iGEM 2024 — Team shsid-china

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{% 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 %}
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![](https://static.igem.wiki/teams/5522/desc-banner.png)

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* 07/01-07/07
* 07/07-07/15
* 07/15-07/30
* 08/01-08/07
* 08/08-08/15
* 08/16-08/22
* 08/23-08/31
* 08/31-09/04

07/01-07/07

1.Making and sterilizing lysogeny broth, preparing agar plates
2.PCR amplification of SUMO-IL-18BPa-Fc, SUMO-IL-18BPc-Fc

![](https://static.igem.wiki/teams/5522/notebook/1.png)

07/07-07/15

1. PCR amplification of SUMO-IL-18BPa-Fc, SUMO-IL-18BPc-Fc
2. pET28a blank plasmid extraction and PCR amplification
3. Enzyme digestion of pET28a and verification that it has been cut
4. Gel-electrophoresis verification of pET28a, SUMO-IL-18BPa-Fc,SUMO-IL-18BPc-Fc
5. DNA extraction of pET28a, SUMO-IL-18BPa-Fc, SUMO-IL-18BPc-Fc from gel

07/15-07/30

1. Restriction digestion and ligation to create recombinant plasmids
2. Transformation of recombinant plasmids into DH5a E. coli

![](https://static.igem.wiki/teams/5522/notebook/2-1.png)
![](https://static.igem.wiki/teams/5522/notebook/2-2.png)

08/01-08/07

1. Selection and verification of monoclonal colonies
2. Send monoclonal colonies' plasmids for gene sequencing for further verification
3. Amplification of DH5a with desired plasmids

08/08-08/15

1. Transformation into BL21 E. coli
2. Selection and verification of monoclonal colonies
3. cultured bacteria

![](https://static.igem.wiki/teams/5522/notebook/3-1.png)
![](https://static.igem.wiki/teams/5522/notebook/3-2.png)

08/16-08/22

1. IPTG induction of protein synthesis
2. Extraction and lysis of BL21 bacteria
3. His-tag purification

08/23-08/31

1. SDS-PAGE verification of extracted proteins
2. BCA Calorimetric method to quantify proteins
3. Western blot for further protein verification
4. Store proteins at different temperatures for 24 hours

![](https://static.igem.wiki/teams/5522/notebook/4.png)

08/31-09/04

1.Cell incubation with proteins (stored at different temperatures) and inflammatory factors
2.ELISA of cell supernatant to detect IFN-gamma levels

![](https://static.igem.wiki/teams/5522/notebook/5.png)

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{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
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![](https://static.igem.wiki/teams/5522/desc-banner.png)

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* Overview
* Cycle 1：BBa\_K5522003
* Cycle 2：BBa\_K5522002
* Cycle 3: Test
* Cycle 4: Learn
* Reference

Overview

The spread of Inflammatory Bowel Disease (IBD) in Asian countries due to the adoption of Western dietary habits have become a severe healthcare problem[1]. Target therapies that turn off specific inflammation-causing genes have been proven as a major treatment in IBD[2]. In this project, a drug that interferes with Interleukin-18 is designed and tested to provide a new possible solution of IBD treatment IL-18 binding protein (IL-18BP) could bind to IL-18 to stop its function[3] (Fig 1). In humans, while there are four types of IL-18BP (IL-18BPa, Pb, Pc, Pd), only IL-18BPa and IL-18BPc could antagonize IL-18 activity[4,5]. Plasmid containing IL-18 BP with SUMO for better stability, Fc for potential targeted drug delivery, and His tag for easier purification is constructed and tested, the protein is then expressed and purified through BL21 E.coli strands. The purified protein was validated using Western Blotting and its activity was confirmed through T cell activation inhibition experiments.

![](https://static.igem.wiki/teams/5522/engineer/1.png)

Fig 1. The IL18 signal pathway diagram[3]

Cycle 1：[BBa\_K5522003](https://parts.igem.org/Part%3ABBa_K5522003) (pET28a-SUMO-IL-18BPa-Fc)

**Design**

The pET28a vector is a highly popular vector in the field of synthetic biology, containing a T7 promoter and lac operator that allows for fine-tuned control over the plasmid's recombinant protein expression (which can be induced by IPTG), a poly-histidine sequence meant to be translated with the gene fragment into the target protein for easier purification, and kanamycin resistance for isolating bacterial colonies that took up the plasmid[1]. After combining the human SUMO, IL-18BPa, and Fc gene fragments (obtained from NCBI), going through codon optimization, and adding NheI and XhoI restriction sites, we plan on ligating the SUMO-IL-18BPa-Fc fragment onto the plasmid using restriction digestion and ligation (Fig 2). We plan on testing this design within BL21 to verify its effectiveness in protein expression.

![](https://static.igem.wiki/teams/5522/engineer/2.png)

Fig 2. The plasmid map of pET28a-IL18-SUMO-BPa-Fc

**Build**

The pET-28alpha blank plasmid was cut by restriction enzymes NheI and XhoI to make them linear and usable for gel electrophoresis. The results of restriction enzyme digestion on the amplification of the pET-28alpha blank plasmid. The concentration and purity of the samples were then measured. The IL18BPa genes fragments were also cut using the same enzymes and verified using gel electrophoresis.

The length of target genes SUMO-IL-18BPa-Fc is 1542bp. Fig 3A showed that the length of the target gene was consistent with the electrophoresis results, indicating that the target gene was successfully amplified. Agarose gel electrophoresis was used to identify the restriction enzyme digestion product of the PET-28α blank plasmid. Fig 3.B shows that the plasmid was successfully digested.

![](https://static.igem.wiki/teams/5522/engineer/3.png)

Fig 3. Identification of PCR amplified gene and enzyme digested pET-28α vector. A. PCR product of SUMO-IL-18BPa-Fc

B.Enzyme digestion product of pET-28α vector

The cut plasmid and the gene were ligated using T4 DNA ligase, and the recombinant plasmid was transformed into DH5alpha(Fig 4 A). The transformants were then identified by colony PCR. The agarose gel electrophoresis results showed that we obtained the expected length of PCR products, indicating the construction were successfully completed (Fig. 4B).

![](https://static.igem.wiki/teams/5522/engineer/4.png)

Fig 4. The transformants form colonies on solid LB medium and colony PCR amplification of IL-18-BPa .

A.pET-28α-Sumo-IL-BP-18-BPa-Fc-DH5α colony B. Verification of presence of Pa gene in DH5a transformants

Then the plasmid pET-28a with Sumo-IL-18BP-Pa-Fc was sent to the biological company for sequencing. The comparison of the sequencing results showed that the target gene sequence was consistent with the sequencing results (Fig 5), indicating that the plasmid was successfully constructed.

![](https://static.igem.wiki/teams/5522/engineer/5.png)

Fig 5. Gene sequencing of IL-18-BPa.

Cycle 2：[BBa\_K5522002](https://parts.igem.org/Part%3ABBa_K5522002)(pET28a-SUMO-IL-18BPc-Fc)

**Design**

The pET28a vector is a highly popular vector in the field of synthetic biology, containing a T7 promoter and lac operator that allows for fine-tuned control over the plasmid's recombinant protein expression (which can be induced by IPTG), a poly-histidine sequence meant to be translated with the gene fragment into the target protein for easier purification, and kanamycin resistance for isolating bacterial colonies that took up the plasmid[1]. After combining the human SUMO, IL-18BPc, and Fc gene fragments (obtained from NCBI), going through codon optimization, and adding NheI and XhoI restriction sites, we plan on ligating the SUMO-IL-18BPc-Fc fragment onto the plasmid using restriction digestion and ligation(Fig 6). We plan on testing this design within BL21 to verify its effectiveness in protein expression.

