iGEM 2024 — Team squirrel-beijing

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{% block title %}Notebook{% endblock %}
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{% block lead %}The Day-to-Day Operations{% endblock %}
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## Notebook

### [cell lab]

**Cell culturing**

We received the cells using for experiment on 7.8, then passaged cells at a 1:4 ratio
Then, we changed the media each day, and passaged cells every 2-3 days.

**Model construction and cell treatment**

In the first step, we divided two types of cells–PC12 cells and N2a cells–into four groups, seeded each in one well of a six-well plate. The four groups are:

1. Group M: r modeling group, refers to the cells treated with Aβ protein 42, to represent cell injury

2. Group C: control group, refers to the cells without any treatment

3. Group M+D: refers to the cells treated with both Aβ protein 42 and rose flavonoid

4. Group C+D: refers to the cells only treated with rose flavonoid.

These are down on 7.13
In the second step, after transcriptomics study clarified the function of gene grin2b in the signaling pathway, we also treated cells with the protein it encodes.

### [molecular lab]

**Western blotting**

We’ve done eight times of wb, with 2 days for each (the first day for gel preparation, electrophoresis, protein transferring, and blotting; the second day for incubating with primary and antibody and ECL)
The dates of each wb experiment are:
7.12-13
7.14-15
7.17-18
7.19-7.20
8.13-14
8.15-16
8.17-18
8.20-21
The samples using for testing are cells and medium mentioned in “cell lab-cell treatment”. We tested several inflammatory factors of each group, including NF-κB, TNF-α and IL-1β.

The results of ECL are listed in the “result and analysis” part

### [preparation room]

**Rose flavonoid extraction**

7.8 Selected, washed, and dried 100g of fresh rose petals with an oven.

7.9 Weight 1g rose petal powder and extracted the powder with distilled water at a ratio of 1:10 (i.e. 10 mL) in a water bath at 65 ℃ for 2 hours, 3 times.

7.10 centrifuged the extraction solution for 15 min (1500 rpm), then collecting the supernatant–containing all rose flavonoid–together

## Detailed procedures and information

### Western blotting

**The concentration of separating gel is 10%**
4X SDS-PAGE separating gel buffer    2.5mL
ddH2O                                    4.1mL
30% acrylamide (29:1)    3.3mL
10% ammonium persulfate    100μL
TEMED                                    10μL

**The concentration of stacking gel is 5%**
4X SDS-PAGE stacking gel buffer    1.25mL
ddH2O                               2.84mL
30% acrylamide (29:1)    0.83mL
10% ammonium persulfate    75μL
TEMED                               7.5μL

**The time and other settings**

1. Electrophoresis, 120V for 15 minutes, then 140V for 30 minutes
2. Prepared PVDF membranes by one-minute immersion in anhydrous methanol
3. the assembly sequences within the transfer apparatus was organized from the negative to positive pole as follows: sponge, filter paper, gel, membrane, filter paper, and sponge
4. protein transfer under 110V for 90 minutes
5. Blot with 10% skim milk powder for at least 1 hour
6. Wash with 1× TBST buffer 3 times for 5 minutes each
7. Incubate with primary antibody (1/1000) for 2 hours
8. Wash with 1× TBST buffer three times for 5 minutes each
9. Incubate with secondary antibody (goat anti-rabbit IgG-HRP) (1/3500) for 1 hours
10. Wash with 1× TBST buffer three times for 7 minutes each
11. Add 100 μl super ECL plus for each membrane to chemiluminescence, then automatically exposing it in the device

### Cell culturing

**culturing**

Use DMEM medium containing 10% FBS and 1% penicillin/streptomycin
culture in a humidified incubator at 37 ℃ with 5% CO2
**cell passage**
1. Wash with 1× PBS buffer for three times
2. Digest with 100μl 0.25% trypsin for 5 minutes
3. Centrifuge at 1500 rpm for 5 minutes
4. Divide the cells into two culturing flasks

