iGEM 2019 — Team LZU-CHINA

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# Team:LZU-CHINA/Notebook

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Daily life~

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* [Team](https://2019.igem.org/Team%3ALZU-CHINA/Team)
  + [Team Members](https://2019.igem.org/Team%3ALZU-CHINA/Team)
  + [Attributions](https://2019.igem.org/Team%3ALZU-CHINA/Attributions)
  + [Collaborations](https://2019.igem.org/Team%3ALZU-CHINA/Collaborations)
* [Project](https://2019.igem.org/Team%3ALZU-CHINA/Description)
  + [Description](https://2019.igem.org/Team%3ALZU-CHINA/Description)
  + [Design](https://2019.igem.org/Team%3ALZU-CHINA/Design)
  + [Safety](https://2019.igem.org/Team%3ALZU-CHINA/Safety)
  + [Notebook](https://2019.igem.org/Team%3ALZU-CHINA/Notebook)
* [Parts](https://2019.igem.org/Team%3ALZU-CHINA/Parts)

  + [Parts Overview](https://2019.igem.org/Team%3ALZU-CHINA/Parts)
  + [Basic Parts](https://2019.igem.org/Team%3ALZU-CHINA/Basic_Part)
  + [Improved Parts](https://2019.igem.org/Team%3ALZU-CHINA/Improved_Part)
  + [Composite Parts](https://2019.igem.org/Team%3ALZU-CHINA/Composite_Part)
* [Results](https://2019.igem.org/Team%3ALZU-CHINA/Results)
  + [Results](https://2019.igem.org/Team%3ALZU-CHINA/Results)
  + [Protocols](https://2019.igem.org/Team%3ALZU-CHINA/Results/Protocols)
* [Model](https://2019.igem.org/Team%3ALZU-CHINA/Model)
* [HP](https://2019.igem.org/Team%3ALZU-CHINA/Human_Practices)
  + [Human Practices](https://2019.igem.org/Team%3ALZU-CHINA/Human_Practices)
  + [Public Education](https://2019.igem.org/Team%3ALZU-CHINA/Public_Engagement)
  + [Entrepreneurship](https://2019.igem.org/Team%3ALZU-CHINA/Entrepreneurship)

![](https://static.igem.org/mediawiki/2019/d/dc/T--LZU-CHINA--projectbg.png)

Notebook

Five magic books~

## May 2019

---

### Abstract

The working laboratory began, and the tasks were scheduled.

### Laboratory

In order to verify the protein-protein interaction between our two target proteins, we used laser energy polarization method. We fused the donor protein CFG and the receptor protein YFG with the two target proteins respectively, and determined whether the two proteins interacted by measuring the loss of fluorescence intensity of CFP. After replacing the medium with fluorescent imaging medium, the imaging begins about 2 hours, and then the image is acquired every 1 hour. We observed the change of SW1990 cells from blue to yellow under 440 nanometer excitation light, which demonstrated the interaction between our two target proteins.

### Human Practice

The Human Practice Committee has been delineated, brainstorming has been carried out and the best ideas have been collected so that they can be realized smoothly in the near future.

## June 2019

---

### Abstract

Wiki page planning started and we contacted the graphic designer for help. We also contacted professors of oncology to gain insights into the project. The hardware and mathematic model are preliminarily designed.

### Laboratory

In order to verify the role of ALKBH5 in migration and creeping of pancreatic cancer cells, scratch experiments were carried out to verify the effect. We altered ALKBH5 by siRNA knockdown and over-expression plasmid (wild-type/mutant) and then examined the migration and creeping ability of pancreatic cancer cells. The migration and crawling ability of siALKBH5 group was stronger than that of Ctrl group (blank control group), P < 0.01. The migration and crawling ability of ALKBH5-WT group was weaker than that of Ctrl group (blank control group), P < 0.01, which indicated that ALKBH5 inhibited the migration and crawling of pancreatic cancer cells. For the accuracy of the experimental results, we conducted Transwell test. AlKBH5 was also altered by siRNA knockdown and over-expression plasmid (wild-type/mutant) to detect the invasion and migration ability of pancreatic cancer cells. The experimental results were the same as scratch test.

### Human Practice

Team leader organizes and plans, tasks have been distributed.

## July 2019

---

### Abstract

Poster design and Wiki page content are under way. The final determination of mathematical model and hardware.