![](https://static.igem.wiki/teams/5522/engineer/6.png)

Fig 6. The plasmid map of pET28a-IL18-SUMO-BPc-Fc

**Build**

The pET-28alpha blank plasmid was cut by restriction enzymes NheI and XhoI to make them linear and usable for gel electrophoresis. The results of restriction enzyme digestion on the amplification of the pET-28alpha blank plasmid. The concentration and purity of the samples were then measured. The IL18BPc genes fragments were also cut using the same enzymes and verified using gel electrophoresis.
The length of target genes SUMO-IL-18BPc-Fc is 1641bp. Fig 7 A showed that the length of the target gene was consistent with the electrophoresis results, indicating that the target gene was successfully amplified. Agarose gel electrophoresis was used to identify the restriction enzyme digestion product of the PET-28α blank plasmid. Fig 7B shows that the plasmid was successfully digested.

![](https://static.igem.wiki/teams/5522/engineer/7.png)

Fig 7. Identification of PCR amplified gene and enzyme digested pET-28α vector. A. PCR product of SUMO-IL-18BPc-Fc

B.Enzyme digestion product of pET-28α vector

The cut plasmid and the gene were ligated using T4 DNA ligase, and the recombinant plasmid was transformed into DH5alpha(Fig 8A). The transformants were then identified by colony PCR. The agarose gel electrophoresis results showed that we obtained the expected length of PCR products, indicating the construction were successfully completed (Fig. 8B).

![](https://static.igem.wiki/teams/5522/engineer/8.png)

Fig 8. The transformants form colonies on solid LB medium and colony PCR amplification of IL-18-BPc .

A.pET-28α-Sumo-IL-BP-18-BPc-Fc-DH5α colony B. Verification of presence of Pc gene in DH5a transformants

Then the plasmid pET-28a with Sumo-IL-18BP-Pc-Fc was sent to the biological company for sequencing. The comparison of the sequencing results showed that the target gene sequence was consistent with the sequencing results (Fig 9), indicating that the plasmid was successfully constructed.

![](https://static.igem.wiki/teams/5522/engineer/9.png)

Fig 9. Gene sequencing of IL-18-BPc.

Cycle 3: Test

**3.1 SDS-PAGE**

The purified IL18-BPa, IL18-BPc, and IL10-BP proteins were 56.9kDa, 61kDa, and 62.1kDa, respectively. The SDS-PAGE successfully verified the IL18-BPa, IL18-BPc, and IL10 proteins extracted and purified from E. coli BL21 (Fig 10).

![](https://static.igem.wiki/teams/5522/engineer/10.png)

Fig 10. SDS-PAGE verification of extracted proteins.

A, pET-28α-Sumo-IL-BP-18-BPa is yellow box, pET-28α-Sumo-IL-BP-18-BPc is red box, pET-28α-Sumo-IL-10 is blue box.

**3.2 Western Blot verification of extracted protein**

Compared to coomassie Brilliant Blue staining, the principle of Western detection is antibody antigen specific reaction, with high detection specificity. The proteins we expressed all carrying His tag, and specific His antibodies can be used to detect purified proteins. As shown in Fig 11, the protein size we obtained is consistent with the expected size, demonstrating successful protein expression.

![](https://static.igem.wiki/teams/5522/engineer/11.png)

Fig 11. Detection of recombination protein expression by western blot. From left to right: pET-28α-Sumo-IL-10, pET-28α-Sumo-IL-BP-18-BPa is yellow box, pET-28α-Sumo-IL-BP-18-BPc. The size of Sumo-IL-18BPa-Fc is about 56.9 kDa; the size of Sumo-IL-18BPc-Fc is 61 kDa.the size of Sumo-IL-10-Fc is 62.1 kDa.

**3.3 Use BCA colorimetric method to determine the protein concentration**

The concentration of the samples was measured using the BCA calorimetric method. We use standard concentration protein solution and BCA colorimetric method to determine the protein concentration. The absorbance value was measured by the enzyme-linked immunosorbent assay (ELISA) reader. We draw a scatter plot, insert a standard curve, and calculate the R-squared value.In the Fig.12, the fitting coefficient is greater than 0.99, indicating that the fitting effect is perfect.

![](https://static.igem.wiki/teams/5522/engineer/12.png)

Fig 12. Protein quantification using BCA colorimetric method.

The protein concentrations of Sumo-IL-18-BPa-Fc, Sumo-IL-18-BPc-Fc and Sumo-IL-10-Fc were calculated according to the standard curve, as shown in Tab 3.

Tab. 3 The concentrations of Sumo-IL-18-BPa-Fc, Sumo-IL-18-BPc-Fc and Sumo-IL-10-Fc protein

| Sample | Concentration |
| --- | --- |
| Sumo-IL-18-BPa-Fc | 2.649213 μg/mL |
| Sumo-IL-18-BPc-Fc | 2.451683 μg/mL |
| Sumo-IL-10-Fc | 2.5152 μg/mL |

**3.4 Verification of Protein Function**

**3.4.1 Measuring the standard curve using an INF-γ standard.**

In the experiment, we need the concentration of INF-γ, and we establish the standard curve of INF-γ.The R squared value is larger than 0.99, which indicates that the curve is accurate (Fig. 13 ).

![](https://static.igem.wiki/teams/5522/engineer/13.png)

Fig 13. INF-γ quantification using ELISA.

**3.4.2 The influence of dose and storage conditions on protein activity**

Store the three proteins (Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10) at different temperatures (-80, -20, 4, 37 ℃) for 24 hours and incubate them with mouse primary T lymphocytes. Stimulate the cells with IL-18 and detect the IFN-γ in the cell supernatant.

In Fig 14, the concentration of IFN-γ gradually decreased with the increase of Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 protein concentrations. It illustrates the inhibitory effect of recombinant Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 proteins on IFN-γ production.

The results are showed in Fig 14,the activity of protein is affected by temperature. The inhibitory effect of Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 on IFN-γwas not affected after 24 hours at -80 °C. After 24 hours at -20 °C and 4 °C, the inhibitory effect of Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 proteins on IFN-γ decreased. After 24 hours at -37°C, the inhibitory effect of Sumo-IL-BP-18-BPc, Sumo-IL-BP-18-BPa and Sumo-IL-10 protein on IFN-γ was significantly decreased. Storing at low-temperature preservation is beneficial for the stability of recombinant proteins. Furthermore, the anti-inflammatory effect of recombinant proteins is dose-dependent.

![](https://static.igem.wiki/teams/5522/engineer/14.png)

Fig 14. The influence of dose and storage temperature on protein activity. Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc, and Sumo-IL-10A protein were stored at different temperatures, then were incubated with cells to determine the concentration of IFN γ in the cell supernatant.

In the fig 15, it can be seen that after storage at 4°C for 24 hours, IL-18BPc's effects are still comparable to that of IL-10 in the 2.0 ug/ml, while IL-18BPa is less stable.

![](https://static.igem.wiki/teams/5522/engineer/15.png)

Fig 15. The protein activity at 4°C. Ratio of IFN-gamma production levels after protein treatment vs control for IL-10 (B), IL-18BPa (C), IL-18BPc

Cycle 4: Learn

The testing in all three stages shows overall positive results, which indicates that our model design was reasonable and accurate. The result of the verifications also suggested that both IL-18BPa and IL-18BPc had similarly significant effect in anti-inflammatory therapies comparable to IL-10, hence, suggesting that these two proteins can serve as a basis for further experiments.

During the process of testing, we used an inefficient primer for the IL-18BPc gene, and redesigning the primers took some trial and error. We also realized two days within the experiment that we needed a positive control, that was why we bought a synthesized pET28a-SUMO-IL-10-Fc plasmid from GenScript and transformed it into BL21 cells along with our constructed plasmids. The end results demonstrated that SUMO-IL-18BPa-Fc and SUMO-IL-18BPc-Fc had great potential to reduce inflammation, which give us future directions: for instance, our functionality tests occurred in vitro, but what would happen to the protein within the environment of the intestines, which is different in terms of pH, temperature, etc? What dose should be taken for different levels of inflammation? More questions are raised from the results, which is how research works.

