iGEM 2024 — Team sdu-denmark

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* [Introduction](#introduction)
* [BSL-1 Notebook](#class-1-notebook)
* [BSL-2 Notebook](#class-2-notebook)

# Introduction

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A laboratory notebook is an essential record-keeping tool for documenting scientific research and experiments. It contains detailed notes on objectives, materials, methods, data, and observations, along with analysis and conclusions. The notebook ensures accuracy, reproducibility, and accountability, serving as a reliable reference for future work or legal protection of intellectual property. We have kept notebooks for both our work in BSL-1 and BSL-2.

# BSL-1 Notebook

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The following notebook contains details of all laboratory experiments performed in BSL-1 laboraotry.

If the PDF does not display correctly or if you're on a mobile device, you can
[download the PDF here](https://static.igem.wiki/teams/5253/notebook-pdfs/notebook-class-i.pdf).

# BSL-2 Notebook

---

The following notebook contains details of all laboratory experiments performed in BSL-2 laboratory.

If the PDF does not display correctly or if you're on a mobile device, you can
[download the PDF here](https://static.igem.wiki/teams/5253/notebook-class-ii.pdf).

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* [Engineering Cycle](#engineering-cycle)
* [Cycle A - Preliminary Plasmid Design](#cycle-1)

+ [Design](#design-a)
+ [Build](#build)
+ [Test and Learn](#test)
+ [Redesign](#design-b)

* [Cycle B – HESC optimization](#cycle-2)

+ [Design](#design-2)
+ [Build](#build-2)
+ [Test](#test-2)
+ [Learn](#learn-2)

# Engineering Cycle

---

**Synthetic biology** can be applied in various fields, such as healthcare, agriculture, industrial biotechnology, and many more. It is consequently important to have a **structured** approach to ensure the best solutions. The engineering cycle is applied specifically to optimize biological systems and organisms.

The engineering cycle has **four stages:** Design, Build, Test, and Learn. This can be seen in Figure 1.

* **Design:** The first step is design. It begins with a brainstorming phase, where problems are identified. Then, a lot of research is conducted to understand the problem and conceptualize possible solutions. The solution can then be visualized by sketching or modeling. In synthetic biology, the design could be a therapeutic compound or a biosensor.
* **Build:** When the design is finalized, the next phase is to build it. This is the practical implementation of the design, where the product is constructed. An example could be the assembling of designed DNA sequences followed by cloning.
* **Test:** When the product has been built, it needs to be tested. This is to ensure that the product functions as expected and meets all the requirements to solve the initial problem. The product can be tested to assess different parameters, such as functionality, durability, and stability.
* **Learn:** After the testing, the results are analyzed to identify what worked and what was unsuccessful. The data is used to pinpoint exactly what can be improved in the design, construct or experimental conditions. This will enhance the product, ensuring an optimal solution to solve the problem (1).

When one cycle is completed, the newfound knowledge can be used to improve the design, thus starting the engineering cycle again.

![Engineering cycle](https://static.igem.wiki/teams/5253/engineering-images/engineering-cycle.png)

Figure 1: Engineering cycle (20).

# Cycle A - Preliminary Plasmid Design

---

Cycle A consists of the preliminary designs for a non-hormonal, minimally invasive treatment for Endometriosis.

# Design

---

We started by defining the **problem:** Endometriosis. A painful disease with a long list of symptoms, making everyday life a challenge for those affected. An even bigger issue is the **lack** of research and treatment methods. We came up with the idea **to kill** the endometrial-like cells, **without** affecting the alike, but healthy, endometrium cells. We started by researching the **differences** in normal endometrial cells and in endometrial-like cells, to find a way to only target endometrial-like cells.

We found out that in endometrial-like cells the estrogen receptor beta (ERβ) is **upregulated**, and estrogen receptor alpha (ERα) is **downregulated** compared to endometrium cells (2). Studies have shown that in the endometriotic-like cells the estrogen receptor β (ERβ) becomes upregulated by **>100 times** (3).

The next thing we then had to Figure out was how we could use that knowledge to create something that could kill the endometrial-like cells but leave the Endometrium cells unharmed. We knew that to kill a cell we could use an **apoptosis-inducing protein**, and that ER’s are nuclear receptors, meaning they can function as **transcription factors**. We began our search to find an apoptosis protein, that could be inhibited by another protein, as well as promotors which were regulated by ERα and ERβ.

We decided to design two different plasmids: **Plasmid design 1**, seen in Figure 2, and **Plasmid design 2**, seen in Figure 6. In plasmid design 1, we would use the concentration difference of both ERα and ERβ by using **two different promoters**, one activated by ERα and the other by ERβ. In plasmid design 2 we would only use the concentration difference of ERβ, where we would use a promoter **activated** by ERβ and a promoter **inhibited** by ERβ.

We designed two different plasmids because we had not yet decided which **cell lines** we were going to use. We thought about testing the plasmids on **lung epithelial cells**, as they were already available in the lab. The plan was to use estrogen in the media, in hope that the extra estrogen would change how much of each receptor that would be expressed, but this seemed unlikely to work. We also investigated the possibilities with using **HeLa cells**, as they have some similarities with endometrial-like cells. Another possibility was engineering the lung epithelial cell line with **lentivirus**, so we could make some express more ERβ and some express more ERα, mimicking endometriosis and endometrium cells respectively. Our last possibility was to order **endometrial cells** and **endometrial-like cells**.

After some research we narrowed the promoters down to the following:

* **CD47 promoter**, which is activated by ERβ (4).
* **BRCA1 promoter**, which is activated by ERα (5).
* **pS2/TFF1 promoter**, which is activated by ERα and **Activating Protein 1 Complex** (AP1) (6).
* **EGFR promoter**, which is inhibited by ERβ (7).
* **ERα promoter**, which is inhibited by ERβ and activated by ERα (8).

After some discussion we decided to use the **CD47 promotor** for both plasmids, the **ERα promoter** for plasmid design 2 and the **BRCA1 promoter** for plasmid design 1.

To ensure that the promoters worked as intended we designed **two control plasmids**, where the apoptosis inducing protein and the apoptosis inhibitor are replaced with a **red fluorescent protein** (DsRed1) and **green fluorescent protein** (GFP). Control Plasmid 1 contains the same promoters as plasmid design 1, and Control Plasmid 2 contains the same promoters as plasmid design 2.

When searching for genes, we prioritized finding a gene for a protein **inducing apoptosis**, which also can be **inhibited** by another protein. We found the following:

* **Tumor protein P53 (p53)**, inhibited by **Mouse double minute 2 homolog (MDM2)** (9).
* **BCL2 Associated X (BAX)**, inhibited by **B-cell leukemia/lymphoma 2 protein (BCL2)** (10).

We did not choose the p53-MDM2 interaction, as it is involved in cancer development, and we wanted to decrease the possibility of the plasmid having negative side effects. Therefore, we decided to use **BAX** as our apoptosis protein and **BCL2** as our inhibitor. In plasmid design 1 the CD47 promoter activates BAX, and the BRCA1 promoter activates BCL2. In plasmid design 2 the CD47 promoter activates BAX, and the ERα promoter activates BCL2. The next step was to find a **terminator**. We accomplished this by seeking assistance from our supervisors who recommended **Simian virus 40 PolyA (SV40 Poly(A))**, which we decided to use for our designs.

Now that we had figured out all our parts, we needed to find a suitable **vector** and develop a cloning strategy for the **assembly**. Upon request were able to get the **pcDNA3.1(+) plasmid** donated from Kim Ravnskjær, Associate Professor at SDU, and we used that as our backbone.

We used **SnapGene** to design our plasmids and together with our supervisors we decided to use **Gibson Assembly** to assemble our plasmids.

# Design 1

---

![Treatment plasmid 1](https://static.igem.wiki/teams/5253/plasmid-designs/pb1-bcl2-cd47-bax.png)

Figure 2: pB1-BCL2-CD47-BAX (plasmid design 1). This design includes two promoters. One promoter is the CD47 followed by the BAX gene, which induces apoptosis. Another promoter is the BRCA1 followed by the BCL2 gene, which inhibits BAX. Both segments ends with the SV40 terminator. (19).

×

![Enlarged image]()

**Plasmid design 1 (pB1-BCL2-CD47-BAX)**, is the design with the BRCA1 promoter coupled to the BCL2 gene followed by the SV40 terminator, and the CD47 promoter coupled to the BAX gene followed by the SV40 terminator in the pcDNA3.1(+) vector, which can be seen in **Figure 2**.
When this plasmid is introduced in an endometrial-like cell, the estrogen will bind to dimerized ERβ. This complex binds to the CD47 promoter, initiating the **transcription** and **translation** of the BAX gene. As the BAX protein is an apoptosis inducing protein, this will ultimately kill the cell. The effect of plasmid design 1 on the endometrial-like cells can be seen in **Figure 3**.
The concentration of ERβ is **low** in healthy endometrium cells, but not zero. For that reason, there is a **safety switch** in the plasmid, where the high concentration of ERα will activate the BCRA1 promoter, leading to the expression of the **BCL2 protein**. BCL2 inhibits BAX, which will ensure the **survival** of the cell. The effect of plasmid design 1 on healthy endometrium cells can be seen in **Figure 4**.

