iGEM 2024 — Team squirrel-beijing-ii

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{% block title %}Notebook{% endblock %}
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# Notebook

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

[Design](#a1)
[Build](#a2)
[Test](#a3)
[Learn](#a4)
[Reference](#a5)

Design

The purpose of this study is to study the affinity of MFG-E8 and Oleic acid/linolic acid/linolic acid by molecular docking technology( 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 used AlphaFold3 to model the target protein MFG-E8, and optimized the structures of MFG-E8 and oleic acid by using 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. Subsequently, according to the semi-flexible docking method, the binding modes of three fatty acid molecules were analyzed. We expect to determine the best binding mode of MFG-E8 with three fatty acids, which will help us to understand their relieving effects on inflammation.

![](https://static.igem.wiki/teams/5456/result/result2.png)

Figure The growth conditions of HEEpiC cells, and the expression levels of inflammatory factors in cells and medium. C stood for control group without any treatment, M stood for model group, D stood for functional protein treatment group.
\* When compare with the control level, p<0.05. # When compare with the model level, p<0.05.

Build

1. Esophageal epithelial cells (HEEpiC) 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(M group),the cells were seeded into 6-well plates and grew for 24 h,then the plates were put into the aseptic cell incubator for 1 h at 45℃with 5%CO2.Then the plates were sent back to the normal culture incubator at 37℃with 5%CO2.In treatment group(M+D group),before the cells were put into the special incubator at 45℃,the embedded functional protein complex was added into the medium with the concentration of 1μM,and the action time of functional protein complex was 1 h. Then, cell samples were collected.

![](https://static.igem.wiki/teams/5456/wetlab/engineer/engineer1.webp)

2. For MFG-E8 is a soluble protein that can be expressed in Escherichia coli(E.coli), which is designed as follows. By cloning into a vector, expression plasmids encoding functional proteins are further generated and transformed into pET283a and Dh5a strains. Then, plasmids are extracted and constructed from positive clones and confirmed by sequencing. Next, extract the protein from the plasmid for purification.( Dong et al , 2023)

![](https://static.igem.wiki/teams/5456/wetlab/engineer/engineer2.webp)

3. 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.Experiment results showed that inflammatory factors including NF-κB,TNF-αand IL-1βwere up-regulated in model group,which were sharply down-regulated with the treatment of three types of fatty acids,indicating that three types of fatty acids could suppress inflammation reaction in some degree,in the present heat injury model.

2. Results demonstrated that MFG-E8 protein could inhibit the expression levels of inflammatory factors,and three types of fatty acids could up-regulate the expression level of MFG-E8 protein,further proved that three types of fatty acids could increase the level of MFG-E8,and the latter factor could suppress inflammation.

![](https://static.igem.wiki/teams/5456/result/result4.png)

3. Through Western blot analysis, we successfully verified the interaction between MFG-E8 and Oleic acid/linolic acid/linolic acid. Results demonstrated that the treatment of MFG-E8 protein complex with the concentration of 1μM significantly alleviated inflammatory reaction,embodying on the down-regulation of inflammatory factors,including TNF-αand IL-1βproteins.

![](https://static.igem.wiki/teams/5456/wetlab/engineer/engineer4.webp)

4. 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 potential interaction model between MFG-E8 and Oleic acid/linolic acid/linolic acid. The expression of functional protein (MFG-E8) in this pathway plays a key role in inhibiting inflammation and malignant lesions of esophageal tissue(Hua et al , 2022). The three fatty acids inhibited the inflammatory reaction in the heat injury model to some extent by reducing the level of inflammatory factors and increasing the level of functional protein (MFG-E8).

2. We found that three kinds of fatty acids can improve the level of MFG-E8, and the treatment of MFG-E8 protein complex with a concentration of 1 μ M can significantly alleviate the inflammatory reaction, which is manifested by the down-regulation of inflammatory factors TNF-α and IL-1 β. Based on these characteristics, we prepared a chemical tablet containing 1 μ M MFG-E8 protein, which is a functional protein with in vitro repair effect.