Reference

[1] Piersiala K, Hjalmarsson E, da Silva PFN, Lagebro V, Kolev A, Starkhammar M, Elliot A, Marklund L, Munck-Wikland E, Margolin G, Georén SK, Cardell LO. Regulatory B cells producing IL-10 are increased in human tumor draining lymph nodes. Int J Cancer. 2023 Aug 15;153(4):854-866. doi: 10.1002/ijc.34555. Epub 2023 May 5. PMID: 37144812.

[2] Liang X, Fan Y. Bidirectional two-sample Mendelian randomization analysis reveals a causal effect of interleukin-18 levels on postherpetic neuralgia risk. Front Immunol. 2023 May 25;14:1183378. doi: 10.3389/fimmu.2023.1183378. PMID: 37304287; PMCID: PMC10247971.

[3]Menachem A ,Alteber Z ,Cojocaru G , et al.Unleashing Natural IL18 Activity Using an Anti-IL18BP Blocker Induces Potent Immune Stimulation and Antitumor Effects.[J].Cancer immunology research,2024,OOF1-OF17.

[4]Yamanishi K ,Hata M ,Gamachi N , et al.Molecular Mechanisms of IL18 in Disease[J].International Journal of Molecular Sciences,2023,24(24):

[5]Ihim, S. A., Abubakar, S. D., Zian, Z., Sasaki, T., Saffarioun, M., Maleknia, S., & Azizi, G. (2022). Interleukin-18 cytokine in immunity, inflammation, and autoimmunity: Biological role in induction, regulation, and treatment. Frontiers in immunology, 13, 919973.

<https://doi.org/10.3389/fimmu.2022.919973>

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

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* 1.Material preparation
* 2.Plasmid construction
* 3. Protein expression
* 4.Functional verification

1. Material preparation

**1.1 Making Lysogeny Broth (LB)**

**Materials**

Tryptone (SolarBio T8490), Yeast Extract (OXOID LP0021B), NaCl (Sangon Biotech A501218-0005), ddH2O, Digital balance, weighing paper, spoon, 1L graduated cylinder, Large flask

**Procedures**

* Wipe digital balance, spoon, wash cylinder with water and rinse with ddH2O
* Diagonally fold weighing paper and place on balance, tare weight; change weighing paper for each different material
* Measure 10g tryptone, 5g yeast extract, 10g NaCl, transfer them all into the large flask
* Measure 1L ddH2O and transfer into large flask
* Seal the flask with autoclavable sealing film wrapped with a rubber band, tape on a strand of autoclave indicator tape, and send to the autoclave room
* Retrieve the flask that is sterilized, then store at 4°C

**1.2 Melting and pouring agar plates**

**Materials**

LB mixed with 1.5% agar, Microwave,35mg/mL Kanamycin (Beyotime),Parafilm Sealing Film, clean bench, alcohol lamp, 75% alcohol spray, Small petri dishes, Large centrifuge tubes
**Procedures**

* 1.5% of agar measured and added into LB medium
* All transferring of growth medium was done within the clean bench, with everything taken into the cabinet sprayed with 75% alcohol; all procedures were conducted near the lighted alcohol lamp, and the openings and caps of bottles/flasks were sterilized using the flame after removing the caps and before replacing the caps
* LB-agar mixture was heated using the microwave on medium heat (with cap turned loose) for two minutes, checked, then resent into the microwave for another two minutes until all medium has melted
* The mixture was left to cool until it was about 60°C as to not affect Kanamycin stability; within large centrifuge tubes, it was mixed with Kanamycin in a 1000:1 ratio (1 uL of Kanamycin for 1 mL of medium)
* An amount of the mixture was quickly transferred into plates (enough to cover the bottom of each plate), then the plates were left with the lid off for the agar to set for 20-30 minutes so that moisture can evaporate
* Parafilm was used to seal all the agar plates, which were labelled with “K+” and date; then the plates were transferred into 4°C refrigerators for later use

2. Plasmid construction

**2.1 Extraction of pET-28a vector from DH5a E. coli that already had the plasmid transformed into them**

**Materials**

Diamond Plasmid Mini-PREPS Kit (Sangon Biotech, B110091-0100) containing: Buffer, SBuffer SP, Buffer SP2, Buffer SP3, Wash Solution,Elusion Buffer,spin column, collection tube, DH5a E, 2 mL EP tubes, Centrifuge (HENGNUO), Microvolume Spectrophotometer (zNano-3 uM20240606015), Pipettes (BIO-DL).
**Procedures**

* Add Buffer S inside spin column which is placed in the collection tube. Centrifuge at 12,000 x g for 1 minute. Discard the liquid in the column and place the column back into the collection tube.
* Add 1.8 mL of the bacterial culture into a 2mL EP tube and centrifuge at 8,000 x g for 2 minutes to collect the bacterial pellet. Discard the liquid and put back the collection tube.
* Repeate step 2 (may not be included）
* Mix pacteria with 250 µL of Buffer SP1. Mix thoroughly with pipette.
* Add 250 µL of Buffer SP2. Gently invert the tube 10 times to mix. Let it stand at room temperature for 5 minutes.
* Add 350 µL of Buffer SP3. Gently invert the tube 10 times to mix.
* Centrifuge the mixture at 12,000 x g for 10 minutes. Transfer the mixture from the EP tube to the spin column and centrifuge at 8,000 x g for 30 seconds. Discard the liquid in collection tube.
* Add 500 µL of Wash Solution to the spin column. Centrifuge at 9,000 x g for 30 seconds. Discard the liquid in collection tube.
* Repeat step 8, but this time centrifuge for 1 minute.
* Centrifuge the tube again for 9000g, 90seconds.
* Place the spin column into a clean 1.5 mL microcentrifuge tube. Add 50 µL of Elution Buffer to the center of the membrane. Let it sit at room temperature for 1 minute, then centrifuge for 1 minute to elute the DNA.
* Expel the spin column and keep the tube. Extract 2uL of the DNA to test out it’s purity and density using a Microvolume spectrophotometer. Collect and store the DNA solution.

**2.2 PCR of pET-28a, Sumo-IL-18BP-Pa-Fc, Sumo-IL-18BP-Pc-Fc**

**Materials**

pET-28a, Sumo-IL-18BP-Pa-Fc, Sumo-IL-18BP-Pc-Fc (from GenScript),Forward/reverse (F/R) primers for each specific template plasmid (Azenta),Yeasen 2X Hieff PCR Master Mix with dye, ddH2O,Beyotime 0.2 mL PCR Tubes (Domed Caps) FTUB002,Vortex shaker and mini centrifuge,MIULAB PR-96E PCR Machine

**Procedures**

* Label all PCR tubes and place all materials on ice
* construct a 20uL experimental system consisting of: 10uL PCR Mix, 7uL ddH2O, 1uL forward primer, 1uL reverse primer, 1uL template DNA
* Place samples on vortex shaker to ensure that it is fully mixed, then place on mini centrifuge to ensure that all liquids are not on the tube walls
* 5 minutes 98°C heating up and denaturation to break DNA double strand
* Repeat 30 times: 30 sec 98°C denaturation, 30 sec 56°C annealing, 1 min/kb 72°C extension
* 10 min 72°C extension to ensure that the DNA strands are properly synthesized
* Lower temperature to 4°C for storage in refrigerator

**2.3 Gel electrophoresis verification of pET-28a, Sumo-IL-18BP-Pa-Fc, Sumo-IL-18BP-Pc-Fc**

Basic Mechanisms and Purpose: By running the material through gel electrophoresis, we can isolate the desired gene fragments from non-DNA components within the PCR system and from other gene fragments that we are not interested in. Larger molecules travel slower through the gel, and vice versa. Higher concentrations of gel are better at seperating smaller molecules, and vice versa.