![](https://static.igem.wiki/teams/5253/plasmid-effect-pathways/t-plasmid-1-endometriosis.png)

Figure 3: Effect of pB1-BCL2-CD47-BAX in endometrial-like cells. Estrogen enters the cell and binds to dimerized estrogen receptor β. This complex is transported to nucleus, and act as a transcription factor for the CD47 promoter. BAX protein is produced, leading to apoptosis in the cell (20).

![Treatment plasmid endometrium cells](https://static.igem.wiki/teams/5253/plasmid-effect-pathways/t-plasmid-1-endometrium.png)

Figure 4: Effect of pB1-BCL2-CD47-BAX in endometrium cells. Estrogen enters the cell and binds to dimerized estrogen receptor α. This complex is transported to nucleus, and act as a transcription factor for the BRCA1 promoter. BCL2 protein is produced, and apoptosis is inhibited (20).

Control Plasmid 1 (pB1-G-CD47-R) can be seen in **Figure 5**. The plasmid consists of the BRCA1 promoter coupled to GFP followed by the SV40 terminator, and CD47 promoter coupled to DsRed1 followed by the SV40 terminator in the pcDNA3.1(+) vector. The Control Plasmid is expected to have the same activation of CD47 promoter as in plasmid design 1 in endometrial-like cells, where there will be produced **red fluorescent protein**. In healthy endometrium cells, the BRCA1 promoter will be activated, and there will be produced **green fluorescent protein**.

![Control plasmid 1](https://static.igem.wiki/teams/5253/plasmid-designs/pb1-g-cd47-r.png)

Figure 5: pB1-G-CD47-R (Control Plasmid design 1). The design includes two promoters. One promoter is the CD47 followed by the gene DsRed1, which is a red fluorescent protein. Another promoter is the BRCA1 followed by GFP, which is a green fluorescent protein gene. Both segments are ended with the SV40 terminator (19).

# Design 2

---

![Treatment plasmid 1](https://static.igem.wiki/teams/5253/plasmid-designs/per-bcl2-cd47-bax.png)

Figure 6: pERα-BCL2-CD47-BAX (plasmid design 2). The design includes two promoters. One promoter is the CD47 followed by the gene BAX, which induces apoptosis. Another promoter is ERα followed by the BCL2 gene, which inhibits BAX. Both segments are ended with the SV40 terminator (19).

**Plasmid design 2 (pERα-BCL2-CD47-BAX)**, consists of the ERα promoter coupled to the BCL2 gene followed by the SV40 terminator, and the CD47 promoter coupled to the BAX gene followed by the SV40 terminator in the pcDNA3.1(+) backbone, which can be seen in **Figure 6**.
This design will have the same effect in endometrial-like cells as plasmid design 1: Dimerized ERβ complex will activate the CD47 promoter, inducing the expression of **BAX protein**, leading to apoptosis in the cells. The ERβ complex will also **inhibit** the ERα promoter, ensuring that no **BCL2** is produced. The effect of plasmid design 2 on endometrial-like cells can be seen in **Figure 7**.
When the plasmid is introduced into **healthy endometrium cells** the ERα will form a complex as well, which activate the ERα promoter, leading to the production of the **BCL2 protein**, and the survival of the cells, as seen in **Figure 8**.

![Treatment plasmid endometriosis cells](https://static.igem.wiki/teams/5253/plasmid-effect-pathways/t-plasmid-2-endometriosis.png)

Figure 7: Effect of pERα -BCL2-CD47-BAX in endometrial-like cells. Estrogen enters the cell and binds to dimerized estrogen receptor β. This complex is transported to nucleus, and act as a transcription factor for the CD47 promoter. BAX protein is produced, leading to apoptosis in the cell (20).

![Treatment plasmid endometrium cells](https://static.igem.wiki/teams/5253/plasmid-effect-pathways/t-plasmid-2-endometrium.png)

Figure 8: Effect of pERα -BCL2-CD47-BAX in Endometrium cells. Estrogen enters the cell and binds to dimerized estrogen receptor α. This complex is transported to nucleus, and act as a transcription factor for the ERα promoter. BCL2 protein is produced, and apoptosis is inhibited (20).

**Control Plasmid 2 (pERα-G-CD47-R)** can be seen in **Figure 9**. This plasmid consists of the ERα promoter coupled to GFP followed by the SV40 terminator, and the CD47 promoter coupled to DsRed1 followed by the SV40 terminator in the pcDNA3.1(+) vector. The expected outcome is activation of CD47 promoter in endometrial-like cells, leading to the expression of **red fluorescent protein**. In healthy Endometrium cells, the ERα promoter will be activated, and there will be expressed **green fluorescent protein**.

![Control plasmid 1](https://static.igem.wiki/teams/5253/plasmid-designs/per-g-cd47-r.png)

Figure 9: pERα-G-CD47-R (Control Plasmid design 2). The design includes two promoters. One promoter is the CD47 followed by DsRed1, which is a red fluorescent protein gene. Another promoter is the ERα followed by GFP, which is a green fluorescent protein gene. Both segments are ended with the SV40 terminator (19).

We preferred **plasmid design 1 (pB1-BCL2-CD47-BAX)**, as the expected outcome is a higher percentage of surviving healthy endometrium cells. Therefore, we decided to order an **endometrial cell line (Human Endometrial Stromal Cells (HESC))** and an **endometrial-like cell line (12Z Human Endometrial Epithelial Cells)**, as this would be the best proof-of-concept. Control Plasmid 1 (pB1-G-CD47-R) will be called Control Plasmid and Treatment Plasmid 1 (pB1-BCL2-CD47-BAX) will be called Treatment Plasmid going forward.

**Table 1:** Parts in Control Plasmid 1 and Treatment Plasmid 1.

| Name Composite Part | Composite Part | Name Basic Part | Basic Part | Type | Designers | Length |
| --- | --- | --- | --- | --- | --- | --- |
| Treatment apoptosis part | BBa\_K5253007 | CD47 promoter | BBa\_K5253000 | Promoter | Anna Kristensen | 1000 bp |
| BAX | BBa\_K5253002 | Coding | Anna Kristensen | 576 bp |
| SV40 terminator | BBa\_K5253006 | Terminator | Anna Kristensen | 122 bp |
| Treatment safety-switch | BBa\_K5253008 | BRCA1 promoter | BBa\_K5253001 | Promoter | Anna Kristensen | 1000 bp |
| BCL2 | BBa\_K5253003 | Coding | Anna Kristensen | 717 bp |
| SV40 terminator | BBa\_K5253006 | Terminator | Anna Kristensen | 122 bp |
| Control red fluorescent | BBa\_K5253009 | CD47 promoter | BBa\_K5253000 | Promoter | Anna Kristensen | 1000 bp |
| RFP | BBa\_K5253004 | Coding | Anna Kristensen | 681 bp |
| SV40 terminator | BBa\_K5253006 | Terminator | Anna Kristensen | 122 bp |
| Control green fluorescent | BBa\_K5253010 | BRCA1 promoter | BBa\_K5253001 | Promoter | Anna Kristensen | 1000 bp |
| GFP | BBa\_K5253005 | Coding | Anna Kristensen | 717 bp |
| SV40 terminator | BBa\_K5253006 | Terminator | Anna Kristensen | 122 bp |

# Build

---

We used **Gibson assembly** to construct the plasmids in the lab. The insertion sequence was cloned into the pcDNA3.1(+) expression vector using the NEB Hifi DNA Assembly Master mix (NEB # M5520). The plasmids were then **transformed** into NEB 5-alpha Competent *E. coli* cells (NEB #C2987) to produce multiple copies. To ensure the transformation was successful, we used **colony PCR**. As the vector has **ampicillin resistance**, this was used to select the transformed colonies. Then **PCR** was performed along with **gel electrophoresis** to ensure the insertion sequence had the right length. Lastly the plasmids were sequenced through **Sanger Sequencing** to ensure there were no mutations in the final products, before the testing.

# Test and Learn

---

To test if our plasmids design worked, we performed **transfection** with our plasmid and cell lines, 12Z and HESC. We decided to begin with the **Control Plasmid**, as it would allow us to use **flow cytometry** to measure the fluorescent levels, making it possible to asses the transfection efficiency and the functionality of the promoters. If our plasmid and transfection were successful, we would expect the **12Z cells** to express red fluorescence, because of the higher concentration of ERβ in these cells. In the **HESC cells** we expected the cells to express green fluorescence, because of the higher concentration of ERα. As shown in **Figure 10**, our expected results were not achieved. A detailed description can be found on the [results](https://2024.igem.wiki/sdu-denmark/results) page. After some reflecting we concluded that either our plasmids had to many **endotoxins**, our cells did not respond well to the transfection, or the transfection process failed, as the plasmid transfection could not be confirmed through **PCR** of the transfected cells.

