iGEM 2020 — Team NOFLS_YZ

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# Team:NOFLS YZ/Notebook

非模块化方式使用layui

Entprenuership

![](https://static.igem.org/mediawiki/2020/7/74/T--NOFLS_YZ--logo.jpeg)

**Counter Sugar**

---

* [Home](https://2020.igem.org/Team%3ANOFLS_YZ)
* Project

  :   [Description](https://2020.igem.org/Team%3ANOFLS_YZ/Description)
  :   [Design](https://2020.igem.org/Team%3ANOFLS_YZ/Design)
  :   [Experiments](https://2020.igem.org/Team%3ANOFLS_YZ/Experiment)
  :   [Notebook](https://2020.igem.org/Team%3ANOFLS_YZ/Notebook)
  :   [Result](https://2020.igem.org/Team%3ANOFLS_YZ/Result)
  :   [Safety](https://2020.igem.org/Team%3ANOFLS_YZ/Safety)
* [Contribution](https://2020.igem.org/Team%3ANOFLS_YZ/Contribution)
* [Engineering](https://2020.igem.org/Team%3ANOFLS_YZ/Engineering)
* [Implementation](https://2020.igem.org/Team%3ANOFLS_YZ/Implementation)
* Parts

  :   [Basic Parts](https://2020.igem.org/Team%3ANOFLS_YZ/Basic%20Parts)
  :   [Composite Parts](https://2020.igem.org/Team%3ANOFLS_YZ/Composite%20Parts)
  :   [Parts Collection](https://2020.igem.org/Team%3ANOFLS_YZ/Parts%20Collection)
* [Model](https://2020.igem.org/Team%3ANOFLS_YZ/Model)
* Human Practice

  :   [Integrated Human Practice](https://2020.igem.org/Team%3ANOFLS_YZ/Human_Practices)
  :   [Fundraising](https://2020.igem.org/Team%3ANOFLS_YZ/Fundraising)
  :   [Entrepreneurship](https://2020.igem.org/Team%3ANOFLS_YZ/Entrepreneurship)
* Team

  :   [Team Member](https://2020.igem.org/Team%3ANOFLS_YZ/Team)
  :   [Collaborations](https://2020.igem.org/Team%3ANOFLS_YZ/Collaborations)
  :   [Attributions](https://2020.igem.org/Team%3ANOFLS_YZ/Attributions)

Notebook

Day 1: Plasmid transformation

In molecular biology, vector is a artificial medium which carries exotic genetic material from one cell to another in order to later express and replicate. There are four major kinds of vectors: plasmids, viral vectors, cosmids, and artificial chromosomes; plasmids are the most common kind. Some characteristics define a vector, such as an origin of replication (where the vector can replicate themselves autonomously inside its host, possessing a compatible site for genetic material, having the ability to clone in great amount, and retain genetic markers which help to distinguish recombinant cells from non-recombinant cells.

Plasmids, the vector chosen in this experiment, are generally circular, dual-standard DNA molecules and are independent from chromosomal DNA of any given cell because they have own origin of replication. Plasmids can not survive out of its host and it can not only replicate independently, but also add genetic information to its host which known as transformation.

When cells are placed in a 0 degrees Celsius CaCl2 solution, E. coli bacteria would become more permeable to the plasmid DNA. Then, after the cells are placed on ice along with the DNA. Then, after the cells are placed on ice along with the DNA, the solution would get heat-shocked at 42 degrees Celsius for about 2 minutes. This would promote the absorbance of the genetic material. The plasmid would then give the bacteria resistance towards a specific type of antibiotics, which could be used in the screening process.

Aim:

The aim of this experiment is to transfer plasmids（hFXRα,FXRE,SV40,RXRα） to competent cells in order to greatly amplify plasmids.

Day 2: Plasmid amplification

Day 3: plasmid extraction

Aim

The aim of this experiment is to amplify the plasmid DNA via the shaking incubator, extract the plasmid DNA from the other proteins that might be present in the solution, and purify the solution in order to isolate the plasmid DNA.