**Materials**

Agarose (Sangon A620014-0100), TAE (Tris acid, acetic acid, EDTA) buffer (Sangon B040123-0100),Yeasen YeaRed Nucleic Acid Gel Stain (Yeasen 10202E576),Trans 15K DNA Marker (Transgen BM161-01),digital balance (Shunyu AE223),weighing paper (Sangon F512112-0001),spoon,gel casting stand, tray, comb

**Procedures**

* mix 1L of ddH2O and 1 premixed pack of TAE and stir to get 1L TAE solution
* measure 100mL of the solution, mix 2g agarose inside to make 2% agarose gel
* Heat in microwave at medium high fire until fully dissolve
* Add 10 uL of Nucleic Acid Gel Stain (0.01%) in a fume hood and mix
* place two small gel casting trays (with the black line on top) within the corresponding slots in the gel casting stand, then place the corresponding comb (with 9 wells per tray) into the trays at the end with the black line
* Pour the liquid gel into the tank, cool it at room temperature for 20min until it is fully solidified
* Slowly remove the comb vertically
* Fill electrophoresis chamber with TAE until the tray is mostly filled
* put the casting tray and gel into the gel electrophoresis system with the end marked with the black line (also where the wells are) facing the cathode (where the black power cable is connected to; cathodes attract cations and therefore have negative charge, so DNA migrates away from it), wait for gel to set
* Add 5 uL of DNA marker into the first slot for reference
* Add 10 uL of PCR product into rest of the slots
* Turn on electricity at 180 volts for 25 minutes

**2.4 DNA extraction from agarose gel of pET-28a, Sumo-IL-18BP-Pa-Fc, Sumo-IL-18BP-Pc-Fc**

**Materials**

Diamond B110092-0100 DNA Gel Extraction Kit (containing buffer B2, wash buffer, elution buffer, spin column, collection tube, etc),1.5 mL EP tubes, Agarose gel, Ultra Slim LED Illuminator (MIULAB), blade,Water bath set at 50°C, Centrifuge, ZUOFEi Microvolume Spectrophotometer

**Procedures**

* Cut the agarose gel containing the band of interest gene using the Illuminator and a razor blade
* Add 500 µL of buffer B2 per 100mg of gel that contains DNA materials within 2mL EP tubes
* Place within 50°C hot water bath until completely dissolved.
* Take out two spin columns and two collection tubes and label one of each Pa and Pc, putting the spin columns into the collection tubes
* Operating with 500 µL of the dissolved gel mixture at a time, add the mixtures into the respective spin columns (Pa melted gel into Pa tube, similarly for Pc), then centrifuge 8000×g for 30 seconds. Discard the liquid within the collection tubes, replace the spin column into the collection tubes, and repeat until all gel mixture has been used.
* Add 500 µL wash buffer into each spin column, centrifuge on 9000×g for 30 seconds, then discard the liquid within the collection tube. Repeat the washing again. Then, without adding any wash buffer, centrifuge the tubes at 9000×g for 1 minute
* Put the spin column into new EP tubes (labelling the EP tubes with Pa and Pc respectively) and add 40 µL elution buffer at the center of the spin column and let it sit for 2 minutes; then centrifuge at 9000×g for 1 minute
* Take the elution buffer and two samples to the microvolume spectrophotometer for determining the DNA concentration
* Wipe the spectrophotometer with paper towels, then load 1 µL of elution buffer and use it as Blank to calibrate the machine
* Wipe the spectrophotometer and load samples, measuring their concentration of DNA

**2.5 Gel electrophoresis verification of pET-28a digested by NheI and XhoI**

**Materials**

pET-28a, NheI, XhoI enzymes, Agarose (Sangon A620014-0100),TAE (Tris acid, acetic acid, EDTA) buffer (Sangon B040123-0100), Yeasen YeaRed Nucleic Acid Gel Stain (Yeasen 10202E576),Trans 15K DNA Marker (Transgen BM161-01),digital balance (Shunyu AE223),weighing paper (Sangon F512112-0001),spoon, gel casting stand, tray, comb

**Procedures**

* Mix 1L of ddH2O and 1 premixed pack of TAE and stir to get 1L TAE solution
* Measure 100mL of the TAE solution, mix 1g agarose inside to make 1% agarose gel
* Heat in microwave at medium high fire until fully dissolve
* Add 10 uL of Nucleic Acid Gel Stain (0.01%) in a fume hood and mix
* Place two small gel casting trays (with the black line on top) within the corresponding slots in the gel casting stand, then place the corresponding comb (with 9 wells per tray) into the trays at the end with the black line
* Pour the liquid gel into the tank, cool it at room temperature for 20min until it is fully solidified
* Slowly remove the comb vertically
* Fill electrophoresis chamber with TAE until the tray is mostly filled
* put the casting tray and gel into the gel electrophoresis system with the end marked with the black line (also where the wells are) facing the cathode (where the black power cable is connected to; cathodes attract cations and therefore have negative charge, so DNA migrates away from it)
* Add 5 uL of DNA marker into the first slot for reference
* Add 2-5 uL of digestion product into rest of the slots
* Turn on electricity at 180 volts for 25 minutes

**2.6 Restriction enzyme digest and ligation of pET-28a with Sumo-IL-18BP-Pa-Fc or Sumo-IL-18BP-Pc-Fc**

**Materials**

pET-28a, Sumo-IL-18BP-Pa, Sumo-IL-18BP-Pc from PCR (within PCR tubes), Nhel enzyme (ThermoScientific), Xhol enzyme (New England Biolabs), rCutSmart Buffer 10X (New England Biolabs), Digestion Procedure, Ligation Materia
Digested pET-28a, Sumo-IL-18BP-Pa, Sumo-IL-18BP-Pc,hermoScientific T4 DNA Ligase (ThermoScientific EL0014),ThermoScientific T4 DNA Ligase Buffer (10X) (ThermoScientific B69), EP tubes
**Procedures**

In order of largest to smallest volume, add 30 µL of the DNA samples, 13 µL ddH2O, 5 µL rCutSmart Buffer, 1 µL Nhel, 1 µL Xhol (so the volume total is 50 µL), then mix using pipette or turning the tube upside down and back for several times (vortex shaking might affect enzyme function), place within 37°C water bath 30 minutes, then 80°C for 5min.
Ligation Procedure:
Within two separate tubes, adding from largest volume to smallest, add 10 µL pET-28a, 7 µL of Pa or Pc, 2 µL of T4 Buffer, and 1 µL T4 DNA Ligase, totaling a volume of 20 µL. Place within 37°C water bath for longer than 30 minutes to 1 hr.

**2.7 Transformation of pET-28a-Sumo-IL-18BP-Pa/Pc-Fc into E. coli type DH5a**

**Materials**

Recombinant plasmid DNA (pET-28a-Sumo-IL-18BPa-Fc, pET-28a-Sumo-IL-18BPc-Fc), Competent DH5α E. coli,LB medium, agar plates containing kanamycinIce, Water Bath, Shaking Incubator

**Procedures**

* Take 50uL of competent DH5α cells (thawed on ice from -80°C) and add 1uL of plasmid DNA. Mix and place the mixture on ice for 30 minutes to allow the plasmid to bind to the cell surface.
* Place the mixture in a 42°C water bath for 90 seconds. This process facilitates the entry of plasmid DNA into the cells.
* Immediately return the tube to ice for 3 minutes.
* Add 1mL of LB medium and shake for 1 hour, allowing the bacteria to recover and express the resistance gene.
* Plate the recovered cells onto agar plates containing Kanamycin. Incubate at 37°C overnight (~16 hours) until distinct colonies form.

