iGEM 2024 — Team um-macau

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## UM-MACAU

#### [Experiment Spaces](#P1)

---

#### [Experiments Calendar](#P2)

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# iGEM 2024 Wet Lab Notebook

## Experiment Spaces

E12-1061&1064 Lab Room

E12-1063 Cell Culture Room

E12-1065 -80℃ Fridge Room

E12-1067 DLS Measurement Room

E12-1072 Sterilization and Ice Taking Room

E12-2068 Apotome Fluorescence Microscope Room

E12-3071 Leo’s Lab

N22-3038 Flow Cytometry (Cell Sorting) Room

## Experiments Calendar

May 30, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Zihan Jiang]:
Sterilizing tips and pipettes. Prepare the LB broth. Writing experimental protocols.

May 31, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Zihan Jiang]:
Bacteria incubation in 100 ml LB broth overnight (1358, 1362, 1364, and 1833). Writing experimental protocols.

Jun 1, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Zihan Jiang]:
Conduct the plasmids midi-purification and DNA concentrations are as below. 1362 and 1833 are qualified for our transfection experiments.

![](https://static.igem.wiki/teams/5353/notebook/screenshot-2024-09-23-at-22-59-46.png)

Jun 2, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Zihan Jiang]:
Find the protocols of medium preparation, cell culture and passage. Prepare the trypsin/EDTA, PBS, FBS, p.s., milli-Q water and sterilize the bottles.

Jun 3, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Gigi Lei, Yuan Tang]:
DMEM preparation and get the HEK293T cells.

Jun 4, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Zihan Jiang]:
Waiting for the HEK293T cells to grow. Search and rewrite the experiment protocols of transfection with HEK293T cells (lentivirus packaging).

Jun 5, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Zihan Jiang]:
Conduct the transfection with HEK293T cells (1108, 1356 and 1833).

![](https://static.igem.wiki/teams/5353/notebook/1.jpg)

Jun 6, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Zihan Jiang]:
Change the medium of the transfected cells. HEK293T cell passage.

Jun 7, 2024 [Daoing MA, Zihan Jiang, Zihan Jiang]:
Lentivirus collection. Change the fresh medium to cells

Jun 8, 2024 [Daoming MA, Zixing Chen]:
Lentivirus collection. Change the fresh medium to cells.

![](https://static.igem.wiki/teams/5353/notebook/2.jpg)

Jun 10, 2024 [Daoming Ma, Zixing Chen, Zihan Jiang]:
Prepare LB broth for more plasmid extraction. HEK293T cell passage.

Jun 11, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
Conduct plasmid midi-purification. The color of the bacteria culture during midi-prep is too turbid, the suggestion given is that we should pick a single bacteria colony and use that for further amplification.

![](https://static.igem.wiki/teams/5353/notebook/3.jpg)

Jun 12, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
Prepare the LB agar and incubate bacteria on them.

Jun 13, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Gigi Lei, QianYu Gu]:
Select the single colon bacteria and incubate in 100 ml LB broth for amplification.
HEK293T cell culture and passage to the six-well plates and 10 cm dish.

![](https://static.igem.wiki/teams/5353/notebook/4.jpg)

Jun 14, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, QianYu Gu, Zihan Jiang]:
Plasmids midi-prep.

![](https://static.igem.wiki/teams/5353/notebook/screenshot-2024-09-23-at-22-59-59.png)

Jun 15, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Gigi Lei]:
HEK293T cell culture and passage.

Jun 16, 2024 [Daoming MA, Zixing Chen, Gigi Lei]:
HEK293T cell infection to generate the stable cell lines with lentivirus (1833 and 1362).

![](https://static.igem.wiki/teams/5353/notebook/5.jpg)

Jun 18, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Gigi Lei, SiCheng Zhou]:
HEK293T cell culture. Change the medium in six-well plates to the fresh one with G418.

Jun 19, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, SiCheng Zhou]:
Transfection with HEK293T cells (1108 and 1358).

Jun 20, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Gigi Lei]:
Change the medium in six-well plates to the fresh one with G418. HEK293T cell culture. Change the medium in the transfected cells to the refresh one.

Jun 21, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Gigi Lei]:
Change the medium in six-well plates to the fresh one with G418. Collect the lentivirus.

Jun 22, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
Change the medium in six-well plates to the fresh one with G418. Collect the lentivirus.

Jun 24, 2024 [Daoming MA, Gigi Lei]:
Prepare the LB broth and incubate the bacterias in 100 ml LB broth overnight for amplification.

Jun 25, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Gigi Lei]:
Conduct plasmids midi-prep.

![](https://static.igem.wiki/teams/5353/notebook/screenshot-2024-09-23-at-23-00-13.png)

Jun 25, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Qianyu Gu]:
Change the medium in six-well plates to the fresh one with G418. HEK293T cell culture.

Jun 26, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Qianyu Gu]:
Fluorescence microscope detection (1833 success, and 1362 fail).

Jun 28, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Qianyu Gu]:
Run PCR to check the DNA.

![](https://static.igem.wiki/teams/5353/notebook/6.jpg)

Jun 29, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Qianyu Gu]:
HEK293T cell culture and passage.

Jul 2, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Qianyu Gu]:
Transfection and Infection.

![](https://static.igem.wiki/teams/5353/notebook/7.jpg)

Jul 3, 2024 [DaoMing MA, Zixing Chen, Zhe Zhang, Qianyu Gu]:
HEK293T cell culture and passage.

Jul 4, 2024 [Daoming MA, Zixing Chen, Qianyu Gu]:
Cell cytometry for cell sorting. (the sorted cell number is too small, and we wait for the cell to grow for about 10 days.)

Jul 5, 2024 [Daoming MA, Zixing Chen, Qianyu Gu]:
Prepare the LB broth and incubate the bacteria in 100 ml LB broth overnight.

Jul 6, 2024 [Daoming MA, Zixing Chen, Qianyu Gu]:
Conduct the midi-prep.

![](https://static.igem.wiki/teams/5353/notebook/screenshot-2024-09-23-at-23-00-28.png)

![](https://static.igem.wiki/teams/5353/notebook/8.jpg)

Jul 7, 2024 [Daoming MA]:
HEK293T cell culture and passage.

Jul 8, 2024 [Daoming MA, Zixing Chen, Gigi Lei]:
Lentivirus collection, cell passage.

Jul 9-15, 2024 [Zixing Chen, Zhe Zhang, Gigi Lei]:
HEK293T cell culture and passage. Transfection and infection.

![](https://static.igem.wiki/teams/5353/notebook/9.jpg)

Jul 17, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
Stable HEK293T cell lines freezing.

Jul 18, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
Change the medium to the Opti-MEM (reduced serum medium) for the exosomes extraction.