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{% block title %}Engineering Success{% endblock %}
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## Design

The purpose of this study is to study the affinity of six main chemicals in rose flavonoids with GRIN2B protein by molecular docking technique (Agu et al., 2023
). We divided the project into four modules: cell damage model building module, protein structure modeling module, small molecule preparation module and docking analysis module. We screened out the specific gene GRIN2B through transcriptomics detection and data analysis, modeled it with the target protein GRIN2B of AlphaFold3, and optimized the structure of the main chemical substances in flavonoids from protein and Rose by Maestro calculation software. The predicted protein structure and small molecules were optimized to ensure stability and minimize energy, which significantly increased the possibility and accuracy of successful docking. Then, according to the semi-flexible docking method, the optimal binding mode and reasonable molecular binding pocket are analyzed. We expect that the six main chemicals in rose flavonoids will closely bind with GRIN2B protein, which will help us understand their effects of relieving inflammation and oxidative damage.

![Human Practices Overview Graphic.](https://static.igem.wiki/teams/5457/drylab/model1.png)

![Human Practices Overview Graphic.](https://static.igem.wiki/teams/5457/wetlab/engineer/engineer2.png)

## Build

1.Fresh rose was purchased, and we selected 100 g petals. After washing with sterile water and drying in oven, the dried petals (about 5 g) were crushed. Accurately 1.0g of rose petal powder was weighed, which was extracted with distilled water at a ratio of 1:10 (i.e. 10 mL) in a water bath at 65 ℃ for 2 hours. The extraction was repeated for three times (for a total of 6 hours), then the extraction solution was centrifuged for 15 min (1500 rpm), and the supernatant (for a total of 20 mL) was combined together. The total amount of flavonoids in the supernatant is the total content of flavonoids in rose petals. (Wu et al., 2022)

![Human Practices Overview Graphic.](https://static.igem.wiki/teams/5457/wetlab/engineer/engineer3.png)

2.Two types of cells (PC12 cells and N2a cells) were cultured in the DMEM medium containing 10% FBS and 1% penicillin/streptomycin, in a humidified incubator at 37 ℃ with 5% CO2. The growth medium was replaced everyday and the cells were propagated every three days. For cells injury model construction, Aβ protein 42 (10 μM) was added into the cells and co-cultured for 24 hours. Then, several inflammatory factors including NF-κB, TNF-α and IL-1β were measured to confirm the successful model construction.

![Human Practices Overview Graphic.](https://static.igem.wiki/teams/5457/wetlab/engineer/engineer4.png)

3.For GRIN2B is membrane protein, we can choose mammalian or insect cell expression system, and the plasmid is designed as follows. By cloning into a vector, expression plasmids encoding functional proteins are further generated and transformed into DH5α-competent cells. Then, plasmids are extracted and constructed from positive clones and confirmed by sequencing. Next, extract the protein from the plasmid for purification. (Li et al., 2022, Li et al., 2024)

![Human Practices Overview Graphic.](https://static.igem.wiki/teams/5457/wetlab/engineer/engineer5.png)

4.Introduce the overexpression vector into expression-deficient cells for the purpose of interaction validation or to apply stress treatment to the overexpressing cells.

## Test

1.To screen out the special sensors (genes) of rose flavonoids extraction in PC12 cells, which was under Aβ protein 42 (10 μM) treatment, we gathered four groups of cells, control without treatment (C), model (M), control+rose flavonoids extraction (C+D), model+rose flavonoids extraction (M+D), and performed transcriptomics detection and data analysis.

![Human Practices Overview Graphic.](https://static.igem.wiki/teams/5457/wetlab/engineer/engineer6.png)

2.To quantify protein expressions of GRIN2B, associated proteins NF-κB, TNF-α and IL-1β, with respect to the control protein β-actin, WB is performed. The relative amounts of proteins in each group were quantified concerning β-actin expression. characterized by the up-regulation of GRIN2B, alongside the down-regulation of NF-κB, TNF-αand IL-1β.