**2.8 Identification of positive clones by colony PCR of monoclonal DH5a**

**Materials**

small pipette tips, plated bacteria (strain DH5a that contain pET28a-SUMO-IL-18BP-Pa, pET28a-SUMO-IL-18BP-Pc),Forward/reverse (F/R) primers for the specific template plasmid (Azenta),Yeasen 2X Hieff PCR Master Mix with dye, ddH2O, Beyotime 0.2 mL PCR Tubes (Domed Caps) FTUB002, Vortex shaker and mini centrifuge, MIULAB PR-96E PCR Machine, Shaking incubators

**Procedures**

* using small pipette tips, scrape several monoclonal bacterial colonies into PCR tubes with 5 uL water.
* PCR amplification of Pa or Pc gene fragment using 1 uL of each bacterial sample, with PCR settings 35 repeats (more details see PCR procedural settings of any step, since we used identical settings for temperature)
* Run gel electrophoresis 168V 25 minutes to see which monoclonal bacteria colony has the desired gene fragment
* Add the correct colonies within sterile culture tubes with 5mL LB + 5uL 1000×Kanamycin
* Shake 37°C 200rpm overnight
* Send some samples off for gene sequencing to verify that the genes' sequences are completely correct

**2.9 Plasmid extraction and transformation of E.Coli BL21(DE3)**

**2.9.1 Extraction of pET-28a-Sumo-IL-18BPa/Pc-Fc from E. coli type DH5a**

**Materials**

Diamond Plasmid Mini-PREPS Kit (Sangon Biotech, B110091-0100) containing;DH5a bacteria with modified plasmids; 2mL EP tubes, 1mL EP tubes

**Procedures**

* Verify that RNase A has been added to Buffer SP1.
* Ensure that anhydrous ethanol has been added to the Wash Solution.
* Check for any precipitates in Buffer SP2 and Buffer SP3.
  1. Add 500 µL of Buffer S to the spin column, which is placed in the collection tube. Centrifuge at 12,000 x g for 1 minute. Discard the flow-through and place the column back into the collection tube.
  2. Add 1.8 mL of the bacterial culture into a 2mL EP tube and centrifuge at 8,000 x g for 2 minutes to collect the bacterial pellet. Discard the supernatant completely.
  3. Resuspend the bacterial pellet with 250 µL of Buffer SP1. Mix thoroughly to ensure complete resuspension.
  4. Add 250 µL of Buffer SP2. Gently invert the tube 10 times to mix. Let it stand at room temperature for 5 minutes.
  5. Add 350 µL of Buffer SP3. Gently invert the tube 10 times to mix.
  6. Centrifuge the mixture at 12,000 x g for 10 minutes. Transfer the supernatant from the 2mL EP tube to the spin column and centrifuge at 8,000 x g for 30 seconds. Discard the flow-through.
  7. Add 500 µL of Wash Solution to the spin column. Centrifuge at 9,000 x g for 30 seconds. Discard the flow-through.
  8. Repeat step 8, but this time centrifuge for 1 minute.
  9. Centrifuge the empty spin column at 9,000 x g for 1 minute to remove any residual Wash Solution.
  10.Place the spin column into a clean 1.5 mL microcentrifuge tube. Add 50 µL of Elution Buffer to the center of the membrane. Let it sit at room temperature for 1 minute, then centrifuge for 1 minute to elute the DNA.
  11.Using the Microvolume Spectrophotometer, measure the 260/280 ratio and concentration within the sample. Collect and store the DNA solution.

**2.9.2 Transformation of pET-28a-Sumo-IL-18BP-Pa/Pc-Fc and pET-28a-Sumo-IL-10-Fc into E. coli type BL21**

**Materials**

Recombinant plasmid DNA (pET-28a-Sumo-IL-18BPa-Fc, pET-28a-Sumo-IL-18BPc-Fc),pET-28a-Sumo-10-Fc, Competent bl21 E. coli, LB medium, agar plates containing kanamycin, Ice, Water Bath, Shaking Incubator

**Procedures**

* Take 50uL of competent BL21 cells (thawed on ice from -80°C) and add 1uL of plasmid DNA. Mix and place the mixture on ice for 30 minutes to allow the plasmid to bind to the cell surface.
* Place the mixture in a 42°C water bath for 90 seconds. This process facilitates the entry of plasmid DNA into the cells.
* Immediately return the tube to ice for 3 minutes.
* Add 1mL of LB medium and shake for 1 hour, allowing the bacteria to recover and express the resistance gene.
* Plate the recovered cells onto agar plates containing Kanamycin. Incubate at 37°C overnight (~16 hours) until distinct colonies form.

**2.9.3 Selection and expansion of positive clones of monoclonal BL21**

**Materials**

small pipette tips, plated bacteria (strain BL21 that contain pET28a-SUMO-IL-18BP-Pa, pET28a-SUMO-IL-18BP-Pc, pET28a-SUMO-IL-10), Shaking incubators

**Procedures**

* Using small pipette tips, scrape several monoclonal bacterial colonies into PCR tubes with 5 uL water.
* Scrape some colonies within sterile culture tubes with 5mL LB + 5uL Kanamycin
* Shake 37°C 200rpm overnight
* monoclonal bacterial colonies containing our desired plasmids are expanded without PCR verification, because the Pa/Pc plasmids were extracted from DH5a already confirmed to have the Pa/Pc genes while the IL-10 plasmid was synthesized by GenScript.

**2.9.4 Expanded cultivation of BL21 containing pET-28a-Sumo-IL-18BP-Pa/Pc-Fc or pET-28a-Sumo-IL-10-Fc**

**Materials**

LB, large flasks that have already been autoclaved, the BL21 strains with desired plasmids, Kuhner Shaker X (large shaking incubator)

**Procedures**

* Add 300 mL of LB and 300 uL Kanamycin
* Add 400 uL BL21 liquid bacterial culture into the 300 mL
* Incubate at 37°C, 220 rpm using the large shaking incubator for seven hours

3. Protein expression

**3.1 IPTG induction of protein expression in BL21 containing pET-28a-Sumo-IL-18BP-Pa/Pc-Fc or pET-28a-Sumo-IL-10-Fc**

**Materials**

IPTG (Isopropyl B-D-1-thiogalactopyranoside) BBI A600168-0005, ddH2O, electric balance, vortex shaker, Kuhner Shaker X (large shaking incubator)

**Procedures**

* Measure 0.238g IPTG and add 1 mL ddH2O and vortex until the solution is clear
* Add 300 uL of 1M IPTG solution (which we just made in the previous step) into each flask with bacteria that contain 300 mL of LB from the previous experiment
* incubate at 20°C 220 rpm overnight

**3.2 Protein extraction & purification from BL21 cultures using His tag**

**Materials**

Beyotime His-tag Protein Purification Kit (Denaturant-Resistant) (Beyotime P2229S) (all unspecified materials used in the procedures are from this kit) large, medium centrifuge tubes and 2mL EP tubes, marker, digital balance, Ultrasonic Cell Crusher Lichen LC-JY92-IIN, centrifuge (HengNuo 2-16R), large centrifuge (Eppendorf Centrifuge 5810R), ice, horizontal shaker