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.Dong Xiaohong, et al."MFG-E8 Alleviates Cognitive Impairments Induced by Chronic Cerebral Hypoperfusion by Phagocytosing Myelin Debris and Promoting Remyelination.."Neuroscience bulletin 40.4(2023):483-499.

3.Hua Xiaomin, et al."Protective Effect of MFG-E8 on Necroptosis-Induced Intestinal Inflammation and Enteroendocrine Cell Function in Diabetes."Nutrients 14.3(2022):604-604.

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[SDS-PAGE Gel Preparation (For Western Blot Setup):](#a1)
[Western Blot (Protein Immunoblotting):](#a2)
[Cell Culture and Subculturing](#a3)
[Polymerase Chain Reaction (PCR)](#a4)

SDS-PAGE Gel Preparation (For Western Blot Setup):

Purpose: To separate and analyze proteins based on molecular weight and charge, essential for protein purification, identification, and functional analysis.
a. Materials
Glass plates, gel mold, gel casting rack, pipettes and tips, test tubes, 30% Acrylamide, 4×SDS-PAGE separation gel buffer, 4×SDS-PAGE stacking gel buffer, 10% Ammonium persulfate (APS), TEMED, ddH2O, waste container, comb.
b. Procedure:
i. Insert thin glass plate into the thick glass plate to form a container for the gel solution and secure the plates in the casting rack.

ii. 10% Separation Gel Preparation:
1. Add 2.5 ml of 4×SDS-PAGE separation gel buffer to a test tube.
2. Add 4.1 ml of ddH2O.
3. Add 3.3 ml of 30% acrylamide.
4. Add 100 µl of 10% APS.
5. Add 10 µl of TEMED, mix quickly.
iii. Transfer the gel solution into the space between the glass plates using a pipette, then cover with methanol to level the gel.

iv. Let the separation gel solidify for 30 minutes, then discard the methanol.

v. 5% Stacking Gel Preparation:
1.Add 1.25 ml of 4×SDS-PAGE stacking gel buffer to a test tube.
2.Add 2.84 ml of ddH2O.
3.Add 0.83 ml of 30% acrylamide.
4.Add 75 µl of 10% APS.
5. Add 7.5 µl of TEMED, mix quickly.

vi. Transfer the stacking gel solution on top of the separation gel using a pipette, insert the comb, and let it solidify for 30 minutes.

vii. Remove the comb before use.

![](https://static.igem.wiki/teams/5456/wetlab/experiment/experiment1.webp)

Western Blot (Protein Immunoblotting)

Purpose:
 The aim of this experiment is to detect specific proteins by using antibodies that specifically recognize the target proteins. It is also used to measure the expression levels of these proteins by comparing the intensity of the bands in different samples, assess the molecular weight of the proteins by comparing them to standard proteins, and evaluate the modification status of proteins, such as phosphorylation or glycosylation, by analyzing changes in their migration under different conditions.
a. Principles:
i. SDS-PAGE: Different proteins carry different charges and molecular weights, so after running the gel, proteins will be distributed across different positions on the gel.
ii. Primary antibodies bind to the target protein, and secondary antibodies bind to the primary antibody. Detection of the emitted signal allows visualization of the protein’s position and expression levels.

b. Procedure:
i. Gel Electrophoresis (Running the Gel)
Prepare the electrophoresis buffer: mix 500 ml deionized water with SDS buffer powder.
Assemble the gel in the electrophoresis tank (red corresponds to the positive electrode, blue to the negative).
Pour electrophoresis buffer into the tank until it reaches the marked line.
Load 20 μL of each protein sample into the wells of the gel (repeat until all samples are loaded).
Load 15 μL of sample loading buffer mixed with 5 μL of protein marker into a separate well.
Connect the power supply and run the gel for 40 minutes, or until the marker reaches the desired position.
ii. Membrane Transfer
Soak two filter papers and two black sponge pads in transfer buffer, and soak one PVDF membrane in methanol.
Remove the gel from the electrophoresis plate, cut the gel based on the protein bands.
Assemble the transfer stack in the following order: black clamp, one black sponge pad, one filter paper, PVDF membrane, the gel, another filter paper, another black sponge pad, and finally, another black clamp.
Insert the assembly into the transfer apparatus.
Pour methanol and transfer buffer into the tank until it reaches the marked line for 4 gels.
Transfer the proteins for 1 hour and 40 minutes.
![](https://static.igem.wiki/teams/5456/wetlab/experiment/experiment2.webp)