Jul 20, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Qianyu Gu]:
Collect the supernatant containing the exosomes and conduct the exosomes extraction experiments.

Jul 22, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Yaolin Shi]:
HEK 293T cell transfection, cell infection, and fluorescent imaging.

Jul 23, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Yaolin Shi]:
Conduct the exosomes extraction experiments.

Jul 24, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Yaolin Shi]:
Run PCR.

![](https://static.igem.wiki/teams/5353/notebook/10.png)

Jul 27, 2024 [Daoming Ma, Yaolin Shi]:
HEK293T cell culture and passage.

Jul 28, 2024 [Daoming Ma, Yaolin Shi]:
Prepare the LB broth and incubate the bacteria in 100 ml LB broth overnight for amplification.

![](https://static.igem.wiki/teams/5353/notebook/11.jpg)

Jul 29, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Yaolin Shi]:
Plasmid midi-prep.

![](https://static.igem.wiki/teams/5353/notebook/screenshot-2024-09-23-at-23-00-37.png)

![](https://static.igem.wiki/teams/5353/notebook/12.jpg)

Jul 30, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Sicheng Zhou]:
1362, 1358, 1108, 1356 cell transfection.

Jul 31, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Sicheng Zhou]:
DMEM medium preparation.

Aug 1, 2024 [Daoming MA, Sicheng Zhou]:
DLS measurement and PCR.

![](https://static.igem.wiki/teams/5353/notebook/13.jpg)

![](https://static.igem.wiki/teams/5353/notebook/14.jpg)

Aug 2, 2024 [Daoming MA, Zixing Chen, Zhe Zhang, Sicheng Zhou]:
HEK293T cell culture and passage.

Aug 4, 2024 [Daoming MA, Sicheng Zhou]:
HEK293T cell transfection and infection.

Aug 6, 2024 [Zixing Chen, Zhe Zhang, Sicheng Zhou]:
Lentivirus collection.

Aug 7, 2024 [Zhe Zhang, Sicheng Zhou]:
Lentivirus collection and HEK293T cell culture.

Aug 9, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
HEk293T cell culture and passage.

Aug 11, 2024 [Daoming MA, Sicheng Zhou]:
HEK293T cell culture and fluorescence microscope imaging.

![](https://static.igem.wiki/teams/5353/notebook/15.jpg)

Aug 12, 2024 [Zhe Zhang]:
Conduct the exosomes extraction experiments.

Aug 13, 2024 [Zhe Zhang, Sicheng Zhou]:
HEK293T cell culture and passage.

![](https://static.igem.wiki/teams/5353/notebook/16.jpg)

Aug 15, 2024 [Zixing Chen, Sicheng Zhou]:
HEK293T cell culture and apotome fluorescence microscope imaging.

Aug 16, 2024[Zhe Zhang, Sicheng Zhou]:
Send the exosomes to do the TEM imaging (one week). HEK293T cell culture and passage.

Aug 19, 2024[Daoming MA, Zixing Chen, Zhe Zhang]:
RPMI1640 medium preparation.

Aug 20, 2024 [Daoming MA, Sicheng Zhou]:
Cancer cell culture.

Aug 21, 2024 [Zixing Chen, Zhe Zhang]:
BCA assay. Cancer cer culture and passage.

Aug 22, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
Western blot.

Aug 23, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
Western blot. Cancer cell culture and passage.

Aug 25, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
Exosome extration.

Aug 26, 2024 [Daoming MA, Sicheng Zhou]:
DLS measurement.

Aug 27, 2024 [Daoming MA, Zhe Zhang, Sicheng Zhou]:
Ultrasound stimulation.

Aug 28, 2024 [Zixing Chen, Zhe Zhang]:
Cancer cell apoptosis assay.

Aug 28, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
Cancer cell apoptosis assay.

Aug 30, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
Meeting with professor Yehuda Assaraf

Sept 1, 2024 [Daoming MA, Sicheng Zhou]:
Cancer cell proliferation assay.

Sept 3, 2024 [Daoming Ma, Zhe Zhang]:
Cell cytometry.

![](https://static.igem.wiki/teams/5353/notebook/17.jpg)

Sept 4, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
Cancer cell culture and passage.

Sept 9, 2024 [Daoming MA, Zhe Zhang, Sicheng Zhou]:
BCA assay and western blot.

Sept 10, 2024 [[Daoming MA, Zixing Chen, Sicheng Zhou]:
Western blot.

Sept 12, 2024 [Zhe Zhang, Zixing Chen]:
Exosome extraction.

![](https://static.igem.wiki/teams/5353/notebook/20240923233943.jpg)

Sept 15, 2024 [Zixing Chen, Sicheng Zhou]:
Infect the cancer cells directly with the lentivirus.

Sept 16, 2024 [Daoming MA]:
Apotome flurescence microscope.

![](https://static.igem.wiki/teams/5353/notebook/19.jpg)

Sept 17, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
Ultrasound stimulation.

Sept 18, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
Cancer cell apoptosis assay.

Sept 19, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
Cancer cell proliferation assay.

Sept 20, 2024 [Daoming MA, Zixing Chen, Zhe Zhang]:
Exosome extraction and DLS measurement.

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{% block headerimg %}
![Hero Image](https://static.igem.wiki/teams/5353/subtitle/header-engineering.png)
![Hero Image](https://static.igem.wiki/teams/5353/subtitle/title-engineering.png)
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## UM-MACAU

#### [Cycle 1](#P1)

* [Define](#P2)
* [Design](#P3)
* [Building](#P4)
* [Test and Experiments](#P5)
* [Learn](#P6)

---

#### [Cycle 2](#P7)

* [Define](#P8)
* [Design](#P9)
* [Building](#P10)

---

#### [Cycle 3](#P11)

* [A. Enhancing the specificity](#P12)
* [B. Increase the yield and concentration](#P13)
* [C. stimulation calcium overload killing](#P14)

---

# Cycle 1

## Define

Cancer remains a global challenge and a persisting leading cause of death despite extensive efforts to combat it. The complexity of the disease, along with the drug resistance of cancer cells has made finding an effective therapeutic approach became an elusive goal. Classical approaches, such as chemotherapy and radiotherapy, are limited by their non-specificity and drug resistance. While recent advancements in immunotherapy have significantly benefited some patients, a substantial percentage of patients do not respond to these treatments. Therefore, a novel strategy that improves specificity and reduces the likelihood of drug resistance is urgently needed.

## Design

Our project focuses on developing a novel approach by engineering exosomes to deliver essential proteins to cancer cells, stimulating calcium overloading and inducing cancer cell death. This strategy involves introducing calcium ion channels (MscS or hTRPC1) and an NO signaling activator (hNOS2) using exosomes as the delivery vehicle.