**Procedures**

* pack some styrofoam boxes with ice; because the enzymes work at 4°C, all procedures are done with the tubes and liquids kept on ice as much as possible to keep cool
* label two large 50mL centrifuge tubes with IL-10BP, IL-18BP-Pa, IL-18BP-Pc respectively (so 6 tubes total); measure their masses and record on the tube
* transfer 50mL of liquid BL21 bacteria culture into each tube, then centrifuge (using Eppendorf) at 5000 rpm for 15 minutes at 4°C; discard the upper medium
* repeat the above step until all the liquid culture has been centrifuged
* measure the mass of the tubes with centrifuged bacteria again, and subtract the mass of the tube from the measured mass to get the mass of the bacteria
* added 60mg of powder enzyme with 600uL of ddH2O to make 100mg/mL enzyme
* prepare 35mL of lysis buffer (1g bacteria=4mL of lysis buffer), mix the lysis buffer with the enzyme solution.
* add appropriate amount of lysis buffer into teach tube. Then, resuspend the bacteria using the pipette.
* put all samples in ice for 30min
* in the meantime, add 1mL purification resin from the kit into two affinity chromatography columns per protein (so six in total) and label the columns with the corresponding protein; open both ends of the column and let the liquid within the resin solution to totally drip out, leaving behind a light blue mass. Repeat three times: cap the column bottom, add 1mL lysis buffer, cap the column top, invert column 20 times to wash what's left of the resin solution with the lysis buffer, then open both ends to get rid of the lysis buffer
* put each tube with bacteria-lysis buffer solution in ultrasonic cell crusher for 3min each, with 3s on and 3s off; with power ratio 35%
* repeat this process 2 times
* transfer the solution now filled with bacteria fragments into 2 mL centrifuge tubes, then centrifuge at 4°C with 12000rpm for 15min using the smaller centrifuge
* using ~2 mL bacterial solution at a time from the centrifuged tubes (not sucking up any precipitate), wash the two chromatography columns (to get the resin off and mixed with the bacterial solution) and add into one 15mL centrifuge tube for each protein
* bury the tubes in ice and put onto horizontal shaker, inverting the tubes 10 times every ten minutes to mix the solution
* after inverting to mix the solution well, add the solution back into the chromatography columns (split the volume evenly between the two columns) and open both ends to allow liquid to drain out; collect some of the liquid draining out initially as control group (protein extracted from BL21 before purification)
* add 2 mL wash buffer, resuspend the resin, then drain the tube
* repeat the above step 4 more times except with 1 mL wash buffer, unless the resin is dirtier than usual (then use 2 mL still)
* label 2 mL centrifuge tubes with protein name + E1/2/3/4 for capturing the eluted protein (each number stands for the nth elution)
* add 500 uL elution buffer and capture the elution liquid flowing out from the bottom using the corresponding centrifuge tube
* store the results within the 4°C refrigerator

**3.3 BCA Colorimetric Method for determining protein concentration of samples**

**Materials**

Pierce BCA Protein Assay Kit, samples from extraction and purification, 96 well plate, horizontal shaker, 37°C incubator, Microplate reader

**Procedures**

* Mix Solution A and Solution B in a 50:1 ratio to make 6mL of solution
* dilute the BSA within the kit for making 6 standard concentrations
* within 3\*9 wells, add 198 uL of the A+B solution mixture
* within 3\*6 wells, add 2uL each of standard concentration
* within the rest of the 3\*3 wells, add 2uL each of the samples (Pa, Pc, IL-10)
* shake 30 seconds to mix, then put into 37°C incubator for 30 minutes
* Using the microplate reader, read the wells' absorbance of 560 nm
* average the three readings of the three standards/samples of the same concentration
* Using Excel's linear regression function, calibrate a standard curve using the standards; if R>0.99, it could be used
* Using the equation determined above and the average absorbance of the samples, determine protein concentration of the samples

**3.4 SDS-PAGE verification of extracted proteins**

**Materials**

BBI 10% SDS-PAGE Color Preparation Kit (BBI C671102-0060), two 1.5 mm glass sandwich plates, two SDS-PAGE gel casting system, fume hood, large pipettor and pipette tips, centrifuge tube for mixing, 5X SDS-PAGE Protein Loading Buffer (Yeasen 20315E505),protein extracted from BL21 (without purification, from elution 1, from elution 2, from elution 3, and from elution 4),hot water bath set at 100°C,Beyotime Color Protein Ladder10-180kDa (Beyotime P0068), medium centrifuge, Coomassie blue (made by the lab), container for containing gel and Coomassie blue dye, horizontal shaker, Coomassie blue destaining solution (50% ddH2O + 40% MeOH + 10% acetic acid)

**Procedures**

* add 10 mL each of the two components of resolving gel (resolving gel solution and buffer) within the kit into a centrifuge tube, using the pipette to mix
* within the fume hood, add 100 uL of catalyst and mix through inverting the centrifuge tube
* add the mixture into the sandwich plates stuck within the casting system, add ethanol to keep the top of the mixture level, and wait for the gel to set
* add 4 mL of stacking gel solution and 4 mL stacking gel buffer, then add 40 uL catalyst* discard the ethanol and add gel solution, then insert the comb, and wait for the gel to set; at this point, two SDS-PAGE gels have been cast
  * in 40uL of protein samples extracted/eluted from BL21, add 10uL SDS-PAGE Protein Loading Buffer and mix
  * put the mixture into the 100°C water bath for 10 minutes
  * centrifuge at 1000g for 10 seconds to get all liquid on tube walls off
  * add 10 uL of protein ladder or sample
  * in Gel 1: 10uL Marker, control of Pa, E1 of Pa, E2 of Pa, E3 of Pa, E4 of Pa, Marker, Marker, control of Pc, E1 of Pc, E2 of Pc, E3 of Pc, E4 of Pc
  * in Gel 2: 10 uL Marker, IL-10 control, IL-10 E1, IL-10 E2, IL-10 E3, IL-10 E4, Marker
    1.run the gel at 70V for 30 minutes
    2.run the gel at 100V until the blue bands from the loading buffer reaches the bottom of the gel
    3.cut off the stacking gel and everything below the blue bands (including the blue bands), then transfer the gel into a container and add Coomassie Blue, put on horizontal shaker and check the gel repeatedly until the gel is visibly very dark and the protein bands are obvious
    4.add destaining solution, shake for 30 minutes, replace destaining solution, and repeat several times until the gel is fairly clear

**3.5 Western Blot verification of extracted protein**

**Materials**

BBI 10% SDS-PAGE Color Preparation Kit (BBI C671102-0060)
two 1.5 mm glass sandwich plates
two SDS-PAGE gel casting system
fume hood
large pipettor and pipette tips
centrifuge tube for mixing
5X SDS-PAGE Protein Loading Buffer (Yeasen 20315E505)
protein extracted from BL21 (without purification, from elution 1, from elution 2, from elution 3, and from elution 4)
hot water bath set at 100°C
Beyotime Color Protein Ladder10-180kDa (Beyotime P0068)
medium centrifuge
rocker
Western Blot buffers from Yeasen
Primary antibody: Anti-His Tag Monoclonal Antibody (SolarBio K200060M)
Secondary antibody: HRP-labelled Goat Anti-Mouse IgG (Beyotime A0216)
Pierce ECL Western Blotting Substrate (ThermoScientific)
**Procedures**

* add 10 mL each of the two components of resolving gel (resolving gel solution and buffer) within the kit into a centrifuge tube, using the pipette to mix
* within the fume hood, add 100 uL of catalyst and mix through inverting the centrifuge tube
* add the mixture into the sandwich plates stuck within the casting system, add ethanol to keep the top of the mixture level, and wait for the gel to set
* add 4 mL of stacking gel solution and 4 mL stacking gel buffer, then add 40 uL catalyst
* discard the ethanol and add gel solution, then insert the comb, and wait for the gel to set; at this point, two SDS-PAGE gels have been cast
* in 40uL of protein samples extracted/eluted from BL21, add 10uL SDS-PAGE Protein Loading Buffer and mix
* put the mixture into the 100°C water bath for 10 minutes
* centrifuge at 1000g for 10 seconds to get all liquid on tube walls off
* add 10 uL of protein ladder or sample
* add samples
* run the gel at 70V for 30 minutes
* run the gel at 100V until the dye front reaches the bottom of the gel
* cut out the gel and place it into transfer buffer
* prepare the transfer stack by stacking in order from black electrode to red electrode within a container filled with transfer buffer: sponge, filter paper, gel, PVDF membrane, filter paper, sponge; remove bubbles between gel and PVDF membrane to ensure that the protein is transferred properly
* put the transfer stack into the tank, add the transfer buffer, put in an ice pack to keep the system cool, run at 100V for 1 hr
  1.put membrane into TBST for 1~2 minutes to wash
  2.block the membrane to prevent non-specific protein binding by soaking into 5% skim milk in TBST and putting on rocker for 1 hr
  3.dilute the primary and secondary antibodies based on the instructions that came with them
  4.pour off the 5% skim milk and add the primary antibodies, rock for 1 hr
  5.recycle the primary antibodies, and wash membrane with TBST on rocker for 10 minutes, repeating three times
  6.add secondary antibody (that has HRP connected) and rock for 1 hr
  7.wash with TBST three times, 10 minutes each time
  8.add ECL substrate and rock for 5 minutes
  9.image the membrane