vi. Signal Detection:
For ECL Substrate (Pumino):
Mix ECL A solution and B solution in a 1:1 ratio.
b) Place the PVDF membrane on the detection equipment and evenly coat with the mixed solution.
c) Activate the equipment, calculate the exposure time, and wait for 3 minutes.
d) Observe the image.

For Super Signal Substrate:
a) Mix Super Signal A solution and B solution in a 1:1 ratio.
b) Place the PVDF membrane on the detection equipment and coat with the mixed solution.
c) Activate the equipment, calculate the exposure time, and wait for 42 seconds.
d) Observe the image.

![](https://static.igem.wiki/teams/5456/wetlab/experiment/experiment4.webp)

Cell Culture and Subculturing

Purpose: Subculturing is a routine method for maintaining cells in culture and is essential for most cell biology experiments. When cells grow to confluence in a flask, they must be diluted and replated in new flasks to continue growing. This process is called subculturing. It is important to maintain strict sterile conditions throughout the process, as any contamination could negatively affect the cells. Suspension cells can be directly transferred, while adherent cells must be digested before subculturing.

a. Medium Change
i. Remove the old culture medium.
ii. Wash the cells.
iii. Add fresh culture medium.
iv. Return the flask to the incubator, set the temperature (e.g., 37°C), gas environment (95% sterile air and 5% CO2), and humidity.

b. Subculturing Cells
i. Preparation: Prepare the culture medium and necessary reagents (e.g., trypsin, PBS). Ensure all equipment and reagents are sterilized.
ii. Observe the cell state: Use a microscope to check the cells' condition, ensuring normal growth and checking for any deformation or contamination (Esophageal epithelial cells should appear elongated).
iii. Remove or pour out the old culture medium from the flask.
iv. Add PBS rinse solution and gently shake to remove residual serum and dead cells (rinse 3–4 times).
v. Add trypsin to cover the cells completely and shake the flask gently.
vi. Add fetal bovine serum to stop the digestion reaction.
vii. Transfer the cell-containing liquid to a 5 ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes (ensure the centrifuge is balanced).
viii. Remove the supernatant, add an appropriate amount of fresh culture medium, and resuspend the cells.
ix. Split the resuspended cells and 5 µl of medium into two new culture flasks. Shake horizontally 15–20 times and vertically 15–20 times, then return the flasks to the incubator.

![](https://static.igem.wiki/teams/5456/wetlab/experiment/experiment5.webp)

Polymerase Chain Reaction (PCR)

Purpose: PCR is a widely used molecular biology technique, primarily for the rapid and specific amplification of a particular DNA fragment in vitro.1. Amplify specific DNA sequences exponentially to obtain sufficient amounts for subsequent analysis.2. Clone genes or DNA fragments into vectors for gene function studies or protein expression. 3. Detect mutations or polymorphisms in genomes, aiding clinical diagnosis and genetic studies.4. Through reverse transcription PCR (RT-PCR), analyze mRNA expression levels to study gene expression under different conditions.

a. Preparing Reaction Components
i. Buffer:
Standard buffer contains 1 pmol/ml Tris ·HCl, pH 8.3-9.0 at room temperature, and near pH 7.2 at the extension temperature (72°C). It includes a divalent cation, usually Mg²⁺, to activate the active site of DNA polymerase.