Referring to the iGEM16\_Slovenia group and we selected the calcium ion channels MscS (bacterial Mechanosensitive Channel ) (BBa\_K1965000) and hTRPC1 (Transient Receptor Potential Channel 1) (BBa\_K1965002) that respond to physical stress on the membrane and can be activated by ultrasound stimulation (Hurst et al., 2007; Kunichika et al., 2004) (Figure 1.1). By introducing these channels into cancer cells, we aim to generate calcium overloading. Ultrasound stimulation will precisely control cell death by opening the channels and leading to calcium ion influx (Hu et al., 2024).

![](https://static.igem.wiki/teams/5353/engineering/figure-1-1.png)

Figure 1.1 MscS and hTRPC1 calcium channels function (iGEM16\_Slovenia).

To deliver our target proteins to cancer cells, we propose using exosomes as the carrier vehicle (Salunkhe et al., 2020) (Figure 1.2). We will utilize the exosomal transmembrane protein lamp2b (BBa\_K5353000), fused with our target proteins, to improve the loading of target proteins into the exosomes. In fact, Lamp2b has been widely used in the manufacture of engineered exosomes for targeted drug or other substances delivery (Qiao et al., 2023) (Figure 1.3).

![](https://static.igem.wiki/teams/5353/engineering/figure-1-2.png)

Figure 1.2 Role of lamp2b in mediating autophagosome-lysosome fusion.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-3.png)

Figure 1.3 Example working model of the engineered exosomes with lamp2b.

## Building

We constructed six plasmids corresponding to MscS, hTRPC1, and hNOS2 respectively (1364, 1833, 1362, 1358, 1108, and 1356 is our plasmid coding number we label).

**MscS Calcium Channel**

1364: LentiV-MscS/HA: EGFP/Neo (BBa\_K5353061)

1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo (BBa\_K5353062)

1362: LentiV-Lamp2b/MscS/mCherry: Neo (BBa\_K5353063)

![](https://static.igem.wiki/teams/5353/engineering/figure-1-4-1.png)

Figure 1.4.1 Plasmids information of the MscS calcium channel.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-4-2.png)

Figure 1.4.2 Plasmid information of 1364.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-4-3.png)

Figure 1.4.3 Plasmid information of 1833.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-4-4.png)

Figure 1.4.4 Plasmid information of 1362.

**hTRPC1 Calcium Channel**

1358: LentiV-hTRPC1/HA: EGFP/Neo (BBa\_K5353064)

1108: LentiV-Lamp2b/hTRPC1/HA: Neo (BBa\_K5353065)

![](https://static.igem.wiki/teams/5353/engineering/figure-1-5-1.png)

Figure 1.5.1 Plasmids information of the hTRPC1 calcium channel.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-5-2.png)

Figure 1.5.2 Plasmid information of 1358.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-5-3.png)

Figure 1.5.3 Plasmid information of 1108.

## Test and Experiments

To achieve the objective of this project and the elaboration, please refer to the “Protocol” and “Results” section for the detailed experimental protocol and results. Here are some important experiments summary and conclusions.

### 1. Midi-Prep Plasmid DNA preparation and PCR

The designed DNA was constructed and we used the midi-prep preparation kit to conduct the plasmid isolation and purification of the plasmid DNA (Table 1). The inserts in each construct were further validated by the PCR reaction (Figure 1.6.1 and Figure 1.6.2).

![](https://static.igem.wiki/teams/5353/engineering/table-1.png)

Table 1. DNA concentrations extracted and purified.

The amplicon size of the four targeted sequences are: Lamp2b (103 bp) , MscS (225 bp), hTRPC1 (162 bp) and hNOS2 (142 bp), respectively.

Plasmids Checked:

1364: LentiV-MscS/HA: EGFP/Neo (BBa\_K5353061)

1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo (BBa\_K5353062)

1362: LentiV-Lamp2b/MscS/mCherry: Neo (BBa\_K5353063)

1358: LentiV-hTRPC1/HA: EGFP/Neo (BBa\_K5353064)

1108: LentiV-Lamp2b/hTRPC1/HA: Neo (BBa\_K5353065)

1356: LentiV-hNOS2/Flag: Puro (BBa\_K5353066)

![](https://static.igem.wiki/teams/5353/engineering/figure-1-6-1.png)

Figure 1.6.1 Gel electrophoresis results of 1833, 1362, 1358, 1108, 1356.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-6-2.png)

Figure 1.6.2 Gel electrophoresis results of 1358 and 1364.

### 2. Transfection, Infection and Fluorescence Microscope Imaging

To validate the infection efficiency, we inserted the EGFP or mCherry into different plasmids. Therefore, the successful infection could be visualized by fluorescence microscopy.

1364: LentiV-MscS/HA: EGFP/Neo

![](https://static.igem.wiki/teams/5353/engineering/figure-1-7-1.png)

Figure 1.7.1 Expression of green fluorescent protein of 1364 HEK293T cell.

1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo

![](https://static.igem.wiki/teams/5353/engineering/figure-1-7-2.png)

Figure 1.7.2 Expression of green fluorescent protein of the 1833 HEK293T cell.

1362: LentiV-Lamp2b/MscS/mCherry: Neo

![](https://static.igem.wiki/teams/5353/engineering/figure-1-7-3.png)

Figure 1.7.3 Expression of red fluorescent protein of 1362 HEK293T cell.

1358: LentiV-hTRPC1/HA: EGFP/Neo

![](https://static.igem.wiki/teams/5353/engineering/figure-1-7-4.png)

Figure 1.7.4 Expression of green fluorescent protein of 1358 HEK293T cell.

### 3. Confirm the production of exosomes by TEM

The size of the exosomes is extremely small, approximately 30–140 nm, membrane-enclosed molecules.

The halo-like structure is the exosomes (Figure 1.8.1 and Figure 1.8.2), and the light dots shown on the background are salts. The normal and well-functioning exosomes are standard round, while the broken and unhealthy exosomes with an elliptical collapse structure were also observed (Figure 1.8.3).

![](https://static.igem.wiki/teams/5353/engineering/figure-1-8-1.png)

Figure 1.8.1 Exosomes with normal structures.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-8-2.png)

Figure 1.8.2 Exosomes tangled together.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-8-3.png)

Figure 1.8.3 Exosomes with elliptical collapse structure.

### 4. DLS Measurement to determine the size of the exosomes

We used DLS to provide valuable insights into the size distribution and polydispersity of exosomes, which typically range in size from 200 to 300 nanometers.

1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo

Exosome Sample 1833-1 (Figure 1.9.1):

The peak of the size (d. nm) is 186.6 nm and more than 70% of exosomes range from 141.8 nm to 220.2 nm, which is similar to the size of TEM imaging.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-9-1.png)

Figure 1.9.1 Exosome size distribution of sample 1833-1.