![Placeholder Image](https://static.igem.wiki/teams/5253/engineering-images/engineering-result.jpg)

Figure 10: Results from transfection both with our plasmids and a standard GFP plasmid. The y-axis show the red fluorescence, and the x-axis shows the green fluorescence. (A) Show the density of cells from the transfection with our Control Plasmid in 12Z cells. Blue dots show the lowest density of cells and red show the highest density of cells. The black box indicates where the cells that express red fluorescent should be. There are only 0.05% of the cells that are in the box. (B) Show the density of cells from the transfection with our Control Plasmid in HESC cells. There are only 1.38% of the cells that are in the box. (C) Show all the cells from the transfection with the standard GFP plasmid. The black box shows where the cells that express green fluorescent should be. (D) The isolated cells that were expressing green fluorescent. In both (C) and (D) the dark blue and green dots represent 12Z cells that went through transfection with the GFP plasmid. The red dots represent 12Z cells that went through transfection without the plasmid. The purple dots represent the negative control for 12Z cells, so they did not go through transfection. The yellow dots represent HESC cells that went through transfection with the plasmid, and the light blue dots represents HESC cells that went through transfection without the plasmid.

# Redesign

---

[Human Practices](https://2024.igem.wiki/sdu-denmark/human-practices#expert-interviews) had an interview with **Martin Rudnicki**, Professor and Doctor in Gynecology and Obstetrics at OUH, Director of Minimal Invasive Surgery. He gave some insights to another approach to target the endometrial-like cells. He informed, that **progesterone receptors** could be another potential **target** for our plasmids, as their intracellular concentrations in endometrial-like cells are more extensively researched. This led us to **research** progesterone receptors’ molecular capabilities and how to implement this into our current Treatment Plasmid.

We learned that **progesterone receptors** are nuclear receptors that can act as **transcription factors** when activated. The levels of progesterone receptors (PR’s) are low in endometrial-like cells compared to healthy Endometrial cells. There are two **isoforms:** PRA and PRB. There are very low levels of **PRA** in endometrial-like cells, whereas there aren’t detected any **PRB** in endometrial-like cells (11).

Therefore, it was possible to make **another survival switch** in the Treatment Plasmid, by inserting a **promoter targeted by the PRB**, as this will ensure, that a higher percentage of healthy endometrium cells will survive, while the plasmid still promotes apoptosis in endometrial-like cells.

We learned that the PRB can activate the **PTEN promoter** (12). When it was time to choose genes, we again wanted to use an apoptosis inhibiting gene. This time, instead of using BCL2, we chose the **X-linked inhibitor of apoptosis (XIAP) gene**. This gene inhibits apoptosis downstream of BAX by inhibiting caspase 3, 7 and 9 (13). The design of this Treatment Plasmid, pB1-BCL2-CD47-BAX-PTEN-XIAP, can be seen in **Figure 11**.

![Treatment plasmid 3](https://static.igem.wiki/teams/5253/plasmid-designs/pb1-bcl2-cd47-bax-pten-xiap.png)

Figure 11: pB1-BCL2-CD47-BAX. The design includes three promoters. One promoter is the CD47 followed by the BAX gene, which induces apoptosis. A second promoter is the BRCA1 followed by the BCL2 gene, which inhibits BAX. A third promoter is the PTEN followed by the XIAP gene, which inhibits apoptosis downstream from BAX. All segments are ended with the SV40 terminator (19).

To **summarize** the effect of the plasmid in endometrial-like cells: **ERβ** will activate the **CD47** promoter, activating the transcription of **BAX**, which ultimately leads to apoptosis of the sick cells. This can be visualized in **Figure 12**.

In the endometrium cells, the plasmid will have the following effect: The low levels of **ERβ** will ensure that only a small amount of **BAX** is produced. **ERα** will activate the **BRCA1 promoter**, activating the transcription of **BCL2**. BCL2 inhibits BAX and therefore apoptosis. **PRB** will activate the **PTEN promoter** and **XIAP** is transcribed. XIAP stops apoptosis downstream of BAX by inhibiting caspase 9 as well as caspase 3 and 7, which can be seen in **Figure 13**. This establishes an even **stronger safety switch** in the Treatment Plasmid, ensuring a higher survival rate in the healthy endometrium cells.

![Treatment plasmid endometriosis cells](https://static.igem.wiki/teams/5253/plasmid-effect-pathways/t-plasmid-3-endometriosis.png)

Figure 12: Effect of pB1-BCL2-CD47-BAX-PTEN-XIAP in endometrial-like cells. Estrogen enters the cell and binds to dimerized estrogen receptor β. This complex is transported to nucleus, and act as a transcription factor for the CD47 promoter. BAX protein is produced, leading to apoptosis in the cell (20).

![Treatment plasmid endometrium cells](https://static.igem.wiki/teams/5253/plasmid-effect-pathways/t-plasmid-3-endometrium.png)

Figure 13: Effect of pB1-BCL2-CD47-BAX-PTEN-XIAP in endometrium cells. Estrogen enters the cell and binds to dimerized estrogen receptor α. This complex is transported to nucleus, and act as a transcription factor for the BRCA1 promoter. BCL2 protein is produced, and apoptosis is inhibited. Progesterone enters the cell and binds to dimerized progesterone receptor b. This complex is transported to nucleus, and act as a transcription factor for the PTEN promoter. XIAP protein is produced, and apoptosis is inhibited (20).

# Cycle B – HESC optimization

---

Cycle B explores the optimization of **growth conditions** for Human Endometrial Stromal Cells (HESC) using the engineering cycle approach.

The optimization process was initiated due to consistent **issues** with our HESC cell lines taking over a week to reach sufficient confluency for experiments. This prolonged growth period significantly **delayed** the experimental timeline, prompting us to investigate potential improvements in the cell culture conditions.

By systematically testing various **media formulations**, we aimed to identify the optimal balance of nutrients, growth factors, and serum components that would enhance **cell proliferation** and reduce the time needed for the cells to reach **confluency**. Through this process, we hoped to **streamline** the culture conditions, allowing for more efficient cell preparation and ultimately improving the overall workflow of our experiments.

# Design

---

In this phase, we aimed to optimize **HESC growth** by testing various culture media. The focus was to explore how different components, particularly the types of **serum** and the use of**charcoal-stripped FBS**, influenced the confluency of HESC cultures. We tested six different media compositions to determine which conditions provided the best growth environment:

**Table 2:** This table gives an overview of the different media combinations used in the experiment. Each flask was assigned a code (e.g. “O2), corresponding to the type of media that they contain.

| Type | Conditions |
| --- | --- |
| Normal | DMEM-F12 + 10% charcoal-stripped FBS + 1% Pen-Strep + 2 mM L-Glutamine |
| O1 | DMEM-F12 + 10% Human AB Serum + 1% Pen-Strep + 2 mM L-Glutamine |
| O2 | DMEM-F12 + 10% charcoal-stripped FBS + 0.5% Pen-Strep + 2 mM L-Glutamine |
| O3 | DMEM-F12 + 10% FBS + 1% Pen-Strep + 2 mM L-Glutamine |
| O4 | Embryomax + 10% charcoal-stripped FBS + 1% Pen-Strep + 2 mM L-Glutamine |
| O5 | Embryomax + 10% Human AB Serum + 1% Pen-Strep + 2 mM L-Glutamine |

# Build

---

The practical setup involved **culturing HESC cells** in each of the six media conditions in the design section. Cells were seeded at a consistent density in T25 culture flasks, ensuring that **media composition** was the only variable. Cultures were **incubated** under identical conditions, including temperature, humidity, and CO2 levels, and regular observations were made to assess cell confluency.

# Test

---

**The growth** of the HESC cells was evaluated by monitoring the confluency of the cells in each condition every 1-2 days. This data was used to determine the growth rates - i.e. the percentage change in quantity the cells over a specified period of time - of the cells with the different media. The following results were observed:

**Table 3:** An overview of how much of a decrease in doubling time each media combination resulted in throughout the experiment.

| Type | Normal (T75) | Normal (T25) | O1 | O2 | O3 | O4 | O5 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Doubling time | 8.87 days | 6.24 days | 2.13 days | 2.55 days | 2.55 days | 1.78 days | 2.24 days |

The following graphs illustrate the growth patterns of cells cultured in **Normal HESC cells (in a T25 flask) media and HESC cells grown in O4 media.**

![Treatment plasmid 3](https://static.igem.wiki/teams/5253/engineering-images/hesc-cells-vs-hesc-o4-cells.jpg)

Figure 14: This figure compares the growth of HESC (T25) cells and HESC O4 cells. On the x-axis is the time from right after a split to being confluent enough for another split, can be seen in number of days. On the y-axis how confluent the cells are from 0-100%.