Day 4: Cell recovery and culture

Aim:

Culture HEK293 cells for 24 hours, prepare for cells transfection.

Day 5: Amplification, Extraction and purification of plasmid

Measurement:

In this experiment, the method of alkali cracking was used to prepare plasmids in small quantity. In the narrow range of pH between 12.0 and 12.5, the linear DNA double helix structure was unraveled and denatured, while the hydrogen bond of covalent closed-loop plasmid DNA would break, but the two complementary chains were coiled around each other and still tightly bound together. When ph4.8 acid potassium acetate was added to reduce the pH value of the solution, the plasmid DNA was rapidly recovered, while the chromosome DNA was large and difficult to be recovered. By centrifugation, most cell fragments, chromosome DNA, RNA and protein will be precipitated (under the action of SDS) to remove, while plasmid DNA remains in the supernatant. The purified plasmid DNA can be obtained by cation column adsorption and enrichment, then precipitation with isopropanol and washing with ethanol.

Result:

A large number of receptor cells and plasmids were obtained and successfully extracted

Day 6: Identification of plasmids by agarose gel electrophoresis

Measurement:

Different DNA, different molecular weight and different configuration, the mobility of electrophoresis is different (the migration rate is inversely proportional to the logarithm of molecular weight), so as to distinguish different zones, namely molecular sieve effect. Other factors affecting mobility include gel concentration, voltage and pH.

Nucleic acid banding observation is to use the combination of nucleic acid and nucleic acid dye, and the color development under ultraviolet light, with the aid of ultraviolet observation instrument.

Result:

It is proved that the molecular weight of the target DNA is equivalent to the theoretical value（hFXRα,FXRE,SV40,RXRα）.

Day 7: Protein expression and purification

Day 8: AlphaScreen-Based Protein-Peptide Interaction Assay

Day 9: Cell transient transfection and reporter gene experiment

![](https://static.igem.org/mediawiki/2020/6/6e/T--NOFLS_YZ--notebook01.jpg)

Contact Email: 3142451726@qq.com

Wechat: Search "2020 IGEM CounterSugar" in WeChat

School Website: https://www.xdfyz.cn/

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:NOFLS YZ/Engineering

非模块化方式使用layui

Entprenuership

![](https://static.igem.org/mediawiki/2020/7/74/T--NOFLS_YZ--logo.jpeg)

**Counter Sugar**

---

* [Home](https://2020.igem.org/Team%3ANOFLS_YZ)
* Project

  :   [Description](https://2020.igem.org/Team%3ANOFLS_YZ/Description)
  :   [Design](https://2020.igem.org/Team%3ANOFLS_YZ/Design)
  :   [Experiments](https://2020.igem.org/Team%3ANOFLS_YZ/Experiment)
  :   [Notebook](https://2020.igem.org/Team%3ANOFLS_YZ/Notebook)
  :   [Result](https://2020.igem.org/Team%3ANOFLS_YZ/Result)
  :   [Safety](https://2020.igem.org/Team%3ANOFLS_YZ/Safety)
* [Contribution](https://2020.igem.org/Team%3ANOFLS_YZ/Contribution)
* [Engineering](https://2020.igem.org/Team%3ANOFLS_YZ/Engineering)
* [Implementation](https://2020.igem.org/Team%3ANOFLS_YZ/Implementation)
* Parts

  :   [Basic Parts](https://2020.igem.org/Team%3ANOFLS_YZ/Basic%20Parts)
  :   [Composite Parts](https://2020.igem.org/Team%3ANOFLS_YZ/Composite%20Parts)
  :   [Parts Collection](https://2020.igem.org/Team%3ANOFLS_YZ/Parts%20Collection)
* [Model](https://2020.igem.org/Team%3ANOFLS_YZ/Model)
* Human Practice

  :   [Integrated Human Practice](https://2020.igem.org/Team%3ANOFLS_YZ/Human_Practices)
  :   [Fundraising](https://2020.igem.org/Team%3ANOFLS_YZ/Fundraising)
  :   [Entrepreneurship](https://2020.igem.org/Team%3ANOFLS_YZ/Entrepreneurship)
* Team