![Human Practices Overview Graphic.](https://static.igem.wiki/teams/5457/wetlab/engineer/engineer7.png)

3.Conduct morphological, protein, and molecular level assessments on cells
successfully transfected with the plasmid to confirm the successful construction of
the vector and the functionality of the protein pathway

## Learn

1.Through experiments, we verified the binding model and different affinities between six main chemical substances in rose flavonoids (Kaempferol, Quercetin, Juglanin, Avicularin, Astragalin, Hyperoside) and GRIN2B protein. Rose flavonoids in this pathway can effectively alleviate the risk of Alzheimer' s disease caused by circadian rhythm disorder.

2.We found that GRIN2B may be a sensitive factor to alleviate inflammation and oxidative damage of PC12 cells, and rose flavonoids can up-regulate PC12 cells.
Expression level of GRIN2B in cells. Based on these characteristics, we prepared a functional tea bag of rose flavonoids.

## Reference

1.Agu P C , Afiukwa C A , Orji O U ,et al.Molecular docking as a tool for the discovery of molecular targets of nutraceuticals in diseases management[J].Scientific Reports, 2023, 13(1).DOI:10.1038/s41598-023-40160-2.

2.Wu Mengqi, et al."Purification and Identification of Flavonoid Molecules from Rosa setate x Rosa rugosa Waste Extracts and Evaluation of Antioxidant, Antiproliferative and Antimicrobial Activities."Molecules 27.14(2022):4379-4379.

3.Li, Y., Liu Y., Huang, M., &Chen, M. (2022). Batch construction and screening recombinant plasmid by connecting DNA solution with plasmid vector. CN113674804A

4.Weiyi Li,Darach Miller,Xianan Liu,Lorenzo Tosi,Lamia Chkaiban,Han Mei... & Sasha F Levy.(2024).Arrayed in vivo barcoding for multiplexed sequence verification of plasmid DNA and demultiplexing of pooled libraries..Nucleic acids research

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{% block title %}Experiments{% endblock %}
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## Background

Circadian Rhythms Disruption (CRD) may increase the risk of Alzheimer's Disease (AD) and trigger an inflammatory response in neurons.
According to omics study, Rose Flavone inhibit the signaling pathway NF-kB/TNF-α by activating the expression of grin2B.

## Experiment procedures

#### Step 1: Create an AD cell model [cell lab]

1. Treat Cells: Create an AD cell model by treating neuronal cells with specific methods (e.g., stimulating inflammatory factors or mimicking CRD).
2. Measure Inflammatory Factors: Record the levels of inflammatory factors in the cells to establish baseline data for the AD cell model.
3. Observe cellular responses: monitor cellular interactions, such as inflammatory signaling, to assess the accuracy of the model.

#### Step 2: Rose Flavonoid Treatment [cell lab]

1. Treat Cells: Treat the AD cell model with rose flavonoids.
2. Test new cell model: Measure the level of inflammatory factors in the treated cells and compare with the standard level in AD cell model.
3. Compare baseline levels: verify the mitigating effect of rose flavonoids on cellular damage.

#### Step 3: omics study [molecule lab & third-party testing company].

1. Sample treatment: treat the cell sample and preparing mRNA samples for omics sequencing and analysis
2. Analysis omics data: use data processing tools to analysis data and identify relevant signaling pathways and genes. Identify the function of proteins and genes.
3. See omics study report： [https://2024.igem.wiki/squirrel-beijing/omicsstudyreport](https://2024.igem.wiki/squirrel-beijing/omicsstudyreport%20)

#### Step 4: Verification of Protein Function

1. Treat cells: treat cells with protein encoded by grin2B.
2. Confirm the gene: Measure the level of inflammatory factors in treated cells and compare with standard level in AD cell model to confirm the protein’s function.