ii. dNTP:Deoxynucleotide triphosphates (dATP, dGTP, dTTP, dCTP) are the building blocks for synthesizing new DNA strands. Their total concentration is typically 200 pmol/ml (saturation level). dNTPs bind to Mg²⁺, reducing free Mg²⁺ concentration, which can affect DNA polymerase activity.

iii. Primers:
Short single-stranded DNA fragments that bind to complementary regions on the template DNA, providing a starting point for DNA polymerase to begin synthesizing the new DNA strand. Primers are usually 18-30 bp long. Shorter primers reduce specificity, while longer ones may cause non-specific binding or primer-dimer formation, increasing synthesis costs.

iv. Template:
A single-stranded or double-stranded DNA template provides the original sequence for PCR amplification. The template’s purity requirements are low, but it should not contain proteases, nucleases, DNA polymerase inhibitors, or DNA-binding proteins. Template DNA should be stored at low temperatures.

v. Taq DNA Polymerase:
DNA polymerase synthesizes DNA from the 5’ to 3’ direction, using the template and primers as a guide. It must be stored at -20°C and contains glycerol as a preservative. The optimal reaction temperature is 72°C (extension temperature). Typically, Taq is added last when preparing the PCR reaction.

![](https://static.igem.wiki/teams/5456/wetlab/experiment/experiment6.webp)
b. Mixing Reaction Components:
i. Using a pipette, add 11.5 µl of ddH2O (in two steps) into a centrifuge tube.
ii. Add 1 µl of primers.
iii. Add 2 µl of PCR buffer.
iv. Add 2 µl of dNTP.
v. Add 2 µl of the template (cDNA).
vi. Finally, add 0.5 µl of Taq DNA polymerase.

c. Setting Up the PCR Program:
i. Denaturation:
Heat the reaction to 94-98°C for 30 seconds to 1 minute, causing the DNA double helix to denature into single strands.

ii. Annealing:
Lower the temperature to 50-65°C for 30 seconds to 1 minute to allow primers to bind to the single-stranded DNA template.

iii. Extension:
Raise the temperature to 72°C, where DNA polymerase synthesizes a new DNA strand by extending the primer along the template.
Steps i-iii are usually repeated for 20-40 cycles to achieve exponential amplification of the DNA fragment. After the final cycle, hold the reaction at 72°C for a final extension of any incomplete fragments, then cool the reaction to 4°C.

Notes:
Ensure the concentration of each component is appropriate to avoid inhibiting the PCR reaction.
Primer design must be specific to prevent dimer formation or non-specific amplification.
Extraction and purification of the PCR product may be critical for subsequent analyses.

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[Result1](#a1)
[Result2](#a2)
[Result3](#a3)
[Result4](#a4)
[Result5](#a5)
[Result6](#a6)
[Result7](#a7)

Result 1: Cells culture and Cells injury model construction

![](https://static.igem.wiki/teams/5456/result/result1.png)

Figure. The growth conditions of HEEpiC cells, and the expression levels of inflammatory factors in cells and medium. C stood for control group without any treatment, M stood for model group, D stood for functional protein treatment group. \* When compare with the control level, p<0.05. # When compare with the model level, p<0.05.

Electrophoresis is a technique that uses an electric field to cause charged particles (such as DNA, RNA, or proteins) to migrate through a gel. Smaller molecules or molecules that are more heavily charged migrate faster.

●Result 2: The affinity prediction of functional protein (MFG-E8) and oleic acid/ linoleic acid/ linolenic acid by molecular docking

![](https://static.igem.wiki/teams/5456/result/result2.png)

Figure. The affinity measurement of MFG-E8 protein and three types of fatty acids by molecular docking.