  :   [Team Member](https://2020.igem.org/Team%3ANOFLS_YZ/Team)
  :   [Collaborations](https://2020.igem.org/Team%3ANOFLS_YZ/Collaborations)
  :   [Attributions](https://2020.igem.org/Team%3ANOFLS_YZ/Attributions)

Overview

Type 2 diabetes (T2DM) is the most common type of diabetes, accounting for around 90% of all diabetes cases. China's diabetic patients rank second in the world, and more than nine hundred thousand people die of diabetes every year, only after cancer and stroke. Moreover, At present, there are still many problems with drugs on the market. Therefore, the research has a great market prospect. Our team aims to build a platform for screening potential small molecular compounds to therapy T2DM. We have testified our platform could working well and screen the potential drugs for T2DM therapy.

Research and Design

Farnesoid X receptor (FXR) belongs to a member of the nuclear receptor Superfamily [3] and has been reported to be associated with glucose metabolism. For example, in FXR−/− mice, the level of hepatic gluconeogenic gene expression was decreased after fasting, while blood glucose level and the hepatic glucose production in response to pyruvate challenge were declined [4]. However, the effect and mechanism of FXR regulating glucose metabolism in vivo are not clear. Thus, we design a platform which can screen a small molecule FXR antagonist as a probe to explore the mechanism of FXR regulating glucose metabolism.

![](https://static.igem.org/mediawiki/2020/c/c3/T--NOFLS_YZ--engineering01.jpg)

![](http://www.cmaj.ca/content/cmaj/172/2/213/F2.large.jpg?download=true)

Build and Test

Part BBa\_K3522001 contains the DNA sequence of the FXR ligand-binding domain (FXR-LBD) , which was cloned to the pET-15b vector and transferred to the BL21 (DE3) competent cells. The transformed cells were inoculated into fresh LB medium for recombinant FXR-LBD production. When the optical density of the culture reached 0.6, IPTG at a final concentration of 0.2 mM was added. The expression cells were harvested by centrifugation and broken by sonication on ice. Proteins in the supernatant was purified by Ni-NTA column using an AKTA FPLC instrument according to the protocol. The protein production and purification were analyzed by SDS-PAGE. As shown in Figure 1, recombinant FXR-LBD was purified to homogeneity.

![](https://static.igem.org/mediawiki/2020/1/15/T--NOFLS_YZ--engineering02.jpg)

Figure 1. SDS-PAGE analysis of recombinant FXR-LBD production and purification. Land 1, 2, 9: protein molecular weight standards. Lane 3: the insoluble proteins in expression cells. Lane 4: the soluble proteins in expression cells. Lane 5: the proteins in the flow through of Ni-NTA column. Lane 6 and 7: eluting fractions of FXR-LBD.

Application of recombinant FXR-LBD for FXR antagonist screening

FXR antagonists screened in Lab in-house compound library by AlphaScreen technology-based assay.

Alphascreen technology is used for in vitro high-throughput detection of protein interactions. In the system we constructed, we used the ability of 6×His-FXR-LBD protein and Biotin-SRC1 small peptide to bind to each other, and added Nickel chelate acceptor beads and Streptavidin donor beads. When protein and small peptide are combined with each other, the distance between the two magnetic beads is close. Under the irradiation of excitation light with wavelength of 680 nm, the photosensitizer on the donor magnetic beads converts the surrounding oxygen into monomer oxygen, and the monomer oxygen diffuses to the receptor. The bulk magnetic beads activate the fluorescent groups on the acceptor magnetic beads. Furthermore, the intensity of the interaction between the protein and the small peptide was characterized by detecting the fluorescence value at the wavelength of 520-620 nm. During the operation, we dissolve 6×His-FXR-LBD protein and SRC1 small peptide on ice, and dilute to a certain concentration with assay buffer (25 mM Hepes, pH 7.4, 100 mM NaCl and 0.1% BSA). Take a white 384 ELISA plate, add 100 nM 6×His-FXR-LBD protein, 30 nM SRC1, 0.5 μM GW4064 (FXR agonist), the selected compound, and 10 μg/mL acceptor beads to each well. Afterwards, shake the plate at 300 rpm for 10 min and incubate at room temperature for 30 min. Then add the final concentration of 10 μg/mL Donor beads 300 rpm shaker for 10 minutes, and incubate for 2 hours at room temperature in the dark. Then use PerkinElmer machine to check the fluorescence value.