1362: LentiV-Lamp2b/MscS/mCherry: Neo

Exosome Sample 1362-3 (Figure 1.9.2):

The peak of the size (d. nm) is 372.2 nm.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-9-2.png)

Figure 1.9.2 Exosome size distribution of sample 1362-3.

1108: LentiV-Lamp2b/hTRPC1/HA: Neo

Exosome Sample 1108-1 (Figure 1.9.3):

The peak of the size (d. nm) is 20.93 nm.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-9-3.png)

Figure 1.9.3 Exosome size distribution of sample 1108-1.

### 5. BCA Assay

The Table 2 shows the absorbance values at 562 nm for various concentrations of BSA standards. By plotting the absorbance values against the known protein concentrations, a standard curve is generated. This curve is then used to determine the protein concentration in unknown samples on the basis of their absorbance readings.

![](https://static.igem.wiki/teams/5353/engineering/table-2.png)

Table 2. The absorbance value (562 nm) of BSA and exosome samples.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-10.png)

Figure 1.10 The BCA standard absorbance curve.

According to the equation of the BCA standard absorbance curve, we calculated the total protein concentration in exosomes (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) samples through their absorbance values. The amount of proteins in three repeat extraction of 1833 is arranged from 0.067 to 0.280 mg/ml. The result suggests the first extraction (1833-1) has the best concentration.

### 6. Western Blot

In our Western blot experiments, we could detect the CD81 exosome marker in the samples 1833-1 (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo).

![](https://static.igem.wiki/teams/5353/engineering/figure-1-11.png)

Figure 1.11. Western Blot of CD81 from 1833 exosome samples.

### 7. Cancer Cell Apoptosis Assay

The Apoptosis Assay by flow cytometry using the Annexin V staining method is a technique to detect and quantify apoptotic cells within a cell population. It depends on Annexin V staining, propidium iodide (PI) staining, and flow cytometry analysis.

Cell apoptosis-A2780

The A2780 ovarian cancer cells treated with the exosome (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) have an increase in necrosis, but there is no difference in apoptosis.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-12-1.png)

Figure 1.12.1 Results of necrosis and apoptosis of A2780 under normal and exosome treated conditions.

Cell apoptosis-NCI-H1299

The NCI-H1299 lung cancer cells treated with the exosome (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) have an increase in apoptosis (although not significant due to a single experiment), but there is no change in necrosis.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-12-2.png)

Figure 1.12.2 Results of necrosis and apoptosis of NCI-H1299 under normal and exosome treated conditions.

## Learn

### 1. Midi-Prep Plasmid DNA preparation

At the beginning, the plasmid DNA extraction yields a low-concentration and impure DNA sample. Therefore, instead of directly amplifying the DH5α, we incubated the DH5α on LB agar and selected the single colony to amplify. This helps us obtain purer and higher-concentration DNA samples.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-13-1.png)

Figure 1.13.1 DH5α Amplification.

![](https://static.igem.wiki/teams/5353/engineering/figure-1-13-2.png)

Figure 1.13.2 Selection of a single colony.

### 2. Checking of the plasmid insert by Polymerase Chain Reaction (PCR)

The gel electrophoresis of the PCR product (1358: LentiV-hTRPC1/HA: EGFP/Neo) in Figure 1.6.1 failed, maybe due to overloading the DNA sample in the PCR reaction or error in electrophoresis.

### 3. Validate the lentivirus infection by an apotome fluorescence microscope imaging

During the transfection process, we encountered multiple failures in our initial attempts. At first, HEK293T cells were on the brink of death due to low density, while in other instances, the lentivirus packaging failed because of the high density of HEK293T cells, which affected the cell-cell interaction. And the optimal cell density for successful lentivirus packaging is typically 70%-80%, ensuring the production of well-functioning lentiviral vectors.

During the infection process, we faced challenges in establishing a stable cell line during the infection process, as G418 did not kill uninfected HEK293T cells. Subsequently, we discovered that G418 requires sustained and prolonged duration to exert its effect, playing a role after approximately the fifth to sixth day with the appropriate concentration of 1000 (μg/ml).

During the imaging process, we discovered that cell density significantly impacts imaging quality, with optimal results achieved when cells are arranged in a single layer (Figure 1.7.2). When cells are closely packed, the fluorescence doesn't show the cell structures (Figure 1.7.3). Additionally, it is crucial to change the medium before capturing images to wash away the dead cells.

### 4. DLS Measurement to determine the size of the exosomes

The results show the significance difference of size distribution between the different samples. The reason may be that we keep the exosomes extracted in the 4℃ refrigerator for a long time, about three weeks, while it is suggested to use the exosomes within one week or keep them in the -80℃ refrigerator for long-term storage. The long time storage may result in abnormal exosome structures or exosomes flocked together to fuse and form larger structures. In addition, because there are different target proteins that are inserted into the exosome, which can also affect the structure and formation of the exosome.

### 5. Exosomes imaging by TEM, measured by BCA Assay

The major challenge at the current stage is the concentration of exosomes. Therefore, we do need to increase the yield of the exosome and to produce a more concentrated exosome. Further optimization must be carried out, such as adjusting the culture environment by using specific media additives, hypoxic conditions, or altering the pH, which can stimulate cells to produce more exosomes. For concentrating exosomes, we could try to perform ultrafiltration or ultracentrifugation.

### 6. Western Blot

Perhaps this is because the exosome concentration is too low, and therefore the protein concentration is too low to be detected by western blot.
Furthermore, the oversized MscS, a weak electric field, and inadequate electrophoresis duration all lead to bands that were too large to migrate fully, resulting in incomplete separation on the gel.

### 7. Cancer cell apoptosis

Cell apoptosis-A2780

With the exosome treated, there are more A2780 cells in necrosis, but there is no difference in apoptosis. Although the exosome concentration in one well of a six-well plate is just approximately 0.056 mg/ml, the results still show that the cancer cell death is affected and more A2780 cells appeared resulting in necrosis. We think with higher exosome concentration and better ultrasound stimulation, there will be more cancer cells to die.

Cell apoptosis-NCI-H1299

With the exosome treated, there are slightly more NCI-H1299 cells in apoptosis (even the results are not significant due to only a single experiment), but there is no change in necrosis. We think the modest response may be due to the low exosome concentration and lack of enough membrane stress stimulation by the ultrasound. Repeat of the experiment is required to confirm the effect of the engineered exosomes.

# Cycle 2

## Define

According to the feedback from different interviews, most opinions suggest that in addition to using the ion channels, we need to look for another alternative to induce calcium overload. Therefore, we proposed to modulate the NO signaling hNOS2 to enhance the calcium overload efficiency.