MFG-E8 does not have a publicly available crystal structure, so Alphafold3 prediction was used. In this study, we employed a semi flexible docking method. Firstly, we downloaded the crystal structure of the target protein MFG-E8 from a protein database or modeled it using AlphaFold3. Then, we used Maestro computational software to prepare the structures of proteins and small molecules separately. During the protein preparation process, optimize the hydrogen bond network and minimize the energy of the protein system. Small molecule preparation: Generate three-dimensional structures and minimize energy, and generate multiple conformations (15) to increase the likelihood and accuracy of successful docking. Next, we use the sitemap module of Maestro software to predict the binding pocket of small molecules (default parameters) and generate a receiver grid file. Based on the predicted pocket location, size, and small molecule docking score, we determine the most likely binding pocket. The other parameters remained at their default values. During molecular docking, select the prepared grid file and small molecules in the small molecule docking ligand docking module for molecular docking. Based on the scoring of docking results and the rationality of small molecule binding conformation, the optimal binding mode is selected through comprehensive analysis and judgment. The docking results were visualized using Pymol software. The human sequence of MFG-E8 was submitted to the server along with oleic acid, and the generated computational model was used for molecular docking and MM-GBSA. Oleic acid combining pattern analysis: The binding mode of the three molecules is the same. The carboxyl group interacts with GLN314 through hydrogen bonding and with ARG377 through salt bridge interaction (also known as electrostatic interaction). The alkyl chain of the molecule occupies the hydrophobic pocket composed of MFG-E8 proteins PHE260, ASN274, ASN275, and ASN309. Linoleic acid combined with pattern analysis: The binding mode of the three molecules is the same. The carboxyl group interacts with GLN314 through hydrogen bonding and with ARG377 through salt bridge interaction (also known as electrostatic interaction). The alkyl chain of the molecule occupies the hydrophobic pocket composed of MFG-E8 proteins PHE260, ASN274, ASN275, and ASN309. Linolenic acid combined with pattern analysis: The binding mode of the three molecules is the same. The carboxyl group interacts with GLN314 through hydrogen bonding and with ARG377 through salt bridge interaction (also known as electrostatic interaction). The alkyl chain of the molecule occupies the hydrophobic pocket composed of MFG-E8 proteins PHE260, ASN274, ASN275, and ASN309.

●Result 3: Special functional protein (MFG-E8) overexpression system design and construction

For MFG-E8 is a soluble protein that can be expressed in Escherichia coli (E. coli), which is designed as follows.
Predicted locationi Secreted, Intracellular (different isoforms)
Introduction of the protein: https://www.proteinatlas.org/ENSG00000140545-MFGE8
mRNA Nucleotide Sequence (1008 nt):
ATGCCGCGCCCCCGCCTGCTGGCCGCGCTGTGCGGCGCGCTGCTCTGCGCCCCCAGCCTCCTCGTCGCCC
TGGATATCTGTTCCAAAAACCCCTGCCACAACGGTGGTTTATGCGAGGAGATTTCCCAAGAAGTGCGAGG
AGATGTCTTCCCCTCGTACACCTGCACGTGCCTTAAGGGCTACGCGGGCAACCACTGTGAGACGAAATGT
GTCGAGCCACTGGGCCTGGAGAATGGGAACATTGCCAACTCACAGATCGCCGCCTCGTCTGTGCGTGTGA
CCTTCTTGGGTTTGCAGCATTGGGTCCCGGAGCTGGCCCGCCTGAACCGCGCAGGCATGGTCAATGCCTG
GACACCCAGCAGCAATGACGATAACCCCTGGATCCAGGTGAACCTGCTGCGGAGGATGTGGGTAACAGGT
GTGGTGACGCAGGGTGCCAGCCGCTTGGCCAGTCATGAGTACCTGAAGGCCTTCAAGGTGGCCTACAGCC
TTAATGGACACGAATTCGATTTCATCCATGATGTTAATAAAAAACACAAGGAGTTTGTGGGTAACTGGAA
CAAAAACGCGGTGCATGTCAACCTGTTTGAGACCCCTGTGGAGGCTCAGTACGTGAGATTGTACCCCACG
AGCTGCCACACGGCCTGCACTCTGCGCTTTGAGCTACTGGGCTGTGAGCTGAACGGATGCGCCAATCCCC
TGGGCCTGAAGAATAACAGCATCCCTGACAAGCAGATCACGGCCTCCAGCAGCTACAAGACCTGGGGCTT
GCATCTCTTCAGCTGGAACCCCTCCTATGCACGGCTGGACAAGCAGGGCAACTTCAACGCCTGGGTTGCG
GGGAGCTACGGTAACGATCAGTGGCTGCAGATCTTCCCTGGCAACTGGGACAACCACTCCCACAAGAAGA
ACTTGTTTGAGACGCCCATCCTGGCTCGCTATGTGCGCATCCTGCCTGTAGCCTGGCACAACCGCATCGC
CCTGCGCCTGGAGCTGCTGGGCTGTTAG
Translation (335 aa):
MPRPRLLAALCGALLCAPSLLVALDICSKNPCHNGGLCEEISQEVRGDVFPSYTCTCLKGYAGNHCETKC
VEPLGLENGNIANSQIAASSVRVTFLGLQHWVPELARLNRAGMVNAWTPSSNDDNPWIQVNLLRRMWVTG
VVTQGASRLASHEYLKAFKVAYSLNGHEFDFIHDVNKKHKEFVGNWNKNAVHVNLFETPVEAQYVRLYPT
SCHTACTLRFELLGCELNGCANPLGLKNNSIPDKQITASSSYKTWGLHLFSWNPSYARLDKQGNFNAWVA
GSYGNDQWLQIFPGNWDNHSHKKNLFETPILARYVRILPVAWHNRIALRLELLGC
Expression vector: pET28A HIS tag at the C-terminus of MFG-E8. NcoI+xhoI enzyme digestion was performed, and the plasmids were cloned into pET283a and Dh5a strains for amplification; Transfer to protein expression using E. coli.

