iGEM 2022 — Team bs-united-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

# ENGINEERING SUCCESS

## Questions which are important to local people

*Staphylococcus aureus* (*S. aureus*) is a common pathogen that the enterotoxins released by it is a sever pathogenic factor of food poisoning cases. *S. aureus* doesn’t need restricted conditions to grow; It has great resistance to arid, heated and saliferous conditions, therefore spreading and surviving widely in the environment around people. *S. aureus* is ubiquitous in nature which can be found in air, water, dust and human and animal’s waste. As a result, People can easily be exposed to *S. aureus* and get infected. Nowadays, *S. aureus* has become a worldly health problem. Reported by the U.S. Centers of Disease Control, about 25% food poisoning cases are caused by *S. aureus* infection in China, about 33% in the U.S., and even about 45% in Canada. As people infected by *S. aureus*, it will cause detrimental diseases as pneumonia, pericarditis, sepsis and death. In the hospital or communities, people catch *S. aureus* through contact infection and the morbidity rate in a developed country is between 100,000 to 300,000 people each year. According to data from the Emerging Infections program (EIP) and Cerner Electronic Health Record databases, 19,832 associated deaths occurred in an estimated 119,247 *S. aureus* bloodstream infections in 2017. To understand the spreading range of *S. aureus*, we construct the following experiments.

**Primers:**

*Escherichia coli* (*E.coli*) primers:

F1: GACGTTACAGCTGCCGGT

R1: TCCCGTGGTTCTGAC

*S. aureus* primers:

F2: TCCTTACTTACACGA

R2: TTATTTCAGGATAT

**Materials**

1. Genome DNA isolation: Kit from Beyotime biotechnology

2. PCR reaction: Phusion enzymes (M0530S)

3. DNA electrophoresis： 100V run for 1-2 hours with 5 ul ER staining for 100 ml 1% agarose gel.

**Experiment Design：**

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/local-design.png)

**Results：**

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/dna-electrophoresis.png)

Fig.1 DNA electrophoresis from PCR.

According to the results above, we can conclude that in soil, river water and fish, they contain both *E.coli* and *S. aureus*’s DNA; In tap water, it contains considerable amount of *S. aureus*’s DNA. To conclude, the *S. aureus* infection is a adverse factor that threaten people’s health and it is very common in our daily life, driving us to inhibit the growth of *S. aureus* in food.

## The model of part 1

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/2.png)

**Fig.1.1 Schematic model of Part1 system.**
If there is no IPTG TurboID will not be activated. If we add IPTG, it will activate TurboID and eventually complete the hole process.

## The model of part 2

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/engineering-success/4-11.jpg)

**Fig.2.1 Schematic model of Part2 system.**
If there is no IPTG, Dethiobiotin synthase will not be activated.

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/engineering-success/3-22.jpg)

**Fig.2.2 Schematic model of Part2 system.**
If we add IPTG, it will activate Dethiobiotin and eventually complete the hole process.

## Materials in the part 1

1. TurboID-AgrD particles
2. ATP powder
3. Biotin powder
4. Engineering Bacteria
5. Strepavdin-568

## Materials in the part 2

1. Dethiobiotin synthase particles
2. ATP powder
3. Biotin powder
4. Engineering Bacteria
5. Strepavdin-568

## Procedures in part 1:

1. Expression of TurboID-AgrD protein in *E. coli*.
2. Staining *Staphylococcus aureus* (*S. aureus*) by using strepavdin-568.
3. Tracking the growth of *S. aureus*.

## Procedures in part 2:

1. Expression of Dethiobiotin synthase in *E. coli*.
2. Staining *S. aureus* by using strepavdin-568.
3. Tracking the growth of *S. aureus*.

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/5.jpg)

**Fig.2.3 TurboID expression in *E. coli* induced by IPTG**

## Results of part part1:

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/6.jpg)

**Fig.1.2 TurboID-AgrD stained *S. aureus*.**
We can clearly know that the one, which TurboID is activated, streptavidin is successfully labeled on the bacteria’s DNA. The one, which TurboID is not activated, nothing happened.