The significant difference in confluency progression highlights the enhanced growth rate observed in the **O4 condition** compared to the Normal **T25 media**. This aligns with the calculated growth rates, where O4 reached confluency much faster than the standard media.
These results provided insight into the **optimal conditions** for HESC growth based on media composition.

# Learn

---

From our observations, the medium that yielded the highest growth rate was **O3**, **O4**, and **O5**, with growth rates of 0,8 days, 1,4 days, and 0,9 days (Table 3). In comparison the original composition used has a growth rate of 8,0 days for the T75 flasks and 3,0 days for the T25 flask.

Each of these formulations contains specific elements that may contribute to their greater performance. This medium provided the best balance of growth and cell health, likely due to factors such as the presence of fetal bovine serum (FBS), and the use of specialized EmbryoMax medium. These factors may explain the enhanced growth observed in these conditions compared to other tested media.

To better understand these results, we can break down the role of key components in the media and their potential effects on HESC proliferation:

*FBS vs. Charcoal-stripped FBS vs. AB Serum:*

* **FBS (Fetal Bovine Serum):**  FBS is a rich source of growth factors, hormones, and nutrients (14), which makes it an ideal supplement for promoting cell growth. The fact that O3 contains FBS may explain why it performed well. FBS provides critical factors that promote cell proliferation, differentiation, and survival.
* **Charcoal-stripped FBS:**  The charcoal stripping process removes hormones such as estrogen (15), which could affect the growth of HESCs. Since **O4** contains charcoal-stripped FBS and still performed well, it suggests that the removal of hormones may have minimized certain unwanted interactions (e.g., excess estrogen) that could otherwise interfere with HESC growth. **O4**’s use of EmbryoMax medium may compensate for the lack of hormones by providing other essential growth factors.
* **Human AB Serum:**  This serum is human-derived and may lack the breadth of growth factors found in FBS (16). However, **O5** performed well despite using AB serum, possibly due to its use of EmbryoMax medium (which will be discussed further below). AB serum might more closely mimic the natural environment of human cells, reducing the variability seen with bovine-derived components.

*DMEM/F-12 vs. EmbryoMax Medium*

* **DMEM/F-12 (Dulbecco's Modified Eagle Medium)**  is a common basal medium that supports a wide range of cells (17) but may not be fully optimized for HESC growth compared to a more specialized medium like EmbryoMax. This could explain why the **Normal** medium, which only uses DMEM/F-12, underperformed compared to **O4** and **O5**.
* Both **O4** and **O5** used **EmbryoMax medium**, which is specially formulated to support the growth of embryonic stem cells (18). For HESCs, EmbryoMax may provide the right balance of nutrients and growth factors that enhance their growth potential.
* **O4:**  The combination of EmbryoMax and charcoal-stripped FBS could explain why this medium performed well. While the charcoal-stripped FBS reduces hormone-induced stress, the EmbryoMax medium compensates with optimal growth conditions.
* **O5:**  The presence of EmbryoMax medium with AB serum may have created a more physiologically relevant environment for HESCs, allowing better growth than media containing DMEM alone.

These factors combined suggest that the specialized components of **O3**, **O4**, and **O5** closer mimic the optimal conditions for HESC growth compared to the **normal**, or other media tested. Moving forward, we plan to optimize the medium further by testing additional variables, to see if the growth rate can be further enhanced.

Collapsible References

References

1. Mortimer J. Plant synthetic biology could drive a revolution in biofuels and medicine. Experimental Biology and Medicine. 2018;244:153537021879389.
2. Chantalat E, Valera MC, Vaysse C, Noirrit E, Rusidze M, Weyl A, et al. Estrogen Receptors and Endometriosis. Int J Mol Sci. 2020;21(8).
3. Rogers PA, D'Hooghe TM, Fazleabas A, Gargett CE, Giudice LC, Montgomery GW, et al. Priorities for endometriosis research: recommendations from an international consensus workshop. Reprod Sci. 2009;16(4):335-46.
4. Hu L, Zhang J, Lu Y, Fu B, Hu W. Estrogen receptor beta promotes Endometriosis progression by upregulating CD47 expression in ectopic Endometrial stromal cells. J Reprod Immunol. 2022;151:103513.
5. Jeffy BD, Hockings JK, Kemp MQ, Morgan SS, Hager JA, Beliakoff J, et al. An estrogen receptor-alpha/p300 complex activates the BRCA-1 promoter at an AP-1 site that binds Jun/Fos transcription factors: repressive effects of p53 on BRCA-1 transcription. Neoplasia. 2005;7(9):873-82.
6. Baron S, Escande A, Albérola G, Bystricky K, Balaguer P, Richard-Foy H. Estrogen receptor alpha and the activating protein-1 complex cooperate during insulin-like growth factor-I-induced transcriptional activation of the pS2/TFF1 gene. J Biol Chem. 2007;282(16):11732-41.
7. Samanta S, Sharma VM, Khan A, Mercurio AM. Regulation of IMP3 by EGFR signaling and repression by ERβ: implications for triple-negative breast cancer. Oncogene. 2012;31(44):4689-97.
8. Hall JM, McDonnell DP. The estrogen receptor beta-isoform (ERbeta) of the human estrogen receptor modulates ERalpha transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology. 1999;140(12):5566-78.
9. Shangary SK, Wang S. Targeting the MDM2-p53 Interaction for Cancer Therapy. Clinical Cancer Research. 2008;14:5318 - 24.
10. Andreu-Férnández V, Sancho M, Genovés A, LucEndo E, Todt F, Lauterwasser J, et al. Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes. Proceedings of the National Academy of Sciences. 2016;114:310 - 5.
11. Yilmaz BD, Bulun SE. Endometriosis and nuclear receptors. Hum Reprod Update. 2019;25(4):473-85.
12. De Amicis F, Guido C, Santoro M, Lanzino M, Panza S, Avena P, et al. A novel functional interplay between Progesterone Receptor-B and PTEN, via AKT, modulates autophagy in breast cancer cells. J Cell Mol Med. 2014;18(11):2252-65.
13. Fraser M, Tsang BK. CHAPTER 37 - The Role of X-Linked Inhibitor of Apoptosis Protein in Chemoresistant Ovarian Cancer. In: Leung PCK, Adashi EY, editors. The Ovary (Second Edition). San Diego: Academic Press; 2004. p. 613-24.
14. ThermoFisher. The Basics of Fetal Bovine Serum Use in Cell Culture [Available from: https://www.thermofisher.com/dk/en/home/references/gibco-cell-culture-basics/cell-culture-environment/culture-media/fbs-basics.html
15. ThermoFisher. Charcoal-Stripped FBS [Available from: https://www.thermofisher.com/dk/en/home/life-science/cell-culture/mammalian-cell-culture/fbs/specialty-serum/charcoal-stripped-fbs.html
16. Sigma-Aldrich. Human Serum for Cell Culture [Available from: https://www.sigmaaldrich.com/DK/en/products/cell-culture-and-analysis/cell-culture-supplements-and-reagents/human-serum
17. ThermoFisher. Dulbecco's Modified Eagle Medium (DMEM) [Available from: https://www.thermofisher.com/dk/en/home/life-science/cell-culture/mammalian-cell-culture/cell-culture-media/dmem.html?gclid=CjwKCAjw9eO3BhBNEiwAoc0-jSWZExy6AOfHDxv9qdZkvr0eQ4in-xdalGNr14u726XPm7Cg4braZxoCfAAQAvD\_BwE&ef\_id=CjwKCAjw9eO3BhBNEiwAoc0-jSWZExy6AOfHDxv9qdZkvr0eQ4in-xdalGNr14u726XPm7Cg4braZxoCfAAQAvD\_BwE:G:s&s\_kwcid=AL!3652!3!591426427323!p!!g!!dulbecco%27s%20modified%20eagle%20medium!1759362858!66460957821&cid=bid\_clb\_cce\_r01\_co\_cp0000\_pjt0000\_bid00000\_0se\_gaw\_nt\_pur\_con&gad\_source=1
18. Sigma-Aldrich. "EmbryoMax® DMEM (1X), liquid, With 4,500mg/L Glucose, 2.25g/L Sodium Bicarb and L-Glut, without Sodium Pyruvate." from https://www.merckmillipore.com/DK/en/product/EmbryoMax-DMEM-1X-liquid-With-4500mg-L-Glucose-2.25g-L-Sodium-Bicarb-and-L-Glut-without-Sodium-Pyruvate,MM\_NF-SLM-120-B#overview.
19. Figures made in SnapGene
20. Figures made in [Biorender.com](https://www.biorender.com/)

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* [Introduction](#introduction)
* [Methods](#methods)
* [Experimental Design](#experimental-design)
* [Protocols](#protocols)

# Introduction

---

As we were working on a Treatment Plasmid that could induce apoptosis in endometrial-like cells and leave the healthy endometrium unaffected, we needed to find a way to test if the plasmid would be able to do that.