## Design

hNOS2 (Nitric Oxide Synthase 2) (BBa\_K5353003) is an enzyme that induces NO signaling by catalyzing the reaction: L-arginine + O2 + NADPH + H+ → NO + L-citrulline + NADP+ + H2O. Up-regulation of the NO signal can induce the opening of the RyR calcium channel in the endoplasmic reticulum, dramatically increasing intracellular calcium levels (Ziolo et al., 2001) (Figure 2.1).

![](https://static.igem.wiki/teams/5353/engineering/figure-2-1.png)

Figure 2.1 NO release to stimulate RyR channel opening.

For hNOS2 (1356: LentiV-hNOS2/Flag: Puro), because of the limitation of the size of the lentivirus plasmid, the insert will be too large, if we combine lamp2b together with hNOS2. Therefore, we aim to carry out a second transfection of hNOS2 after generating the stable HEK293T cell line with the 1833, 1362, or 1108 plasmid. In this way, our objective is to observe whether hNOS2 can enhance the effect of MscS or hTRPC1 on calcium overload.

## Building

**hNOS2 (Nitric Oxide Synthase 2)**

1356: LentiV-hNOS2/Flag: Puro (BBa\_K5353066)

![](https://static.igem.wiki/teams/5353/engineering/figure-2-2-1.png)

Figure 2.2.1 Plasmid information of the hNOS2.

![](https://static.igem.wiki/teams/5353/engineering/figure-2-2-2.png)

Figure 2.2.2 Plasmid information of 1356.

We have extracted the plasmid DNA and checked the plasmid insert by **PCR**. We will continue the experiments following the two to three three weeks.

# Cycle 3

## A. Enhancing the specificity of exosome in targeting cancer cells

### Define

Precisely targeting cancer cells is crucial in effective therapy. Enhancing exosome targeting poses a significant challenge. Strategies to improve exosome targeting efficiency could involve exploring advanced delivery mechanisms, enhancing specificity through surface modifications, optimizing targeting ligands, and leveraging innovative technologies to ensure accurate and effective delivery to cancer cells.

### Design

To achieve the precision, we plan to introduce the iRGD peptide tag to our exosomes. Known for its tumor-penetrating properties, iRGD facilitates improved targeting specificity and efficiency in the delivery of payloads to cancer cells. By incorporating the iRGD tag, we aim to optimize the targeting ability of exosomes, potentially enhancing therapeutic outcomes through the delivery of more calcium channels from MscS and hTRPC1 to cancer cells.

### Building

![](https://static.igem.wiki/teams/5353/engineering/figure-3-1.png)

Figure 3.1 Plasmid information of exosomes with iRGD.

## B. Increase the yield and concentration of the exosome collection

### Define

As we mentioned before, because of the low concentration of exosomes, we cannot detect the CD63 and HA-tag successfully by western blot. More importantly, higher exosome concentrations can lead to more potent therapeutic effects. Therefore, how to improve the exosome production and exosome concentration is an important content.

### Design

Enhance exosome production by optimizing cell culture conditions such as media composition, pH, and temperature. Utilize efficient isolation methods like ultracentrifugation, size exclusion chromatography, or commercial kits to concentrate exosomes from biological samples. Moreover, ultrafiltration techniques are employed to concentrate exosomes by selectively passing them through membranes based on size.

Tips for Ultracentrifugation Process:

Choose an appropriate rotor and centrifuge tubes that can withstand the high speeds and forces generated during ultracentrifugation.

Carefully load the sample into the centrifuge tubes without introducing air bubbles, ensuring balanced loading across all tubes.

Start the ultracentrifuge and gradually increase the speed to the desired level, typically reaching speeds exceeding 100,000 x g or higher.

## C. ​​The use of ultrasound stimulation to control the calcium overload killing

### Define

We assume the MscS and hTRPC1 could be precisely controlled by the ultrasound stimulation. However, the optimal frequency of the ultrasound to open the two calcium channels is 310kHz, while ultrasound frequencies that are typically above 20 kHz can potentially damage cancer cells. Typically, these frequencies fall within the range of high-intensity focused ultrasound (HIFU). Furthermore, ultrasound frequencies can potentially cause damage to normal cells in the range of high-intensity focused ultrasound (HIFU) above 20 kHz. Therefore, how to improve the sensitivity of the MscS and hTRPC1 to the ultrasound is very important.

### Design

Use pulsed ultrasound instead of continuous waves to modulate calcium channel sensitivity. Pulsed ultrasound can exert controlled effects on calcium channels, enhancing their response to stimulation.

Employ focused ultrasound techniques to target specific areas more precisely. Focused ultrasound can concentrate energy on the target region, potentially increasing the sensitivity of calcium channels in that area.

{% endblock %}


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## UM-MACAU

#### [Overview](#P1)

---

#### [Design](#P2)

* [MscS Calcium Channel](#P3)
* [hTRPC1 Calcium Channel](#P4)
* [hNOS2 (Nitric Oxide Synthase 2)](#P5)

---

#### [Results](#P6)

* [Midi-Prep Plasmid DNA](#P7)
* [Checking of the plasmid insert](#P8)
* [Validate the lentivirus infection](#P9)
* [Confirm the production](#P10)
* [DLS Measurement](#P11)
* [BCA Assay](#P12)
* [Western Blot](#P13)
* [Cancer Cell Apoptosis Assay](#P14)

---

#### [Future Works](#P15)

---

# Overview

Our project focuses on developing a novel approach by engineering exosomes to deliver essential proteins to cancer cells, stimulating calcium overloading and inducing cancer cell death. This strategy involves introducing calcium ion channels (MscS or hTRPC1) and an NO signaling activator (hNOS2) using exosomes as the delivery vehicle.

We utilized the exosomal transmembrane protein lamp2b, fused with our target proteins, to enhance the loading of MscS, hTRPC1, and hNOS2 into the exosomes. Indeed, Lamp2b has been widely used in the manufacture of engineered exosomes for targeted drug delivery (Qiao et al., 2023).

# Design

## MscS Calcium Channel

By referencing the project of iGEM16\_Slovenia group, we selected the calcium ion channels MscS (bacterial Mechanosensitive Channel ) (BBa\_K1965000) and hTRPC1 (Transient Receptor Potential Channel 1) (BBa\_K1965002) to induce calcium overload. These receptors respond to physical stress on the membrane and can be activated by ultrasound stimulation (Hurst et al., 2007; Kunichika et al., 2004). By introducing the channels into cancer cells, we aim to generate calcium overload upon ultrasound stimulation. The ultrasound stimulation could precisely control cell death in tumors by opening the channels and leading to calcium ion influx (Hu et al., 2024). Hence, reduce the nonspecific killing effect. According to this objective, we designed three plasmids to investigate how the MscS calcium channel influences cancer cell growth.