4. Functional verification

**4.1 Treatment of our protein at different temperatures**

**Materials**

Extracted proteins
Refrigerators (-80°C, -20°C, 4°C)
Incubator (37°C)
**Procedure**

* divide our protein samples into four equal parts, storing one part each in -80°C, -20°C, 4°C, and 37°C for 24 hours
* take out for use on cells

**4.2 Plating of mouse CD8+ T cells into 96 well plates**

**Materials:**

mouse CD8+ T cells (from IPHASE company)
proteins stored at different temperatures
cell culture plates
RPMI 1640
37°C incubator
96 well plate
**Procedure:**

* After thawing the cells, dilute cell suspension with culture medium and centrifuge, removing the supernatant; resuspend cells using fresh RPMI 1640 and incubate 24 hr in plate to let cells recover and grow
* Centrifuge and resuspend cells, then plate onto plate, incubate 24 hr
* Centrifuge and resuspend cells using 1mL T cell culture medium, take 10uL and dilute 10fold, count number of cells using hemocytometer and calculate cell density in original 1mL cell suspension
* Dilute cell suspension to 3\*10^6 cells/mL, add 100uL into the middle 60 wells in 96 well plates; for the remaining 36 outer wells, add 200uL PBS each
* Incubate 24 hours

**4.3 Incubation of our proteins with T cells**

**Material**

96 well plate with T cells
proteins treated at different temperatures (extracted IL-10, IL-18BPa, IL-18BPc)
IL-18
TNF-alpha
RPMI 1640
**Procedure**

* there are three variables in this experiment: protein type (IL-10, IL-18BPa, IL-18BPc), protein treatment temperature (-80, -20, 4, 37°C), concentration of protein (0, 0.5, 1.0, 1.5, 2.0 ug/mL). Three repeats were made for each, amounting to 180 wells (thus three plates) needed for the experiment.
* Based on the measured concentration of our extracted proteins, the appropriate amount of protein solution was added. Because our protein concentration was high, the amount of solution added was not large, and thus didn't affect volume much. 
* For the functional test of IL-18BPa and IL-18BPc, 20ng/mL of TNF-alpha and 100ng/mL IL-18 was added to T cell culture medium to create an inflammation environment.
* Cells were incubated for 24 hours with the proteins.

**4.4 ELISA to detect IFN-gamma levels of the T cells**

**Materials**

Yeasen Human IFN-gamma ELISA kit (all materials within the procedures are from here unless indicated otherwise)
ddH2O, pipettes, pipette tips
**Procedure**

* Centrifuge cells within wells and collect supernatant
* Mix 20X wash solution and ddH2O in 1:19 volume ratio; mix 5X dilution buffer and ddH2O in 1:4 volume ratio; leave them both to stabilize at room temperature before using (keep the bottles of reagents on ice)
* Dilute the detection antibody by 50 times, and enzyme-conjugated detection reagent by 20 times
* To produce the standard samples: add 1mL ddH2O into an unopened protein standard; label 8 EP tubes, one with B for blank and S1-S7 for different standard concentrations
* Add 300uL dilution buffer into blank and six other tubes; add 300uL of protein standard into S1; add 300uL protein standard into S2, mix, draw 300uL solution from S2 into S3, mix, then add 300uL S3 solution into S4...etc to produce the samples for making the standard curve
* Wash ELISA plate wells with 350uL wash solution, discard, then dry facedown by tapping lightly on paper towel
* For three rows of wells, add 100uL of B, S1~S7. The measurements from them will act as a standard for detection of IFN-gamma levels.
* In the rest of the wells, add the samples.
* Incubate (cover the wells) for two hours to allow IFN-gamma to bind to the stationary capture antibody
* Wash wells five times with wash solution
* Add 100uL of detection antibody and incubate another 2 hours
* Wash wells five times with wash solution
* Add 100uL detection conjugate and incubate 20 minutes
* Wash five times with wash solution
* Add 100uL substrate and incubate (avoid light) for 15 minutes
* Measure absorbance at 450nm wavelength light
* Using the standard samples to get the linear relationship between absorbance values and the concentration of IFN-gamma. Based on this relationship, the quantity of IFN-gamma in the tested samples can be caculated.

{% 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 %}

![](https://static.igem.wiki/teams/5522/desc-banner.png)

{{ self.title() }}

![](https://static.igem.wiki/teams/5522/cloud.png)

* Overview
* 1. Construction of Plasmid
* 2. Protein Expression
* 3. Verification of Protein Function
* Reference

Overview

The spread of Inflammatory Bowel Disease (IBD) in Asian countries due to the adoption of Western dietary habits have become a severe healthcare problem in recent years[1]. Target therapies that turn off specific inflammation-causing genes have been proven as a major treatment in IBD[2]. In this project, a drug that interferes with Interleukin-18 is designed and tested to provide a new possible solution of IBD treatment IL-18 binding protein (IL-18BP) could bind to IL-18 to stop its function[3] (Figure 1). In humans, while there are four types of IL-18BP (IL-18BPa, Pb, Pc, Pd), only IL-18BPa and IL-18BPc could antagonize IL-18 activity[4,5]. Plasmid containing IL-18 BP with SUMO for better stability, Fc for potential targeted drug delivery, and His tag for easier purification is constructed and tested, the protein is then expressed and purified through BL21 E.coli strands. The purified protein was validated using Western Blotting and its activity was confirmed through T cell activation inhibition experiments.

![](https://static.igem.wiki/teams/5522/result/1.png)

Figure 1. The IL18 signal pathway diagram3

1. Construction of Plasmid

**1.1 Double enzyme digestion of pET-28a vector and PCR amplified the target gene**

The pET-28α blank plasmid was cut by restriction enzymes to make them linear. Agarose gel electrophoresis was used to identify the restriction enzyme digestion product of the pET-28α blank plasmid. After the DNA was recovered from gel, the concentration and purity of the samples were then measured (Tab 1).

Tab 1. Concentration and purity of blank plasmid samples

| Experimenter | Concentration | 260/280 |
| --- | --- | --- |
| Annie | 135.4 µg/µL | 1.7 |
| Nicole | 58 µg/µL | 2.15 |
| Gloria | 69.2 µg/µL | 1.94 |
| Jonathan | 26 µg/µL | 2.13 |
| Wells | 79.4 µg/µL | 2.12 |

The length of target genes SUMO-IL-18BPa-Fc and SUMO-IL-18BPc-F are 1542bp and 1641bp, respectively. Figure 2 A showed that the length of the target gene was consistent with the electrophoresis results, indicating that the target gene was successfully amplified. Agarose gel electrophoresis was used to identify the restriction enzyme digestion product of the pET-28α blank plasmid. Figure 2.B shows that the plasmid was successfully digested.

![](https://static.igem.wiki/teams/5522/result/2.png)

Fig 2 Identification of PCR amplified gene and enzyme digested pET-28α vector. A. PCR product of SUMO-IL-18BPa-Fc and SUMO-IL-18BPc-Fc
B. Enzyme digestion product of pET-28α vector

**1.2 Construction and Extraction of Recombinant plasmids**

After restriction enzyme digest and ligation of pET-28a with Sumo-IL-18BP-Pa-Fc and Sumo-IL-18BP-Pc-Fc , the recombinant plasmids were transformed into E.coli DH5α competent (Fig 3A and B). The transformants were then identified by colony PCR. The agarose gel electrophoresis results showed that we obtained the expected length of PCR products, indicating the construction were successfully completed (Fig3 C).