Our Treatment Plasmid contains two different composite parts [BBa\_K5253007](https://parts.igem.org/Part%3ABBa_K5253007) and [BBa\_K5253008](https://parts.igem.org/Part%3ABBa_K5253008):

* BBa\_K5253007 consist of promoter: CD47’s promoter, Gene: BAX, Terminator: SV40
* BBa\_K5253008 consist of promoter: BRCA1’s promoter, Gene: BCL2, Terminator: SV40

We also made a Control Plasmid to ensure that our promoters perform as expected.

Our Control Plasmid contains two different composite parts [BBa\_K5253009](https://parts.igem.org/Part%3ABBa_K5253009) and [BBa\_K5253010](https://parts.igem.org/Part%3ABBa_K5253010):

* BBa\_K5253009 consist of promoter: CD47’s promoter, Gene: DsRed1, Terminator: SV40
* BBa\_K5253010 consist of promoter: BRCA1’s promoter, Gene: GFP, Terminator: SV40

With a high enough level of ERβ, the CD47 promoter is activated the BAX protein will be expressed, which will start the pathway that will induce apoptosis in the cell. However, with a high enough level of ERα, the BRCA1 promoter is activated and the BCL2 protein will be expressed, which will bind to BAX and inhibit apoptosis in the cell.

# Methods

---

The design of our plasmid dictated the methods used in the project. Below are descriptions of the methods employed and their purposes.

##### Detailed Methods

*▼*

**Construction of the plasmids using Gibson assembly**

We used NEBuilder® Hifi DNA Assembly Master Mix for Gibson Assembly to insert two fragments into our plasmid backbone, which was digested with the restriction enzymes MluI and BbsI before assembly. For the Gibson Assembly to be successful, we added 25-base pair (bp) overhangs to both sides of each fragment. These overhangs were identical to the DNA regions where the assembly would occur, allowing the 5’ exonuclease to create single-stranded overhangs that were complementary, hereby facilitating the seamless assembly of our fragments into the plasmid backbone.

**Transformation into competent cells using heat-shock**

We used heat-shock to make competent E.coli DH5α able to take in our plasmids assembled by Gibson Assembly. The heat-shock makes the E.coli want to adapt to the sudden change in temperature and therefore more willing to take in our plasmids. After the heat-shock, the cells are plated onto selection plates with 50 µg/mL ampicillin.

**Colony PCR**

We used colony PCR to verify that the plasmids were correctly assembled. This was done by selecting some of the colonies from the selection plates and run a PCR with primers that bind to the flanking sequences of the insertion segment. If the Gibson Assembly was successful, a band with the same length as the insertion piece would be revealed on a gel. In our case the length of the insertion piece for the Control Plasmid was 3720 bp and the length for the Treatment Plasmid was 3627 bp.

**Western blot**

We used western blot to see if our antibodies bind specifically to the ERα and ERβ receptors. The process began by extracting proteins from our two cell lines, which were then denatured to ensure they unfold. These proteins were separated by size using SDS-PAGE, a form of gel electrophoresis. After separation, the proteins were transferred onto a membrane. To prevent nonspecific binding, the membrane is blocked with a protein solution. Next, the membrane was incubated with one of our antibodies that specifically binds to the target protein following a second antibody that binds to the other target. The antibodies were conjugated with a fluorescent dye allowing the visualization of the target proteins.

**Media Change**

The media was changed to ensure the cells had the proper nutrients needed for continued growth. The old media was removed from the cells, and new media was prewarmed at 37°C before it was added to the cells.

**Subculturing**

Cells were subcultured to provide them with enough space to grow. This was done when the confluency was around 70-90%. The old media was removed, and the cells were washed with prewarmed PBS. The cells were then treated with using 0.25% Trypsin to detach the cells from the flask. New media was added to the cells, which were then transferred to a Falcon tube. The cells were centrifuged at 120xg for 5 min, and the supernatant was discarded after which the cells were resuspended in culture media and split into a suitable ratio (1:3, 1:6, or 1:10).

**Electroporation**

Cells were harvested and resuspended in PBS and transferred to a chilled cuvette. The plasmid was added to a final concentration of 5 μM. The electroporator's parameters (voltage, pulse length, and number of pulses) were optimized for the specific cell type. The pulse button was pressed to activate the automatic charge and pulse sequence. Following electroporation, the cuvette was incubated for 2 minutes on ice, followed by 8 minutes at 37°C. After incubation, the cell suspension was carefully removed from the cuvette and transferred to 5 mL of culture medium in a T25 flask for cultivation.

**Transient transfection**

Cells were harvested at an optimal density (70-90% confluence), and the desired number of cells was transferred to an Eppendorf tube. The transfection reagent was mixed with culture media and then added to the cells, after which the mixture was incubated for approximately 24 hours at 37°C. After incubation, the transfection mixture was removed, and fresh media was added to the cells. After a recovery time of 24 to 48 hours, transfection efficiency was assessed by measuring fluorescence using flow cytometry.

**Filtration and fixation (prep for flow cytometry)**

To obtain optimal results from flow cytometry analysis, the cells needed to be in a single-cell suspension, which required filtration. This was done by cutting the end of a P1000 pipette tip, then placing a small piece of nylon filter on top of another P1000 pipette tip while pushing the cut pipette tip down into the filter. A third pipette tip was used with a pipette to add the sample to the filter. The pipette tip was then discarded, the pipette was attached to the filter, and gentle pressure was applied to expel the last bit of sample.

Once filtered, the cells needed to be fixed before flow cytometry analysis. This was done by adding 4% formaldehyde to the filtered cell sample and were directly after washed three times with PBS. The cells were then fixated and could be taken out of the Class II lab for flow cytometry.

# Experimental Design

---

To get an overview of the different experiments we have performed within the timeframe of the project, and what experiments we would have liked to have performed if we had more time, try exploring our experiment tree! To know more about the different methods, click on each spot on the tree.

**NOTE**: The experiments which we did not perform in time, were Dead Cell Stain, Caspase 3/7 assay and Transfection with Treatment Plasmid on both 12Z and HESC cells.

* Assembly of designed plasmids
  + Transformation into *E. coli* DH5α
    - Colony PCR
      * Sequencing
        + Transfecting 12Z w. Control Plasmid
          - Flow Cytometry
            * Antibody Staining of 12Z
              + Transfecting 12Z w. Treatment Plasmid
                - Deadcell Staining
                - Caspase Activity Assay
        + Western Blotting
        + Transfecting HESC w. Control Plasmid
          - Flow Cytometry
            * Antibody Staining of HESC
              + Transfecting HESC w. Treatment Plasmid
                - Deadcell Staining
                - Caspase Activity Assay

×

##

# Protocols

---

The following protocols are for the experiments preformed in the BSL-1 laboratory.

If the PDF does not display correctly or if you're on a mobile device, you can
[download the PDF here](https://static.igem.wiki/teams/5253/protocols-for-bsl-1.pdf).

The following protocols are for the experiments preformed in the BSL-2 laboratory.

If the PDF does not display correctly or if you're on a mobile device, you can
[download the PDF here](https://static.igem.wiki/teams/5253/protocols-for-bsl-2.pdf).

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* [Introduction](#Introduction)
* [Gibson Assembly](#experiment-1)
* [Electroporation](#experiment-2)
* [Transfection](#experiment-3)
* [HESC Optimization](#experiment-4)
* [Western Blot](#experiment-5)
* [Antibody Staining](#experiment-6)
* [Fluorescence Microscopy](#experiment-7)
* [Future plans](#Future-plans)

# Introduction

---

To test whether our plasmids were functioning properly and if our cell lines had the same properties as we expected, we developed an experimental plan. This plan included plasmid assembly, transfection and antibody staining - among other experiments.

In addition to these steps, we included various controls, such as non-transfected cells, to ensure the reliability of our results. Through this experimental approach, we were able to thoroughly assess the functionality of the plasmids and the behavior of the cell lines, providing a foundation for further analysis.

# Gibson Assembly

---

In order to assemble both our Control and Treatment Plasmid, we used Gibson assembly as described in [experiments](https://2024.igem.wiki/sdu-denmark/experiments).

**Assembly of Treatment Plasmid:**

After the Gibson assembly of our Treatment Plasmid we performed colony PCR, to ensure the selection of our plasmid within the different E. coli colonies. As shown in Figure 1, our insertion piece was 3627 bp long.