1364: LentiV-MscS/HA: EGFP/Neo (BBa\_K5353061)

1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo (BBa\_K5353062)

1362: LentiV-Lamp2b/MscS/mCherry: Neo (BBa\_K5353063)

![](https://static.igem.wiki/teams/5353/results/figure-1-1.png)

Figure 1.1 Plasmid information for the MscS calcium channel.

## hTRPC1 Calcium Channel

Since MscS is a bacteria protein, we also used hTRPC1 (Transient Receptor Potential Channel 1) (BBa\_K1965002) as an alternative to induce the calcium overload. hTRPC1 could also respond to physical stress on the membrane and lead to calcium ion influx (Hurst et al., 2007; Kunichika et al., 2004). By introducing this calcium ion channel into cancer cells, it could also generate calcium overload after ultrasound stimulation. We designed two plasmids to investigate how the calcium channel hTRPC1 influences cancer cell growth in this project.

1358: LentiV-hTRPC1/HA: EGFP/Neo (BBa\_K5353064)

1108: LentiV-Lamp2b/hTRPC1/HA: Neo (BBa\_K5353065)

![](https://static.igem.wiki/teams/5353/results/figure-1-2.png)

Figure 1.2 Plasmid information for the hTRPC1calcium channel.

## hNOS2 (Nitric Oxide Synthase 2)

In addition to directly exploiting the calcium ion channels, we also propose to use a signaling molecule to increase the intracellular calcium level. hNOS2 (Nitric Oxide Synthase 2) is an enzyme that induces NO signaling by catalyzing the reaction: L-arginine + O2 + NADPH + H + NO + L-citrulline + NADP+ + H2O. Up-regulation of the NO signal can induce the opening of the RyR calcium channel in the endoplasmic reticulum, dramatically increasing intracellular calcium levels (Ziolo et al., 2001). We designed this plasmid to investigate how hNOS2 influences cancer cell growth.

1356: LentiV-hNOS2/Flag: Puro (BBa\_K5353066)

![](https://static.igem.wiki/teams/5353/results/figure-1-3.png)

Figure 1.3 Plasmid information for hNOS2.

# Results

## 1. Midi-Prep Plasmid DNA preparation

To amplify the plasmids to generate sufficient amounts of genetic material for further analysis and manipulation, we used the midi-prep preparation kit. After plasmid isolation and purification. We qualified DNA concentrations and the results are shown in Table 1. The results suggested the plasmids were successfully produced. The concentrations were good enough for further experiments (generally >100 ng/ml). The A260/A280 and A260/A230 ratio also suggested the purity of the plasmids was excellent.

![](https://static.igem.wiki/teams/5353/results/table-1.png	)

Table 1. DNA concentrations extracted and purified.

## 2. Checking of the plasmid insert by Polymerase Chain Reaction (PCR)

We tested the target DNA segments in the plasmids by PCR. After PCR amplification, we conducted gel electrophoresis. The amplicon size of the four targeted sequences are: Lamp2b (103 bp) , MscS (225 bp), hTRPC1 (162 bp) and hNOS2 (142 bp), respectively.

Primer information:
**Lamp2b**

F: TGACGACAACTTCCTTGTGC

R: AGCATGATGGTGCTTGAGAC

**MscS**

F: TGAATCGCCTGATGATCTCC

R: ACATGACAAGTAACACGCC

**hTRPC1**

F: GTGATGGCGCTGAAGGATGT

R: CCCAAGCACATCTACGCAAT

**hNOS2**

F: CGCATGACCTTGGTGTTTGG

R: CATAGACCTTGGGCTTGCCA

Plasmids Checked:

1364: LentiV-MscS/HA: EGFP/Neo (BBa\_K5353061)

1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo (BBa\_K5353062)

1362: LentiV-Lamp2b/MscS/mCherry: Neo (BBa\_K5353063)

1358: LentiV-hTRPC1/HA: EGFP/Neo (BBa\_K5353064)

1108: LentiV-Lamp2b/hTRPC1/HA: Neo (BBa\_K5353065)

1356: LentiV-hNOS2/Flag: Puro (BBa\_K5353066)

Compare the size of the bands to the fast DNA ladder running alongside the samples (Figure 2.1 and Figure 2.2), helping us to determine the amplified DNA fragment sizes practically. Theoretically, the amplicon size of the four targeted sequences are: Lamp2b (103 bp) , MscS (225 bp), hTRPC1 (162 bp) and hNOS2 (142 bp), respectively, which is aligned to our results.

The PCR product (1358: LentiV-hTRPC1/HA: EGFP/Neo) gel electrophoresis failed (Figure 2.1) in the first trial. In the second attempt, we can observe the pcr products (1358: LentiV-hTRPC1/HA: EGFP/Neo; 1364: LentiV-MscS/HA: EGFP/Neo) with correct band sizes (Figure 2.2). In summary, the result from PCR confirmed all plasmid inserts are correct.

![](https://static.igem.wiki/teams/5353/results/figure-2-1.png	)

Figure 2.1 Gel electrophoresis results of 1833, 1362, 1358, 1108, 1356.

![](https://static.igem.wiki/teams/5353/results/figure-2-2.png	)

Figure 2.2 Gel electrophoresis results of 1358 and 1364.

## 3. Validate the lentivirus infection by an apotome fluorescence microscope imaging

After plasmid preparation and PCR verification, we transfected the plasmids together with DVPR and VSV-G into the HEK293T cells for the packaging of lentivirus. The transfection process begins with the assembly of the lentiviral components, including the target DNA, the DVPR (packaging plasmid) and the VSV-G (envelope plasmid) within HEK293T cells. These components work synergistically to encapsulate the genetic material within the lentiviral vector, a crucial step in ensuring successful gene delivery to target cells.

Once the lentivirus is generated, we collect and utilize them to infect a fresh batch of HEK293T cells. This infection process is carefully orchestrated to introduce the modified genetic material into the recipient cells. By infecting the new HEK293T cells with the lentivirus, our objective is to create the genetically modified exosome with the target protein that we have designed.

To validate the infection efficiency, we inserted the EGFP or mCherry into different plasmids. Therefore, the successful infection could be visualized through fluorescence microscopy. As shown in Figure 3.1 to 3.4, the fluorescence signal confirms that the lentiviral vectors (1364, 1833, 1362, and 1358) had effectively delivered the desired genetic material to the target cells, leading to the expression of the gene construct.

1364: LentiV-MscS/HA: EGFP/Neo

![](https://static.igem.wiki/teams/5353/results/figure-3-1.png	)

Figure 3.1 Expression of green fluorescent protein of 1364 HEK293T cell.

