex in vivo microenvironments. The ultimate goal is to advance this technology toward preclinical translation, providing a feasible solution for monitoring of breast cancer occurence.

Reference
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Reference

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| 2 | Gayet RV, Ilia K, Razavi S, Tippens ND, Lalwani MA, Zhang K, Chen JX, Chen JC, Vargas-Asencio J, Collins JJ. Autocatalytic base editing for RNA-responsive translational control. Nat Commun. 2023 Mar 11;14(1):1339. |
| 3 | Wang T, Chen Y, Goodale D, Allan AL, Ronald JA. A survivin-driven, tumor-activatable minicircle system for prostate cancer theranostics. Mol Ther Oncolytics. 2021 Jan 20;20:209-219. |
| 4 | Wei X, Li S, He J, Du H, Liu Y, Yu W, Hu H, Han L, Wang C, Li H, Shi X, Zhan M, Lu L, Yuan S, Sun L. Tumor-secreted PAI-1 promotes breast cancer metastasis via the induction of adipocyte-derived collagen remodeling. Cell Commun Signal. 2019 Jun 6;17(1):58. |
| 5 | Zhou C, He X, Tong C, Li H, Xie C, Wu Y, Wang L, Yan X, Luo D, Tang Y, Cheng Z, Xiong X. Cancer-associated adipocytes promote the invasion and metastasis in breast cancer through LIF/CXCLs positive feedback loop. Int J Biol Sci. 2022 Jan 16;18(4):1363-1380. |

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