![Placeholder image](https://static.igem.wiki/teams/5253/results/gibson-result-1.jpg)

Figure 1: PCR product from transformation of *E. coli* DH5α with our Treatment Plasmid. The outer wells of the gel contains ladders. Well 2 contains a negative control and the following 8 wells contain different colonies from the *E. coli* DH5αwith the insertion piece of the plasmid amplified by PCR. As our insertion piece was about 3627 bp long, we chose to purify the plasmid of well 3, 4 and 8, marked with the respective numbers, as these were therefore the correct length.

Based on these results of the PCR reaction we decided to continue with the assembled plasmids from well 3, 4 and 8, as these contained the entire insertion piece. We changed the names of the plasmids to T1, T2 and T3. Upon purification, the plasmids were sent for Sanger sequencing analysis. Analyzing the sequencing results, we discovered that none of the plasmids had mutations, thereby allowing us to continue testing with the chosen plasmids.

**Assembly of Control Plasmid:**

The control plasmids were also assembled using Gibson assembly, where our insertion piece is 3721 bp long. We used the ladder band that was 3000 bp, as seen in Figure 2 and 3, we used as a guideline to choose the colonies. We chose to continue working with the plasmids that have the same length of our insertion piece, which is 3721 bp long.

![Placeholder image](https://static.igem.wiki/teams/5253/results/gibson-result-2.jpg)

Figure 2: PCR product from transformation of *E. coli* DH5α with our Control Plasmid. The Figure shows ladders in both ends of the gel, and a negative control in the second well. The next 12 wells show different insertions from the E.
coli DH5α amplified plasmid. As our insertion piece was about 3721 bp long we chose to use the colonies C, F and L, marked with the respective letters, as these had a similar length.

![Placeholder image](https://static.igem.wiki/teams/5253/results/gibson-result-3.jpg)

Figure 3: PCR product from transformation of *E. coli* DH5α with our control plasmid. The four wells in the middle show different insertions from the *E. coli* DH5αamplified plasmid. As our insertion piece is about 4000 bp long, we chose to use the colony M, which is marked with the letter, as this has the same approximate length.

Based on the results of the PCR reaction we sent the plasmids marked as C, F, L and M for Sanger sequencing analysis. We changed their names to C1, C2, C3 and C4 and after analyzing the sequencing results, we concluded that C3 and C4 have mutations and could not be used for further testing. C1 and C2 have no mutations and can therefore be used for further testing.

# Electroporation

---

We performed electroporation on our 12Z cell lines with our Control Plasmid, as described in [experiments](https://2024.igem.wiki/sdu-denmark/experiments). The control plasmid is supposed to emit green fluorescence if there is a high concentration of ERα in the cells and emit red fluorescence if there is a high concentration of ERβ.

![Placeholder image](https://static.igem.wiki/teams/5253/results/electroporation-1.jpg)

Figure 4: Results from the elctroporation was analyzed via flow cytometry. On the x-axis there is green fluorescent intensity and on the y-axis, there is red fluorescent intesity. The green dots represent the negative control, and the blue dots represent the 12Z cells that have gone through the electroporation with the control plasmid

The graph presented in Figure 4 compares 12Z cells that have been transfected with the Control Plasmid to a negative control of non-transfected 12Z cells, with measurements based on green and red fluorescence emitted by the respective cells. As mentioned, the blue dots represent the negative control sample, meaning that the 12Z cells were not subjected to electroporation, while the green dots represent the 12Z cells that were subjected to electroporation and were introduced to the plasmid. We expected to see that the blue dots would be higher on the y-axis than the green dots, because the red fluorescence should be activated by the high concentration of ERβ in the 12Z cells. However, as seen in Figure 4 both blue and green dots are concentrated in the same location. We later discovered mutations in the BRCA1 promoter of the control plasmid, so we are unsure as to if the promoter was functioning properly. If not, this could have caused the lack of fluorescence.

Electroporation optimization of 12Z and HESC cells with pmaxGFP Vector

After the previous failed attempts, we decided to optimize the electroporation process for 12Z cells with the pmaxGFP Vector, a standard GFP plasmid. We conducted an experiment with 48 different reactions with 3 Nucleofector Solutions (SE, SF, and SG) alongside 15 different electroporation programs, plus a control. Our goal was to find the best combination that would maximize cell transfection efficiency while minimizing cell death. Once we identified the optimal conditions, we aimed to apply them to all future experiments, to transfect our plasmids.

![Placeholder image](https://static.igem.wiki/teams/5253/results/electrporation-2.jpg)

Figure 5: Results from electroporation on 12Z cells. On the y-axis is the percentage of GFP+ cells. On the x-axis are the different programs, that were run on the samples. The blue pillars show the live cells. The PI- stand for propidium iodide negative. Propidium iodide is used to dye dead cells. The Orange pillars show the GFP+ cells. (A) The electroporation is done with a SE solution. There was an error due to too low an event rate in the control sample. (B) The electroporation is done with a SF solution. There was an error due to too low an event rate in the sample that got run with the CM-150 program. (C) The electroporation is done with a SG solution.

Based on the results from the electroporation experiments, we observed distinct transfection efficiencies across the different Nucleofector solutions and electroporation programs. For the SE solution (Figure 5A), transfection efficiency varied significantly between programs, but a noticeable issue arose with the control sample, which had too low an event rate, rendering its results inconclusive. With the SF solution (Figure 5B), the transfection profile followed a similar pattern, but the sample treated with the CM-150 program also experienced an error caused by cells absence, affecting the reliability of that specific condition. However, despite these errors, we identified notable trends, including an increased percentage of GFP+ cells in certain programs. Finally, in the SG solution (Figure 5C), we achieved relatively consistent results across programs, though with varying levels of transfection efficiency. Overall, while no singular solution-program combination produced optimal results across all metrics, the data enabled us to narrow down the most effective conditions for maximizing GFP expression while minimizing cell death. These findings guided the selection of the optimal conditions for subsequent plasmid transfections in 12Z cells.Due to the low cell number in the performed experiment we decided to repeat the electroporation optimization experiment as a general trend was observed favoring SF solution in the subsequent experiment. Due to the low cell number in the performed experiment we decided to repeat the electroporation optimization experiment as a general trend was observed favoring SF solution in the subsequent experiment.

![Placeholder image](https://static.igem.wiki/teams/5253/results/electrporation-3.jpg)

Figure 6: Results from electroporation on 12Z cells with SF solution. On the y-axis is the percentage of GFP+ cells. On the x-axis is the different programs, that were run on the different samples. The blue pillars show the live cells. The PI- stand for propidium iodide negative. Propidium iodide was used to dye dead cells. The Orange pillars show the GFP+ cells. The sample that was run with the CA-137 did not receive any electric impulses. The sample that was run with the DS-130 program had too few cells.

In the optimized electroporation experiment using the SF solution (Figure 6), we observed improvements in transfection efficiency and cell viability across the tested programs. Most notably, programs that applied electric impulses generally showed a higher percentage of GFP+ cells, indicating successful transfection. The CA-137 and control program, which did not receive any electric impulses, predictably showed no GFP+ cells, serving as negative controls for the experiment. Meanwhile, the DS-130 program had an insufficient number of cells, leading to unreliable results for that condition. Despite this, the majority of the remaining programs demonstrated a balance between GFP expression and cell viability, with the blue pillars indicating a healthy population of live cells. These results confirmed that the optimized conditions for electroporation with the SF solution significantly improved both transfection efficiency and cell survival, allowing us to select the most favorable parameters for future experiments.

It is important to note that the percentage of live cells, as indicated by the blue pillars, represents only about 5% of the total number of cells that were initially added to each well. This means that a big portion of cells were lost during the electroporation process, possibly due to stress or suboptimal conditions. Despite this, most of the remaining programs demonstrated a balance between GFP expression and cell viability, highlighting their potential for effective transfection. These results confirmed that the optimized conditions for electroporation with the SF solution significantly improved both transfection efficiency and cell survival, allowing us to select the most favorable parameters for future experiments.

# Transfection

---

We did multiple transfection attempts, as described in [experiments](https://2024.igem.wiki/sdu-denmark/experiments), but unfortunately none of the attempts were successful. In our very first transfection attempt we used a 6-well plate, where two wells contained 12Z cells transfected with the Control Plasmid and one well with HESC cells transfected with the control plasmid. Besides this, we also had a negative control sample of the 12Z cells. The Control Plasmid is designed to emit green fluorescence if there is a high concentration of ERα in the cells and emit red fluorescence if there is a high concentration of ERβ. Generally, there is a higher concentration of ERα in endometrium cells, while there is a higher concentration of ERβ in the endometrial-like cells. Therefore, we would expect similar concentration patterns in our HESC and 12Z cell lines. In a later attempt we used a standard GFP plasmid to determine whether the issue was with our designed plasmid or with the transfection process itself, since the transfections kept being unsuccessful.