### Laboratory

ALKBH5 plays an inhibitory role in Wnt/beta catenin signaling pathway. In order to know how ALKBH5 inhibits the Wnt/beta catenin signaling pathway, four important proteins (beta-catenin, p-GSK-3beta, GSK-3beta, beta-tubulin) and ALKBH5 were Western blotted. ALKBH5 was used as independent variable, high expression and low expression were set as independent variables, and four important proteins were used as dependent variables to observe the changes of their respective expression levels. Our results showed that up-regulation of ALKBH5 expression in SW1990 pancreatic cancer cells significantly decreased the expression of beta-catenin, p-GSK-3 beta protein in Wnt/beta-catenin signaling pathway, and the total expression of GSK-3 beta protein remained unchanged. These results indicate that up-regulation of ALKBH5 can enhance the activity of GSK-3 beta from to promote the phosphorylation of beta-catenin, leading to the phosphorylation of beta-c-catenin in pancreatic cancer cells. Atenin decreased and inhibited Wnt/beta catenin signaling pathway.

### Human Practice

Team members publicize projects in schools, governments, drug institutions and hospitals respectively, and popularize knowledge about synthetic drugs to the masses.

## August 2019

---

### Abstract

Wiki pages are constantly improving. The team participated in the 6th China International Genetically Engineered Machinery Competition (iGEM) Team Exchange Meeting CCiC, and communicated with the participating teams.

### Laboratory

In order to understand the induction effect of Hif-1, we used CopGFP to test it. The experimental results show that the fluorescence intensity of CopGFP increases with the increase of CoCl (II) concentration. At 500 uM concentration, the fluorescence intensity decreased significantly. This is the result of high concentration of CoCl (II) inhibiting cell activity. Similar effects were observed in SW1990 cells.

## September 2019

---

### Abstract

The Wiki page has been preliminarily completed.

### Laboratory

In order to demonstrate that Casp3 can cause death of pancreatic cancer cells by randomly cutting nucleosomes under hypoxia, we conducted a hypoxia-sensing system experiment.

## October 2019

---

### Abstract

All plans have been completed.

### Laboratory

In order to observe this phenomenon, we use fluorescence microscopy for microscopic examination because different cells can produce different degrees of apoptosis under different conditions.

![](https://static.igem.org/mediawiki/2019/e/ee/T--LZU-CHINA--xiaohui.png)Lanzhou University

![](https://static.igem.org/mediawiki/2019/c/c8/T--LZU-CHINA--jiaowuchu.png)Academic Affair Office of LZU

![](https://static.igem.org/mediawiki/2019/4/41/T--LZU-CHINA--shengming.png)School of Life Science , LZU

![](https://static.igem.org/mediawiki/2019/d/df/T--LZU-CHINA--yiyuan.png)The First Hospital of LZU

Contact Us

E-mail：1746921258@qq.com 752786236@qq.com

Address：No. 222, Tianshui South Road, chengguan district, Lanzhou City, Gansu Province,730000, P. R. China.

Copyright ©2019 iGEM LZU-CHINA Team All rights reserved.


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# Team:LZU-CHINA/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.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### 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://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:LZU-CHINA/Results

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* [Team](https://2019.igem.org/Team%3ALZU-CHINA/Team)
  + [Team Members](https://2019.igem.org/Team%3ALZU-CHINA/Team)
  + [Attributions](https://2019.igem.org/Team%3ALZU-CHINA/Attributions)
  + [Collaborations](https://2019.igem.org/Team%3ALZU-CHINA/Collaborations)
* [Project](https://2019.igem.org/Team%3ALZU-CHINA/Description)
  + [Description](https://2019.igem.org/Team%3ALZU-CHINA/Description)
  + [Design](https://2019.igem.org/Team%3ALZU-CHINA/Design)
  + [Safety](https://2019.igem.org/Team%3ALZU-CHINA/Safety)
  + [Notebook](https://2019.igem.org/Team%3ALZU-CHINA/Notebook)
* [Parts](https://2019.igem.org/Team%3ALZU-CHINA/Parts)