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/7.jpg)

**Fig.1.3 The growth curve of TurboID-AgrD stained *S. aureus*.**
As we can see from the data, the growth of TurboID-AgrD stained *S. aureus* is inhibited in growth.

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/8.jpg)

**Fig.1.4 The growth rate of TurboID-AgrD stained *S. aureus* is 2 folded slower than unstained S.**

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/9.jpg)

**Fig.2.4 TurboID-AgrD stained *S. aureus*.**
We can clearly know that the one, which part 1 and part 2combine together, generate a greater effect.

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/10.jpg)

**Fig.2.5 The growth curve of TurboID-AgrD stained *S. aureus*.**
As we can see from the data, the growth of TurboID-AgrD stained *S. aureus* is inhibited in growth.

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/11.jpg)

**Fig.2.6 The growth rate of TurboID-AgrD stained *S. aureus* is 5 folded slower than unstained *S. aureus*.**

The engineering success since when we compare the result is more significant when Part 1 and Part 2 combine.

## Testing TurboID-AIP toxicity to human cells

**Purpose:**

The purpose of the experiment is to evaluate the damage of *S. aureus* enterotoxin toward human cells, and the amount of enterotoxin produced by Biotinylated *S. aureus*. We intended to use the apoptosis rate of epithelial cell to reflect the production of enterotoxin.

**Design:**

We divided *S. aureus* into two groups. One is the control group with lysate extracted only from *S. aureus*. Another is a lysate extracted from TurboID-AIP and biotin blocked S.aureus.

**Materials:**

1. Inner lining epithelial cell sample in human mouth

2. Culture media containing bovine serum

3. *S. aureus* DNA

4. Biotin powder

5. *S. aureus* Lysate

6. Biotin labeled S aureus Lysate

**S. aureus Lysate preparation:**

1. Extract 1ml of *S. aureus* (OD0.3)

2. *S. aureus* in bovine serum lysed via supersonic schizolysis method.

**Biotin labeled *S. aureus* Lysate preparation:**

3. Let Biotin reacts with S.aureus.

4. Collect 1ml of *S. aureus* (OD0.3) after centrifugation.

5. *S. aureus* in bovine serum lysed via supersonic schizolysis method.

**Fluid preparation for flow cytometry:**

1. Scrape epithelial cells with a dentiscalprum.

2. Separate the sample into two parts.

3. Mix the samples with *S. aureus* Lysate and Biotin labeled *S. aureus* lysate respectively (10 minutes).

4. Centrifugation.

5. Discard the supernatant.

6. Resuspend the cell pellet for 15 minutes.

7. Collect precipitated cells (1400rpm) after centrifugation.

8. Analyze the fluid with flow cytometry.

**Result:**

It is presented that in figure 3, 5, 7, the enterotoxin damages the epithelial cells. In figure 4, 6, 8, the apoptosis rate of cells declined indicating that the damage of enterotoxin decreased. Thus, we can conclude that our project can successfully block the production of enterotoxin in *S. aureus*.

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/new-16.jpg
)

Fig.3 Analysis of epithelial cell of subject 1 with *S. aureus* lysate from (A) to (C);
(A) size of the cells. (B) width of cells. It eliminates the duplicates which are useless to the Analysis. (C) The apoptosis rate of cells.

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/new-26.jpg
)

Fig.4 Analysis of epithelial cell of subject 1 with biotin labeled *S. aureus* lysate from (A) to (C);
 (A) size of the cells. (B) width of cells. It eliminates the duplicates which are useless to the Analysis. (C) The apoptosis rate of cells.

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/new-36.jpg
)

Fig.5 Analysis of epithelial cell of subject 2 with *S. aureus* lysate from (A) to (C);
(A) size of the cells. (B) width of cells. It eliminates the duplicates which are useless to the Analysis. (C) The apoptosis rate of cells.

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/new-46.jpg
)

Fig.6 Analysis of epithelial cell of subject 2 with biotin labeled *S. aureus* lysate from (A) to (C);
(A) size of the cells. (B) width of cells. It eliminates the duplicates which are useless to the Analysis. (C) The apoptosis rate of cells.