1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo

![](https://static.igem.wiki/teams/5353/results/figure-3-2.png	)

Figure 3.2 Expression of green fluorescent protein of the 1833 HEK293T cell.

1362: LentiV-Lamp2b/MscS/mCherry: Neo

![](https://static.igem.wiki/teams/5353/results/figure-3-3.png	)

Figure 3.3 Expression of red fluorescent protein of 1362 HEK293T cell.

1358: LentiV-hTRPC1/HA: EGFP/Neo

![](https://static.igem.wiki/teams/5353/results/figure-3-4.png	)

Figure 3.4 Expression of green fluorescent protein of 1358 HEK293T cell.

For the plasmid 1356 and 1108, because the target protein we insert is too large, we did not add the fluorescence protein on them. We would detect them with western blotting through the HA tag (1108: LentiV-Lamp2b/hTRPC1/HA: Neo) and flag tag (1356: LentiV-hNOS2/Flag: Puro).

## 4. Confirm the production of exosomes by TEM

Since the TEM imaging process is complicated and expensive, we just sent our exosome samples coding 1833 (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) to the company in Guangzhou, China to do the TEM imaging.

We used the supernatant of the successfully infected HEK293T cell to isolate the exosomes. The size of the exosomes is extremely small, approximately 30–140 nm, membrane-enclosed molecules. Also, since we used the exoEasy Maxi Kit (QIAGEN, cat. no. 76064) to extract them and did not concentrate the exosomes using ultrafiltration, the exosome concentration is a little low.

The figures below show the shape and size of our exosomes, the halo-like structure is the exosomes (Figure 4.1 and Figure 4.2), and the light dots shown on the background are salts. The normal and well-functioning exosomes are standard round, while the broken and unhealthy exosomes with an elliptical collapse structure were also observed (Figure 4.3).

![](https://static.igem.wiki/teams/5353/results/figure-4-1.png	)

Figure 4.1 Exosomes with normal structures.

![](https://static.igem.wiki/teams/5353/results/figure-4-2.png	)

Figure 4.2 Exosomes tangled together.

![](https://static.igem.wiki/teams/5353/results/figure-4-3.png	)

Figure 4.3 Exosomes with elliptical collapse structure.

## 5. DLS Measurement to determine the size of the exosomes

Dynamic light scattering (DLS) is a powerful technique used in the field of nanotechnology and biophysics to measure the size of particles in a solution, and Dynamic light scattering (DLS) is an optical analysis method for measuring the size and distribution of sub-micron particles. Therefore we used DLS to provide valuable insights into the size distribution and polydispersity of exosomes.

In DLS, a laser beam is directed into a sample containing exosomes. As these particles move randomly as a result of Brownian motion, they scatter light. When the fluctuations in scattered light intensity are analyzed over time, DLS can determine the hydrodynamic size of exosomes in the sample. The technique is highly sensitive to small particles, making it ideal for studying exosomes, which typically range in size from 30 to 150 nanometers.

The results of the different samples of exosome size distribution are as follows (Figure 5.1 to Figure 5.3).

1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo

Exosome Sample 1833-1 (Figure 5.1.1):
The peak of the size (d. nm) is 186.6 nm and more than 70% of exosomes range from 141.8 nm to 220.2 nm, which is similar to the size of TEM imaging.

![](https://static.igem.wiki/teams/5353/results/figure-5-1-1.png	)

Figure 5.1.1 Exosome size distribution of sample 1833-1.

Exosome Sample 1833-2 (Figure 5.1.2):
The peak of the size (d. nm) is 390.8 nm.

![](https://static.igem.wiki/teams/5353/results/figure-5-1-2.png	)

Figure 5.1.2 Exosome size distribution of sample 1833-2.

Exosome Sample 1833-3 (Figure 5.1.3):
The peak of the size (d. nm) is 14.23 nm.

![](https://static.igem.wiki/teams/5353/results/figure-5-1-3.png	)

Figure 5.1.3 Exosome size distribution of sample 1833-3.

Exosome Sample 1833-4 (Figure 5.1.4):
The peak of the size (d. nm) is 43.22 nm.

![](https://static.igem.wiki/teams/5353/results/figure-5-1-4.png	)

Figure 5.1.4 Exosome size distribution of sample 1833-4.

1362: LentiV-Lamp2b/MscS/mCherry: Neo

Exosome Sample 1362-1 (Figure 5.2.1):
The peak of the size (d. nm) is 493.7 nm.

![](https://static.igem.wiki/teams/5353/results/figure-5-2-1.png	)

Figure 5.2.1 Exosome size distribution of sample 1362-1.

Exosome Sample 1362-2 (Figure 5.2.2):
The peak of the size (d. nm) is 31.14 nm.

![](https://static.igem.wiki/teams/5353/results/figure-5-2-2.png	)

Figure 5.2.2 Exosome size distribution of sample 1362-2.

Exosome Sample 1362-3 (Figure 5.2.3):
The peak of the size (d. nm) is 372.2 nm.

![](https://static.igem.wiki/teams/5353/results/figure-5-2-3.png	)

Figure 5.2.3 Exosome size distribution of sample 1362-3.

11108-1: LentiV-Lamp2b/hTRPC1/HA: Neo

Exosome Sample 1108-1 (Figure 5.3.1 ):
The peak of the size (d. nm) is 20.93 nm.

![](https://static.igem.wiki/teams/5353/results/figure-5-3-1.png	)

Figure 5.3.1 Exosome size distribution of sample 1108-1.

Exosome Sample 1108-2 (Figure 5.3.2):
The peak of the size (d. nm) is 10.24 nm.

![](https://static.igem.wiki/teams/5353/results/figure-5-3-2.png	)

Figure 5.3.2 Exosome size distribution of sample 1108-2.

Exosome Sample 1108-3 (Figure 5.3.3):
The peak of the size (d. nm) is 16.14 nm.

![](https://static.igem.wiki/teams/5353/results/figure-5-3-3.png	)

Figure 5.3.3 Exosome size distribution of sample 1108-3.

From the results, we can confirm that the modified HEK293T cell can produce the exosomes successfully and we can also extract the exosomes from the supernatant using the exoEasy Maxi Kit.

However, the results show the significance difference of size distribution between the different samples. The reason may be that we keep the exosomes extracted in the 4℃ refrigerator for a long time, about three weeks, while it is suggested to use the exosomes within one week or keep them in the -80℃ fridge for long-term storage. Keeping them for a long time may result in abnormal structure of the exosome or in the exosomes flocked together to fuse and form larger structures. In addition, because there are different target proteins that are inserted into the exosome, which may also affect the structure and formation of the exosome.

Overall, the DLS measurement and TEM imaging confirm the existence of exosomes, and we then conducted the BCA assay to measure the exosome concentration in our different samples.