![](https://static.igem.wiki/teams/5456/result/result3.jpg)

Figure Protein expression design using E. coli

●Result 4: The effects of oleic acid/ linoleic acid/ linolenic acid in decreasing the levels of inflammatory factors and increasing the level of functional protein (MFG-E8)

![](https://static.igem.wiki/teams/5456/result/result4.png)

Figure The levels of inflammatory factors and increasing the level of functional protein (MFG-E8)

MFG-E8 does not have a publicly available crystal structure, so Alphafold3 prediction was used. In this study, we employed a semi flexible docking method. Firstly, we downloaded the crystal structure of the target protein MFG-E8 from a protein database or modeled it using AlphaFold3. Then, we used Maestro computational software to prepare the structures of proteins and small molecules separately. During the protein preparation process, optimize the hydrogen bond network and minimize the energy of the protein system. Small molecule preparation: Generate three-dimensional structures and minimize energy, and generate multiple conformations (15) to increase the likelihood and accuracy of successful docking. Next, we use the sitemap module of Maestro software to predict the binding pocket of small molecules (default parameters) and generate a receiver grid file. Based on the predicted pocket location, size, and small molecule docking score, we determine the most likely binding pocket. The other parameters remained at their default values. During molecular docking, select the prepared grid file and small molecules in the small molecule docking ligand docking module for molecular docking. Based on the scoring of docking results and the rationality of small molecule binding conformation, the optimal binding mode is selected through comprehensive analysis and judgment. The docking results were visualized using Pymol software. The human sequence of MFG-E8 was submitted to the server along with oleic acid, and the generated computational model was used for molecular docking and MM-GBSA. Oleic acid combining pattern analysis: The binding mode of the three molecules is the same. The carboxyl group interacts with GLN314 through hydrogen bonding and with ARG377 through salt bridge interaction (also known as electrostatic interaction). The alkyl chain of the molecule occupies the hydrophobic pocket composed of MFG-E8 proteins PHE260, ASN274, ASN275, and ASN309. Linoleic acid combined with pattern analysis: The binding mode of the three molecules is the same. The carboxyl group interacts with GLN314 through hydrogen bonding and with ARG377 through salt bridge interaction (also known as electrostatic interaction). The alkyl chain of the molecule occupies the hydrophobic pocket composed of MFG-E8 proteins PHE260, ASN274, ASN275, and ASN309. Linolenic acid combined with pattern analysis: The binding mode of the three molecules is the same. The carboxyl group interacts with GLN314 through hydrogen bonding and with ARG377 through salt bridge interaction (also known as electrostatic interaction). The alkyl chain of the molecule occupies the hydrophobic pocket composed of MFG-E8 proteins PHE260, ASN274, ASN275, and ASN309.