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/new-56.jpg
)

Fig.7 Analysis of epithelial cell of subject 3 with *S. aureus* lysate from (A) to (C);
 (A) size of the cells. (B) width of cells. It eliminates the duplicates which are useless to the Analysis. (C) The apoptosis rate of cells.

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/new-66.jpg
)

Fig.8 Analysis of epithelial cell of subject 3 with biotin labeled *S. aureus* lysate from (A) to (C);
(A) size of the cells. (B) width of cells. It eliminates the duplicates which are useless to the Analysis. (C) The apoptosis rate of cells

## Protecting food from *S. aureus* contamination

Description: This experiment is conducted to testify the effectiveness of our products to inhibit the growth of *S. aureus*. Our samples have been made by mixing TurboID-biotin fusion protein with food DNA extracting solutions. After that, we use PCR to amplify DNA of our samples and confirm the existence of *S. aureus* and E. coli in electrophoresis process.

**Design:**

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/design.png)

**Procedure：**

1. Genome DNA isolation: Beyotime

a. Lysozyme solution was prepared: 20 mM Tris, pH 8.0, 2 mM EDTA, 1.2% Triton x-100, 20 mg/ml lysozyme. Lysozyme was added before use. Centrifugation will collect up to 2 billion bacteria and discard the supernatant.

b. Bacteria were fully resuspended with 180 microliters of lysozyme solution. Incubate at 37 ° C for 30 min to lyse bacteria. Centrifuge as the same speed to remove the supernatant. Add 200 microliters of absolute ethanol and Vortex mix.

c. (Ethanol must be thoroughly mixed after addition, otherwise it will seriously affect the extraction effect. White precipitate may be produced after adding ethanol, which is a normal phenomenon. In the following steps, the white precipitate and solution must be transferred to the purification column).

d. Add the mixture from step c to the DNA purification column. Centrifuge at least 6000g (Approximately ≥ 8000rpm) for 1 minute. Discard the liquid in the waste liquid collection tube. Add 500 microliters of washing liquid I, centrifuge at least 6000g (about ≥ 8000rpm) for 1 minute. Discard the liquid in the waste liquid collection tube. Add 600 microliters of washing solution II, centrifuge at least 18000g (about ≥ 12000 rpm) for 1 minute. Discard the liquid in the waste liquid collection tube. Then centrifuge at least 18000g (about ≥12000rpm) for 1 min to remove the residual ethanol.

e. The DNA purification column was placed on a clean 1.5-ml centrifuge tube and 50-200 microliters of eluate were added. Leave at room temperature for 1-3 minutes. Centrifuge at ≥12000rpm for 1 min. The resulting liquid is the purified total DNA.

**PCR reaction: Phusion emzymes**

Solution:20ul reaction:10 uM forward primer 1ul, 10uM reverse primer 1ul, taq enzyme mix 8ul, template DNA 2ul,Nuclease-free water 8ul; for experimental group, 10 uM forward primer 1ul, 10uM reverse primer 1ul, taq enzyme mix 8ul, template DNA 2ul, TurboID-Biotin fusion protein 1 ul, Nuclease-free water 8ul
Condition:initial denaturation:98 30seconds,30 cycles 98 10seconds,68 30seconds,72 30seconds,final extension 72 10seconds,hold 4
DNA electrophoresis：200V run for 30-60min, solution:5 ul ER staining for 100 ml 1% agarose gel.

**Results:**

![](https://static.igem.wiki/teams/4329/wiki/engineering-success/fig10.png)

Fig.9 DNA electrophoresis of control groups and experimental groups from PCR

**Conclusion:**

The results shown above reveal that the amount of *S. aureus* contained in samples treated with TurboID-biotin is less than that of the groups without any treatment. For example, we can clearly notice that the group of shrimp DNA with TurboID-biotin mixture reacted for 60 minutes have more vague figure of stripes ,the one contains same base pairs with *S. aureus*, compared with the group includes sorely shrimp DNA. We can conclude that our products have effects on preventing *S. aureus* growth.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}