## 6. BCA Assay

The Bicinchoninic Acid (BCA) assay is a widely used method to quantify the total protein concentration in a sample. This colorimetric assay is based on the ability of protein molecules to reduce Cu2+ to Cu+ in an alkaline medium, forming a purple complex with BCA. The intensity of this complex is directly proportional to the protein concentration and can be measured spectrophotometrically.

The Table 2 shows the absorbance values at 562 nm for various concentrations of BSA standards. By plotting the absorbance values against the known protein concentrations, a standard curve is generated. This curve is then used to determine the protein concentration in unknown samples on the basis of their absorbance readings.

![](https://static.igem.wiki/teams/5353/results/table-2.png)

Table 2. The absorbance value (562 nm) of BSA and exosome samples.

![](https://static.igem.wiki/teams/5353/results/figure-6.png	)

Figure 6. The BCA standard absorbance curve.

According to the equation of the BCA standard absorbance curve, we calculated the total protein concentration in exosomes (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) samples through their absorbance values. The amount of proteins in three repeat extraction of 1833 is arranged from 0.067 to 0.280 mg/ml. The result suggests the first extraction (1833-1) has the best concentration.

Moreover, according to research related to exosome measurement, we can assume the total protein concentration in the sample reflects the amount of proteins on the exosomes. However, the results do not reflect the target protein expression such as MscS, lamp2b, and hNOS2 in the exosomes.

## 7. Western Blot

Western blotting, also known as protein immunoblotting, is a widely used technique in molecular biology for detecting specific proteins in a complex sample. This method allows us to identify and quantify proteins based on their size and antigenicity.

In our Western blot experiments, we detected CD63 and CD81, which are exosome marker proteins, and also the HA-tag , which is linked to the MscS (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) and hTRPC1 proteins. We were able to detect the CD81 marker protein successfully (1833-1), this suggests the existence of the exosome (Figure 7). However, possibly due to the low exosome or target protein concentration, we did not detect the CD63 and HA tag successfully. Because our laboratory does not have the antibody to target the Flag-tag yet, the detection of Flag will be performed later.

![](https://static.igem.wiki/teams/5353/results/figure-7.png	)

Figure 7. Western Blot of CD81 from 1833 exosome samples.

## 8. Cancer Cell Apoptosis Assay

The Apoptosis Assay by flow cytometry using the Annexin V staining method is a technique to detect and quantify apoptotic cells within a cell population. It depends on Annexin V staining, propidium iodide (PI) staining, and flow cytometry analysis.

Annexin V Staining:
Apoptotic cells exhibit phosphatidylserine (PS) residues on their outer membrane, a hallmark of early apoptosis. Annexin V, a calcium-dependent phospholipid-binding protein with high affinity for PS.

Propidium iodide (PI) staining:
PI is often used in conjunction with Annexin V to differentiate between apoptotic cells and necrotic cells. PI stains necrotic cells with compromised membrane integrity. Because propidium iodide does not penetrate living cells, it is commonly used to detect dead cells in a population. PI binds to DNA by intercalating between bases with little or no sequence preference.

Flow Cytometry Analysis:
After staining with Annexin V and PI, the cells were analyzed using flow cytometry. Flow cytometers detect fluorescence emissions from stained cells and provide quantitative data on the cell population.

**Cell apoptosis-A2780**

Figures (Figure 8.1.1, Figure 8.1.2 and Figure 8.1.3) below show the result of ovarian cancer cells A2780 treated with or without the exosome treatment (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo). With the exosome treated, there are more A2780 cells during necrosis, but there is no difference in apoptosis.

Since the number of exosomes produced by HEK293T cells is low, the exosome concentration in one well of a six-well plate is approximately 0.056 mg/ml. However, the results still show that the cancer cell death is affected and more A2780 cells appeared resulting in necrosis.

![](https://static.igem.wiki/teams/5353/results/figure-8-1-1.png	)

Figure 8.1.1 Flow cytometry of the control group of A2780.

![](https://static.igem.wiki/teams/5353/results/figure-8-1-2.png	)

Figure 8.1.2 Flow cytometry of the exosome treated group of A2780.

![](https://static.igem.wiki/teams/5353/results/figure-8-1-3.png	)

Figure 8.1.3 Results of necrosis and apoptosis of A2780 under normal and exosome treated conditions.

**Cell apoptosis-NCI-H1299**

The figures (Figure 8.2.1, Figure 8.2.2 and Figure 8.2.3) below show the result of the growth of lung cancer cells, NCI-H1299, and the cancer cells treated with/without our exosomes (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo). With the exosome treated, there are slightly more NCI-H1299 cells in apoptosis (even the results are not significant due to only a single experiment), but there is no change in necrosis. We believe the modest response may be due to the low exosome concentration and lack of enough membrane stress stimulation by the ultrasound.

![](https://static.igem.wiki/teams/5353/results/figure-8-2-1.png		)

Figure 8.2.1 Flow cytometry of the control group of NCI-H1299.

![](https://static.igem.wiki/teams/5353/results/figure-8-2-2.png		)

Figure 8.2.2 Flow cytometry of the exosome treated group of NCI-H1299.

![](https://static.igem.wiki/teams/5353/results/figure-8-2-3.png		)

Figure 8.2.3 Results of necrosis and apoptosis of NCI-H1299 under normal and exosome treated conditions.

Due to the time limit, we just investigated how the exosome containing MscS (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) affects cancer cell death. Also due to the very specialized instruments required for the ultrasound stimulation, we cannot induce the opening of the MscS and hTRPC1 calcium channels precisely as we proposed. But to our surprise, the A2780 group was treated with 0.056 mg/ml exosome (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) seems to show that cancer cell death has been affected. We will try the other two kinds of the exosomes (1108: LentiV-Lamp2b/hTRPC1/HA: Neo), and (1362: LentiV-Lamp2b/MscS/mCherry: Neo) to treat the cancer cells later.

# Future Works

The major challenge at the current stage is the concentration of exosomes. Therefore, we do need to increase the yield of the exosome and to produce more concentrated exosome. Further optimization must be carried out, such as adjusting the culture environment by using specific media additives, hypoxic conditions, or altering the pH, which can stimulate cells to produce more exosomes. For concentrating exosomes, we could try to perform ultrafiltration or ultracentrifugation.

For hNOS2 (1356: LentiV-hNOS2/Flag: Puro), because of the lentivirus plasmid size limitation, the insert will be too large, if we combine the lamp2b with hNOS2. Therefore, our plan is to perform a second transfection of hNOS2 after generating the stable HEK293T cell line with the 1833, 1362, or 1108 plasmid. In this way, our objective is to observe whether hNOS2 can enhance the effect of MscS or hTRPC1 to perform calcium overload.

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