●Result 5: Expression detection of the special functional protein and the related inflammatory pathway by western blot

![](https://static.igem.wiki/teams/5456/result/result5.jpg)

Figure. The expression levels of MFG-E8, mTOR, TNF-α and IL-1β proteins. C stood for control group without any treatment, M stood for model group, D stood for functional protein treatment group.

The treated cells in Part 1 were collected and treated with lysis buffer, 300 μL of the 5× loadding buffer was added into each cell lysis sample, and we boiled and denatured the samples for 15 min. To quantify protein expressions of MFG-E8, associated proteins of mTOR, TNF-α and IL-1β, with respect to the control protein β-actin, western blot experiment was performed. 1× Tris-glycine electrophoresis buffer was filled into the electrophoresis unit, samples and pre-stained rainbow protein marker were loaded onto the gel plates and electrophoresed initially at 110V for 20 min, then at 135V for about 30 min until the protein marker was completely separated. The PVDF membranes were prepared by 40 s immersion in anhydrous methanol. The membranes, sponges, and filter paper were submerged in a 1× transfer buffer, and 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, and the membranes then underwent a 4W transfer for 1.5 h. After transfer, the membranes were rinsed and incubated in 10% skimmed milk for an hour. The membranes were then washed using 1× TBST with 5 min for three times, which were further incubated with the primary antibodies for two hours. Later, the membranes were washed using 1× TBST buffer for three times, and next incubated with goat anti-rabbit IgG-HRP and 1× TBST for 1 h. After three times of 1× TBST washing, 1 mL ECL developer for the membrane was prepared in darkness and added to the membrane, and chemiluminescence signals were captured with a designated imaging system, and the relative amounts of proteins in each group were quantified concerning β-actin expression. Results demonstrated that the treatment of MFG-E8 protein complex with the concentration of 1 μM significantly alleviated inflammatory reaction, embodying on the down-regulation of inflammatory factors, including TNF-α and IL-1β proteins.

●Result 6: Statistical Analysis

All data are presented as mean ± standard deviation (SD). Statistical analyses were performed using SPSS version 19.0 and GraphPad Prism version 7.0 (GraphPad Software Inc., San Diego, CA, USA). Comparisons between control and intervention groups were made using t-tests, with statistical significance set at p < 0.05.

●Result 7 Production of chemical tablets containing functional protein

![](https://static.igem.wiki/teams/5456/result/result7.png)

Figure Electron microscopy image of nanoparticles (NPs) and product of chemical tablets complex

Oleanolic acid (OA) nanoparticles were prepared by ultrasonic emulsification method, and the morphology was observed by SEM. Overexpressed MFG-E8 protein sample was successfully prepared into OA nanoparticles. Agilent 1290 liquid chromatography was used to detect encapsulated protein samples, with the following liquid phase conditions: Mobile phase: 0.5% formic acid water (A) - acetonitrile (B), flow rate of 0.3 mL/min Gradient elution conditions: 0-1 min, 30% B; 1-3 min, 30-80% B; 3-5 min，80-98% B; 5-6 min，98% B; 6-7.5 min, 98-30% B; 7.5-9.5 min, 30% B; 9.5 min, stop. Injection volume: 2 μL, column temperature: 35 ℃, detection wavelength: 350 nm.
Production of chemical tablets: the concentration of OA embedded MFG-E8 protein was 1 μM, the accessory ingredient was natural peppermint extract.

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