![Placeholder image](https://static.igem.wiki/teams/5253/results/transfection-results-1.jpg)

Figure 7: Results from transfection both with our plasmid and a standard GFP plasmid. The y-axes show the red fluorescent intensity, and the x-axes show the green fluorescent intensity. (A) Show the density of cells from the transfection with our control plasmid in 12Z cells. Blue dots show the lowest density of cells and red dots show the highest density of cells. The black box indicates where the cells that emit red fluorescent should be seen, which in this case is 0.05% of the sample. (B) Show the density of cells from the transfection with our control plasmid in HESC cells. Only 1.38% of the cells that are in the box. (C) Show all cells from the transfection with the standard GFP plasmid. The black box shows where cells that emit green fluorescent should be seen. (D) Shows the isolated cells that were expressing green fluorescence. In both (C) and (D) the dark blue and green dots represent 12Z cells that have gone through transfection with the GFP plasmid. The red dots represent 12Z cells that went through transfection without the plasmid. The purple dots represent the negative control for 12Z cells, so they have not gone through transfection. The yellow dots represent HESC cells that went through transfection with the plasmid, and the light blue dots represent HESC cells that went through transfection without the plasmid.

×

![Enlarged image]()

The graphs, seen in Figure 7, visualizes the results from two different transfection experiments. In Figure 7A the density of 12Z cells that underwent transfection with our control plasmid are shown. We expected to observe a large number of cells within the black box, which indicates the area for cells that expressed red fluorescent. Unfortunately, only 0.05% of cells are located within the black box, indicating an unsuccessful transfection. In Figure 7B the density of HESC cells that underwent transfection with our control plasmid is shown. We expected to observe no cells in the black box, and a large number of cells positioned lower on the y-axis and further to the right on the x-axis, as these cells were expected to emit green fluorescence. This expectation is based on the function of the control plasmid, as the BRCA1 promotor in the control plasmid responds to the higher concentration of ERα, which promotes the expression of GFP - thereby emission of green fluorescence would be expected. Unfortunately, this is not the case as only 1.38% of the cells are located in the black box.

In Figure 7C and D the results from the transfection with the standard GFP plasmid are shown. In Figure 7C all the cells transfected with the standard GFP plasmid are shown. Here we expected to see a large number of dark blue, green and yellow dots within the black box, as these cells were transfected with the standard GFP plasmid. However, this was not the case, as there are more cells outside the box than within the area. In Figure 7D the cells within the black box, seen in Figure 7C, are isolated. As mentioned earlier, we expected to see a lot of dark blue, green and yellow dots in this area. Unfortunately, there are lots of every color in Figure 7D, indicating that cells from almost all of the samples are found there. However, there is mostly dark blue dots, which represent the 12Z cells transfected with the standard GFP plasmid, but there are too few to confirm whether the plasmid was successfully transfected into the cells.

# HESC Optimization

---

One of the challenges that we faced during our project was that our HESC cell line was growing very slowly. The prolonged growth period caused an issue as it delayed our experimental timeline, due to the fact that the cells needed to be confluent enough (70-90%) to actually be used for an experiment. Therefore, we decided to try and optimize the HESC cells conditions, and thereby their growth, through various media combinations.

The average time between splits in our original HESC cell lines was 11.3 days, before we began our growth optimization experiment. Our cell lines had a doubling time of approximately 8.87 days, which was considerably longer than the typical scenario, where cells are split every 3 days upon reaching a confluency of about 70-90%. Prior to our optimization experiment, the cells were cultured in T75 flasks, but as shown in Figure 8A, transferring the cell lines to a T25 results in a slight change in the growth rate compared to cells grown in the standard T75 flasks, as shown in Figure 8B.

![Placeholder image](https://static.igem.wiki/teams/5253/results/hesc-1.jpg)

Figure 8: (A) shows the growth rate of original HESC cells in T75 flasks. (B) Shows the growth rate of original HESC cells in T25 flasks.

In the experiment we used several different media combinations, as shown in the table below.

**Table 1:** This table gives an overview of the different media combinations used in the experiment. Each flask was assigned a code (e.g. “O2”), differentiating the type of media that they contain.

| Type | Conditions |
| --- | --- |
| Normal | DMEM-F12 + 10% charcoal-stripped FBS + 1% Pen-Strep + 1% L-glutamine |
| O1 | DMEM-F12 + 10% Human AB Serum + 1% Pen-Strep + 1% L-glutamine |
| O2 | DMEM-F12 + 10% charcoal-stripped FBS + 0.5% Pen-Strep + 1% L-glutamine |
| O3 | DMEM-F12 + 10% FBS + 1% Pen-Strep + 1% L-glutamine |
| O4 | Embryomax + 10% charcoal-stripped FBS + 1% Pen-Strep + 1% L-glutamine |
| O5 | Embryomax + 10% Human AB Serum + 1% Pen-Strep + 1% L-glutamine |

Replacing 10% charcoal-stripped FBS with 10% Human AB Serum results in an improved growth rate of the cells in O1 and O5. The doubling time of the HESC cells growing in O1 media, when compared to the original HESC cell line in a T75 flask, is decreased by 6,74 days in comparison to the original doubling time of 8.87 days, which was initially positive for our experiment. However, in the following days it was observed that the O1 media appeared cloudy, and it was therefore checked for contamination. Although the test did not confirm any contamination, the cells growing in the O1 media looked anomalous and were therefore discarded. The media was no longer used throughout the experiment.

In O2 media, the concentration of Pen-strep was reduced to 0.5% from the recommended 1%. This change in the concentration of Pen-strep is also seen to have a positive effect on the growth rate of the O2 cells, reducing the doubling time by 6.32 days.

The O5 media consists mainly of EmbryoMax and 10% Human AB Serum instead of DMEM-F12 and 10% charcoal-stripped FBS. The media combination shows good improvement in growth rate, decreasing the doubling time by 6.63 days,compared to the original 8.87 days. The O3 media results in a decrease of 6.74 days in doubling time, compared to the original 8.87 days. The single change in the O3 media, is the use of normal 10% FBS instead of 10% charcoal-stripped FBS.

The media combination O4 shows the best results out of all. We switched out the recommended DMEM-F12 with Embryomax, which is the media recommended for our 12Z cell line. This shows a very noticeable increase in cell growth, thereby indicating that Embryomax could have a positive effect on the growth of our HESC cells. The doubling time is decreased by 6.4 days in comparison to the original 8.87 days.

![Placeholder image](https://static.igem.wiki/teams/5253/results/hesc-2-result.jpg)

Figure 9: (A) shows the growth rate of O4 HESC cells, (B) shows the growth rate of O3 HESC cells and (C) shows the O1 HESC cells.

In all, our HESC growth optimization experiment results in the O4 media being the most successful media combination, resulting in a decrease in doubling time by 7.09 days as opposed to the original 8,87 days. O1 and O3 are closely following it, each decreasing the cells doubling time by 6.71 days, as compared to the original 8.87 days. To learn more about our HESC optimization experiment, take a look under [experiments](https://2024.igem.wiki/sdu-denmark/experiments).

# Western Blot

---

To test if our antibodies are specific for only the ERα and Erβ, we used western blot as described in [experiments](https://2024.igem.wiki/sdu-denmark/experiments). The Nanodrop in our lab was broken so we could not measure the protein concentration of our lysed cells. Therefore, we decided to make five different samples for each cell line. The cell lines were lysed and diluted with PBS. When loading the gel, we used a protein ladder, a 12Z sample, and a HESC sample. Afterwards we added our antibodies one after the other, so both antibodies were distributed all over the membrane. The fluorescence was measured at the same time.

![Placeholder image](https://static.igem.wiki/teams/5253/results/western-blot-new.jpg)

Figure 10: Western blot results from antibodies that bind to ERα or ERβ. (A) Results for Estrogen Receptor alpha, the fluorescent antibody that binds to ERα. The red squares show the bands from the antibody. (B) results for ESR2 Monoclonal Antibody, the fluorescent antibody that binds to ERβ. The red squares show the bands form the antibody. The yellow square marks two ladder bands. The upper band is ~70 kDa and the lower band is ~55 kDa.

The red squares, seen in Figure 10A, are indicating where the bands of the ERα are located. ERα is 67 kDa (1). Therefore, we expected to see the ERα band near the ladder band with the length of ~70 kDa. This was also the case as shown in Figure 10A, where the ERα band is located next to the ladder band. This could confirm that the antibody binds specifically, and thereby only to ERα. In Figure 10B the red squared is indicating where the bands of ERβ are located. ERβ is 59 kDa (1), and we therefore expected that the ERβ band would appear between the two ladder bands marked in the yellow square in Figure 10B, as the lower band has a length of ~55 kDa and the upper band has a length of ~70 kDa. However, it appears that there was an error with the staining process, as the ERβ band is next to the ladder band with the size of ~70 kDa and not in between the two ladder bands. Thereby we cannot 100% confirm the specificity of this antibody.