As indicated in Figure 2A and 2B, compound H7 antagonized the GW4064-induced promotion of FXR-LBD binding to its coactivator SRC-1. These results thus implied the antagonistic feature of H7 against FXR.

![](https://static.igem.org/mediawiki/2020/0/0f/T--NOFLS_YZ--engineering03.jpg)

Figure 2. H7 as an FXR antagonist inhibited FXR activity. The effect of compounds (20μM) on the combination of FXR-LBD and SRC-1 was detected by AlphaScreen-based protein-peptide interaction assay. A. Among the compounds, H7 was finally selected for its highly antipathogenic activity against FXR. B. H7 antagonized GW4064-induced AlphaScreen signal. All data were presented as mean ± S.E.M (\*P<0.05, \*\*P< 0.01, \*\*\*P< 0.001).

Learn, research and improvement

According to the recent articles, the FXR typical functional domains include DNA binding domain and ligand-binding domain. After binding its ligand, FXR forms a heterodimer with another nuclear receptor retinoid X receptor, and then the receptor dimer binds to the FXR response element (FXRE) located in the promoter region of the FXR target gene, thereby, regulating the transcription of these genes. Therefore, we design an improved platform working in the mammalian cell HEK293T to screen the potential drugs for T2DM therapy.

Design, Build and Test

Part BBa\_K3522013 consists of a FXRE fragment and luciferase gene. When presence of FXR and RXRα, the FXRE is activated, subsequently, the luciferase gene is transcribed. Such a transactivation assay cam be used to verify the antagonistic effect of H7 on FXR.

![](https://static.igem.org/mediawiki/2020/1/11/T--NOFLS_YZ--engineering04.jpg)

Figure 1. H7 as an FXR antagonist inhibited transactivation activity.

HEK293T cells were transiently transfected with pcDNA3.1a-FXR, pcDNA3.1a-RXRα, pGL3-FXRE-Luc and pRL-SV40. After 6 h, cells were treated with different concentrations of H7 with GW4064 for 24 h. Then transactivation activity of FXR was detected by luciferase reporter assay. GW4064: FXR agonist, GS: FXR antagonist. All data were presented as mean ± S.E.M (\*P<0.05, \*\*P< 0.01, \*\*\*P< 0.001).

Finally, transactivation assay was further carried out to verify the antagonistic effect of H7 on FXR. As shown in Figure 3, FXR agonist GW4064 efficiently activated reporter gene expression, and H7 antagonized GW4064-induced reporter gene stimulation in transactivation assay. Thus, these results confirmed the antagonism of H7 against FXR transactivation activity.

References

[1] Chatterjee S, Khunti K, Davies MJ. Type 2 diabetes. Lancet 2017;389:2239–51.

[2] Rines AK, Sharabi K, Tavares CD, Puigserver P. Targeting hepatic glucose metabolism in the treatment of type 2 diabetes. Nat Rev Drug Discov 2016;15:786–804.

[3] Lee FY, Lee H, Hubbert ML, Edwards PA, Zhang Y. FXR, a multipurpose nuclear receptor. Trends Biochem Sci 2006;31:572-80.

[4] Renga B, Mencarelli A, D'Amore C, Cipriani S, Baldelli F, Zampella A, et al. Glucocorticoid receptor mediates the gluconeogenic activity ofthe farnesoid X receptor in the fasting condition. FASEB J 2012;26:3021-31.

Contact Email: 3142451726@qq.com

Wechat: Search "2020 IGEM CounterSugar" in WeChat

School Website: https://www.xdfyz.cn/

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:NOFLS YZ/Experiments

# Experiments

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

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

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:NOFLS YZ/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

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

### Describe what your results mean

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

### Project Achievements

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

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

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)