![](https://static.igem.wiki/teams/5522/result/3.png)

Fig 3. The transformants form colonies on solid LB medium and colony PCR amplification of IL-18-BPa and IL-18-BPc.
A.pET-28α-Sumo-IL-BP-18-BPa-Fc-DH5α colony B.pET-28α-Sumo-IL-BP-18-BPc-Fc-DH5α colony. C. Verification of presence of Pa and Pc gene in DH5a transformants

**1.3 Plasmid extraction and sequencing**

The correct clones were cultured in LB medium, then we extracted plasmids for sequencing. The concentration and purity of plasmids were also measured (Tab 2).

Tab 2. Concentration and purity of extracted recombinant plasmid samples

| Sample | Concentration | 260/280 |
| --- | --- | --- |
| BPa (sample 1) | 97.95 ng/µL | over 2.1 |
| BPa (sample 2) | 144.2 ng/µL | over 2.1 |
| BPc (sample 1) | 90.65 ng/µL | over 2.1 |
| BPc (sample 2) | 126.6 ng/µL | over 2.1 |

Then the plasmid pET-28a with Sumo-IL-18BP-Pa-Fc and Sumo-IL-18BP-Pc-Fc were sent to the biological company for sequencing. The comparison of the sequencing results showed that the target gene sequence was consistent with the sequencing results (Fig 4A and B ), indicating that the plasmid was successfully constructed.

![](https://static.igem.wiki/teams/5522/result/4.png)

Fig 4. Gene sequencing of IL-18-BPa and IL-18-BPc.A. Gene sequencing of IL-18BPa showed no mutation has occurred. B.. Gene sequencing of IL-18BPc showed no mutation has occurred

2. Protein Expression

**2.1 protein induced expression**

We transformed the correct recombinant plasmids into E.coli BL21 competent cells to promote protein expression. The positive clones were selected out by kanamycin resistance in the solid LB medium.The BL21 colony cultures were expanded into large flasks for growth. After IPTG induction, the colonies were shaken overnight for protein production. The proteins were extracted then purified use the His tag.

At the same time, we transformed the pET28a-Sumo-IL10-Fc synthesized by the company into E.coli BL21 to promote protein expression as our subsequent positive control. In the fig 5 A, the agarose gel electrophoresis results showed that we obtained the expected length of sumo-IL10-Fc, indicating the construction were successfully completed. And the target gene sequence was consistent with the sequencing results(Fig. 5 B).

![](https://static.igem.wiki/teams/5522/result/5.png)

Fig 5. Gene sequencing and colony PCR amplification of sumo-IL10-Fc

**2.2 Protein verification after purification**

The purified IL18-BPa, IL18-BPc, and IL10-BP proteins were 56.9kDa, 61kDa, and 62.1kDa, respectively. The SDS-PAGE successfully verified the IL18-BPa, IL18-BPc, and IL10 proteins extracted and purified from E. coli BL21 (Fig 6).

![](https://static.igem.wiki/teams/5522/result/6.png)

Fig 6. SDS-PAGE verification of extracted proteins.
A, pET-28α-Sumo-IL-BP-18-BPa is yellow box, pET-28α-Sumo-IL-BP-18-BPc is red box, pET-28α-Sumo-IL-10 is blue box.

**2.3 Western Blot verification of extracted protein**

Compared to coomassie Brilliant Blue staining, the principle of Western detection is antibody antigen specific reaction, with high detection specificity. The proteins we expressed all carrying His tag, and specific His antibodies can be used to detect purified proteins. As shown in Figure 7, the protein size we obtained is consistent with the expected size, demonstrating successful protein expression.

![](https://static.igem.wiki/teams/5522/result/7.png)

Fig 7. Detection of recombination protein expression by western blot. From left to right: pET-28α-Sumo-IL-10, pET-28α-Sumo-IL-BP-18-BPa is yellow box, pET-28α-Sumo-IL-BP-18-BPc. The size of Sumo-IL-18BPa-Fc is about 56.9 kDa; the size of Sumo-IL-18BPc-Fc is 61 kDa.the size of Sumo-IL-10-Fc is 62.1 kDa.

**2.3 Use BCA colorimetric method to determine the protein concentration**

The concentration of the samples was measured using the BCA calorimetric method. We use standard concentration protein solution and BCA colorimetric method to determine the protein concentration. The absorbance value was measured by the enzyme-linked immunosorbent assay (ELISA) reader. We draw a scatter plot, insert a standard curve, and calculate the R-squared value.In the Fig.8, the fitting coefficient is greater than 0.99, indicating that the fitting effect is perfect.

![](https://static.igem.wiki/teams/5522/result/8.png)

Fig 8. Protein quantification using BCA colorimetric method.

The protein concentrations of Sumo-IL-18-BPa-Fc, Sumo-IL-18-BPc-Fc and Sumo-IL-10-Fc were calculated according to the standard curve, as shown in Tab 3.

Tab. 3 The concentrations of Sumo-IL-18-BPa-Fc, Sumo-IL-18-BPc-Fc and Sumo-IL-10-Fc protein

| Sample | Concentration |
| --- | --- |
| Sumo-IL-18-BPa-Fc | 2.649213 μg/mL |
| Sumo-IL-18-BPc-Fc | 2.451683 μg/mL |
| Sumo-IL-10-Fc | 2.5152 μg/mL |

3. Verification of Protein Function

**3.1 Measuring the standard curve using an INF-γ standard.**

In the experiment, we need the concentration of INF-γ, and we establish the standard curve of INF-γ.The R squared value is larger than 0.99, which indicates that the curve is accurate (Fig. 9 ).

![](https://static.igem.wiki/teams/5522/result/9.png)

Fig 9 INF-γ quantification using ELISA.

**3.2 The influence of dose and storage conditions on protein activity**

Store the three proteins (Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10) at different temperatures (-80, -20, 4, 37 ℃) for 24 hours and incubate them with mouse primary T lymphocytes. Stimulate the cells with IL-18 and detect the IFN-γ in the cell supernatant.

In Figure 10, the concentration of IFN-γ gradually decreased with the increase of Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 protein concentrations. It illustrates the inhibitory effect of recombinant Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 proteins on IFN-γ production.

The results are showed in Fig 10,the activity of protein is affected by temperature. The inhibitory effect of Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 on IFN-γwas not affected after 24 hours at -80 °C. After 24 hours at -20 °C and 4 °C, the inhibitory effect of Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc and Sumo-IL-10 proteins on IFN-γ decreased. After 24 hours at -37°C, the inhibitory effect of Sumo-IL-BP-18-BPc, Sumo-IL-BP-18-BPa and Sumo-IL-10 protein on IFN-γ was significantly decreased. Storing at low-temperature preservation is beneficial for the stability of recombinant proteins. Furthermore, the anti-inflammatory effect of recombinant proteins is dose-dependent.

![](https://static.igem.wiki/teams/5522/result/10.png)

Fig 10 The influence of dose and storage temperature on protein activity. Sumo-IL-BP-18-BPa, Sumo-IL-BP-18-BPc, and Sumo-IL-10A protein were stored at different temperatures, then were incubated with cells to determine the concentration of IFN γ in the cell supernatant.

In the fig 11, it can be seen that after storage at 4°C for 24 hours, IL-18BPc's effects are still comparable to that of IL-10 in the 2.0 ug/ml, while IL-18BPa is less stable.

![](https://static.igem.wiki/teams/5522/result/11.png)

Fig 11 The protein activity at 4°C. Ratio of IFN-gamma production levels after protein treatment vs control for IL-10 (B), IL-18BPa (C), IL-18BPc

Reference

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