# Antibody Staining

---

In this experiment, 12Z and HESC are stained for ERα and ERβ with the use of ESR2 Monoclonal Antibody and Estrogen Receptor alpha Antibody coupled with fluorescence. The goal was to titrate the antibodies used for staining, in order to optimize detection of ERα and Erβ in these specific cell lines. ERα staining was performed as an extracellular stain without permeabilizing the cells, while ERβ staining was performed intracellularly after permeabilization. The stained cells were analyzed using flow cytometry to assess the percentage of receptor-positive cells, and to evaluate the antibody titrations.

![Placeholder image](https://static.igem.wiki/teams/5253/results/ab-staining.jpg)

Figure 11: Results from antibody staining for both 12Z- and HESC cells. (A) Results from antibody staining on 12Z cells with the antibody that binds to ERα. The y-axis is ERα antibody fluorescence, and the x-axis shows the antibody dilution, highest (left) to lowest (right). Blue dots show the individual population and red dots show the highly dense population. (B) A graph visualizing the percentage of cells with positive ERα signal after staining with the different antibody concentrations. The y-axis shows the percentage of ERα+ cells present, and the x-axis shows the concentration of antibody at different dilutions. (C) Results from antibody staining on HESC cells showing the amount of ERα signaling in each dilution range. (D) Results from antibody staining on 12Z cells, showing single cells with ERβ signals. (E) A graph visualizing the affect antibody concentration had on the amount of ERβ+ cells. The y-axis shows the percentage of ERβ + cells present, and the x-axis shows the concentration of antibody at different dilutions. (F) Results of the ERβ stain on HESC cells, showing single cells with ERβ signals.

**12Z Cells - ERα Staining (Figure 11A)**

For ERα staining in 12Z cells, the antibody was titrated at different concentrations between 1:12.5 and 1:200, doubling at each dilution. 5 wells were stained with ERα antibody extracellularly while 1 additional well remained unstained on the surface. The flow cytometry plot, seen in Figure 12A, was obtained by creating live cells channel by using zombie violet dye and the ERα antibody channel to detect the cells with positive cells with ERα antibody.

The results show a gradient from high concentration / low dilution (left) toward low concentration / high dilution (right). For most samples with antibody staining, the majority of the cell population show low fluorescent signal intensity; therefore, only a small fraction was considered as ERα positive. Only with the highest antibody concentration (least diluted), around 85% of cells were Era positive, but still with relatively low signal.

**12Z Cells - ERβ Staining (Figure 11D)**

For ERβ intracellular staining in 12Z cells, the antibody is titrated at different concentrations between 1:25 and 1:400 into 5 wells and 2 further wells are used as intracellular unstained cells. The flow cytometry data, seen in Figure 11D, show the majority of the cells exhibiting strong fluorescence signal even at highest dilutions (lowest antibody concentrations), resulting in more than 90% of cells being ERβ positive. These findings support the upregulation of ERβ in endometrial-like cells stated in literature.

**HESC Cells - ERα Staining (Figure 11E)**

In HESC cells, ERα staining was titrated between 1:12.5 and 1:25 in 2 wells and 1 well for surface unstained control. As shown in Figure 11C, the fluorescence signal for ERα is weak and sporadic, with only a small population of cells showing positive staining. The low amount HESC samples tested restraints the accuracy of the titration curve for these dilutions and limits the descending gradient as well. More samples should be tested to refine the graph and ensure the optimal dilution is accurately identified.

**HESC Cells - ERβ Staining (Figure 11F)**

For HESC cells, ERβ staining is titrated between 1:50 and 1:100 in 2 wells and 1 additional well is used for intracellular unstained control. Similar to the ERα staining, the small number of samples limits the precision of this titration. More HESC samples across a wider range of dilutions would provide a more accurate assessment of the optimal ERβ antibody concentration, and higher dilutions should be tested to avoid the saturation seen in the current data.

 **Conclusion**

Based on the performed antibody titration, it was found that the optimal dilution of the ERα antibody on 12Z cells was 1:50, as shown in Figures 11A and 11B. For the ERβ antibody titration on the 12Z cells, no optimal dilution factor could be identified, as all investigated dilutions resulted in saturated staining, which can be seen in Figures 11C and 11D. For the antibody titration on the HESC cells, no successful results were obtained due to insufficient cell numbers. However, it was observed that the ERα and ERβ antibodies successfully bound to their targets on the HESC cells, as both positive and negative populations were detected, as shown in Figures 11E and 11F.

# Fluorescence Microscopy

---

We have based our whole project around the fact that research shows, that the cells involved in endometriosis express up to 100x more ERβ than ERα (2), so we wanted to see if this was true for the two cell lines which we have used throughout this experiment: 12Z and HESC. We did this by performing an antibody staining with two antibodies: ESR2 Monoclonal Antibody and Estrogen Receptor alpha Antibody. The ESR2 Monoclonal Antibody is conjugated with CoraLite Plus 488, which emits a green-fluorescent light. The Estrogen Receptor alpha Antibody, on the other hand, is conjugated with PerCP, which emits a red-fluorescent light.

![Placeholder image](https://static.igem.wiki/teams/5253/results/fluorescence-result-1.jpg)

Figure 12: This figure shows the results of staining 12Z cells (A-C) and HESC cells (D-F) with both ESR2 Monoclonal Antibody (green) and Estrogen Receptor alpha Antibody (red). Additionally, the cells were stained with DAPI to visualize the nucleus.

In Figure 12 it can be seen that the 12Z cells (picture A-C) show a slightly clearer expression of ERβ, than in the HESC cells (picture D-F), as ER-β can be seen as a kind of green hue around the cells. On the other hand, it looks like the ERα, in the 12Z cells, has a tendency to lay more around the nuclei of the cells, which can be seen as the slightly red hue “covering” the nuclei. In the HESC cells, it looks like there is way less ERβ present in the cytoplasm of the cells, as not much green fluorescent can be seen in pictures D-F. The ERα is only slightly present in the HESC cells, not showing a very strong signal. These findings roughly align with what we expected to find, which is that the endometrial-like cells (here: 12Z) express noticeably more ERβ than endometrium cells (here: HESC), while the endometrium cells should express more ERα than the endometrial-like cells.

From this experiment alone, it is not possible to conclude the precise localizations of the two receptors, or if there is more of one receptor type present than the other in the cells. If our experiment with transfecting our two cell lines with the Control Plasmid had been successful, those results could have been used to confirm and/or disconfirm our theory on which receptor is expressed more in which cell line. The western blot performed on the antibodies (see Figure 10), confirm that the Estrogen Receptor alpha Antibody is specific for the ERα receptor only. This tells us that the signal of red fluorescent is only coming from ERα receptors in both cell lines. The western blot performed on the ESR2 Monoclonal Antibody, did not confirm its specificity (see Figure 10), so that makes it harder for us to trust that all the green fluorescence in pictures A-F comes only from the ERβ receptors.

![Placeholder image](https://static.igem.wiki/teams/5253/results/fluorescence-results-2.jpg)

Figure 13: The excitation and emission spectrums of the conjugates which the two antibodies contain, respectively. The excitation spectrum is only outlined, while the emission spectrum is shown colored in.

As it can be seen in figure 13 the distance between the two antibodies emission spectrums is not that far, which could cause some trouble. While the emission spectrums are shown as the colored-in graphs in figure 13, it is important to note that the filter of the microscope starts to emit 100 nm before and after the spectrum. Due to this, there is going to be a slight overlap between the two antibodies, which could possibly be the reason why the red fluorescence signal is not that clear. The samples were checked for “bleed-through” - a fantom that occurs when multiple lasers are used at once, causing them to leak into the other channels - and this was not the case. Another reason for the weak red fluorescent signal could be, that the antibodies which we have chosen to work with, are just not compatible with the confocal microscopy used to analyze the samples.

# Future plans

---

For future experiments, we plan to expand our electroporation studies by using our own control and treatment plasmids, leveraging the optimized parameters identified in electroporation tests. By utilizing the Nucleofector solutions and programs that demonstrated best efficiency and lowest cell mortality, we aim to enhance transfection outcomes in subsequent trials.

Additionally, we aim to repeat the antibody titration, expanding the number of samples for each cell line and increasing the dilution range. This will provide a more comprehensive understanding of antibody performance across different conditions.

Furthermore, we intend to redesign our plasmid to incorporate new elements, including more "safety switches" such as a progesterone-responsive system. Since higher levels of progesterone are associated with healthy endometrial cells, and lower levels correlate with endometriosis, incorporating this switch could allow for better regulation of our plasmid expression based on cellular health status. More of this can be read in [Engineering](https://2024.igem.wiki/sdu-denmark/engineering).

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