#### Step 5: Product Design

1. Protein Production: synthesize the protein with E. coli.
2. Products designing: based on the experimental results, develop products containing rose flavone or the protein as an alternative to rose flavonoids.
3. See details in Entrepreneurship: <https://2024.igem.wiki/squirrel-beijing/entrepreneurship>

## Operational details added

1. See our daily records： [https://2024.igem.wiki/squirrel-beijing/notebook](https://2024.igem.wiki/squirrel-beijing/notebook%20)
2. R\*Blue sentences refer to detailed information of each step.
3. RCellular experiments were responsible for simulating nerve cell damage due to circadian rhythm disruption and verifying the protective effects of rose flavonoids at the cellular level. Molecular experiments were then performed to further reveal the signaling pathways activated by rose flavone and the synthesis of specific proteins.

#### Details of procedure in molecular lab

Conducted to analyze the level of specific proteins to help verify inflammatory level and confirm the effect of different treatement

##### SDS-PAGE Electrophoresis

###### The concentration of separating gel is 10%

1. 4X SDS-PAGE separating gel buffer 2.5mL
2. ddH2O 4.1mL
3. 30% acrylamide (29:1) 3.3mL
4. 10% ammonium persulfate 100μL
5. TEMED 10μL

###### The concentration of stacking gel is 5%

1. 4X SDS-PAGE stacking gel buffer 1.25mL
2. ddH2O 2.84mL
3. 30% acrylamide (29:1) 0.83mL
4. 10% ammonium persulfate 75μL10% ammonium persulfate 100μL
5. TEMED 7.5μL

##### immunohistochemical reaction and ECL

###### Detailed procedures

1. Electrophoresis, 22V for 90 minutes
2. protein transfer, 4.5W for 90 minutes
3. Blot with 5% skim milk powder for 2-4 hours
4. Wash with 1× TBST buffer 3 times for 5 minutes each
5. Incubate with primary antibody (1/1000) for 2 hours
6. Wash with 1× TBST buffer three times for 5 minutes each
7. Incubate with secondary antibody (1/3500) for 1 hours
8. Wash with 1× TBST buffer three times for 7 minutes each
9. Add 100 μl super ecl plus for each membrane to chemiluminescence, then
10. automatically exposing in the device

#### Details of procedure in cell lab

##### cell media change

During the cultivation process, regularly replace the culture medium to maintain cell growth, and observe cellular morphology under the microscope to ensure cells meet experimental requirements.

###### Detailed procedures

1. Disinfect and take out the cells from the incubator
2. Examine the survival and growth status of each culture flask
3. Disinfect the culture flasks and put them into the laminar flow cabinet
4. Discard the old medium and rinse with 1× PBS buffer, taking care not to dislodge the cells
5. Add 6 mL of fresh medium
6. Seal the cap, covering the bottle opening with a sealing film
7. Disinfecting culture flasks and put them back to the incubator

##### cell passaging

By performing cell passaging, increase the number of cells available for the experiment to ensure its smooth progression.

###### Detailed procedures

1. Disinfect the culture flask of neurons and take it into the benchtop
2. Suck the medium into a centrifuge tube
3. Wash with 1× PBS buffer for three times, in order to remove remaining medium
4. Digest with 100μl 0.5% trypsin for 5 minutes
5. Add serum to stop the digestion, resuspending, and collect the cell suspension into a centrifuge tube
6. Take out the centrifuge tube from the benchtop, and centrifuge at 1500 rpm for 5 minutes
7. Disinfect the tube and return it to the benchtop, aspirating the supernatant and discarding it, adding fresh medium, and resuspending the cells
8. Divide the cell suspension evenly between two culture flasks, toping up the medium to 6 mL in each culture flask, sealing and label each
9. Take the culture flasks out, disinfecting and putting them back to the incubator.

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