  + [Parts Overview](https://2019.igem.org/Team%3ALZU-CHINA/Parts)
  + [Basic Parts](https://2019.igem.org/Team%3ALZU-CHINA/Basic_Part)
  + [Improved Parts](https://2019.igem.org/Team%3ALZU-CHINA/Improved_Part)
  + [Composite Parts](https://2019.igem.org/Team%3ALZU-CHINA/Composite_Part)
* [Results](https://2019.igem.org/Team%3ALZU-CHINA/Results)
  + [Results](https://2019.igem.org/Team%3ALZU-CHINA/Results)
  + [Protocols](https://2019.igem.org/Team%3ALZU-CHINA/Results/Protocols)
* [Model](https://2019.igem.org/Team%3ALZU-CHINA/Model)
* [HP](https://2019.igem.org/Team%3ALZU-CHINA/Human_Practices)
  + [Human Practices](https://2019.igem.org/Team%3ALZU-CHINA/Human_Practices)
  + [Public Education](https://2019.igem.org/Team%3ALZU-CHINA/Public_Engagement)
  + [Entrepreneurship](https://2019.igem.org/Team%3ALZU-CHINA/Entrepreneurship)

![](https://static.igem.org/mediawiki/2019/8/86/T--LZU-CHINA--wetlabbg.png)

Results

Facts speak louder than words~

[Metastasis System](#l1)
[Infiltration System](#l3)
[Delivery System](#l2)
[Basic Progress](#l4)

## Metastasis System

---

### Sensor verification

![](https://static.igem.org/mediawiki/2019/e/e5/T--LZU-CHINA--result1.png)

Imaging begins approximately 2 hours after the media is replaced with a fluorescent imaging medium. Get images every 1 hour. SW1990 cells were observed using 440 nm excitation light, and the cells turned from cyan to yellow.

### ALKBH5

#### Scratch test to verify transfer ability

![](https://static.igem.org/mediawiki/2019/f/fc/T--LZU-CHINA--res13.png)
![](https://static.igem.org/mediawiki/2019/2/2d/T--LZU-CHINA--res14.png)

In the pancreatic cancer cell line SW1990, we modified ALKBH5 by siRNA knockdown and overexpression plasmids and performed a scratch test to detect the migration and crawling ability of pancreatic cancer cells.

A and D are blank control groups. B and E overexpress the ALKBH5 cell scratch assay; panels C and F are knockdown ALKBH5 cell scratch assays.

The results showed that the migration ability of the siALKBH5 (knockdown expression) group was stronger than that of the Ctrl group (blank control group), p<0.01. The migration ability of the ALKBH5-WT (overexpression) group was higher than that of the Ctrl group (blank control group).

#### Transwell experiment to verify invasion ability

![](https://static.igem.org/mediawiki/2019/f/f6/T--LZU-CHINA--res15.png)
![](https://static.igem.org/mediawiki/2019/7/73/T--LZU-CHINA--res16.png)

In the pancreatic cancer cell line SW1990, we modified ALKBH5 by siRNA knockdown and overexpression plasmids and Transwell to detect the invasive ability of pancreatic cancer cells.

Ctrl is the blank control group, ALKBH5-WT is the ALKBH5 overexpression group, and siALKBH5 is the knockdown expression group.

The results showed that the invasive ability of ALKBH5-WT group was weaker than that of Ctrl group, p<0.01; the invasive ability of siALKBH5 group was stronger than that of Ctrl group, p>0.01.

#### WB experiment verification action pathway

![](https://static.igem.org/mediawiki/2019/3/3b/T--LZU-CHINA--res17.png)
Western blot analysis showed that the expression levels of β-catenin and p-GSK-3β in Wnt/β-catenin signaling pathway were up-regulated in the pancreatic cancer cell SW1990 compared with the control group (3xFlag-Vector). Significantly decreased, the total GSK-3β protein expression was unchanged, this result indicates that up-regulation of ALKBH5 can enhance the activity of GSK-3β and promote the phosphorylation of β-catenin, resulting in a decrease in intracellular β-catenin and inhibition of Wnt/ --catenin signaling pathway.

## Infiltration System

---

### Hypoxia induction system

We tested the induced expression of pHRE with CopGFP. CoCl(II) is used to produce anoxic conditions. The above figure shows the effect of pHRE on HEK 293T cells. The experimental results show that the fluorescence intensity of CopGFP increases with the increase of CoCl(II) concentration. At a concentration of 500 uM, the fluorescence intensity decreased significantly. This is the result of inhibition of cell viability by high concentrations of CoCl(II). A similar effect was observed in SW1990 cells. In addition, the fluorescence intensity of SW1990 is usually lower than that of HEK293T, which may be due to the poor expression efficiency of SW1990 as a differentiated cell.

![](https://static.igem.org/mediawiki/2019/c/c3/T--LZU-CHINA--res31.png)
![](https://static.igem.org/mediawiki/2019/8/89/T--LZU-CHINA--res32.png)

### Casp3 system

#### WB experiment verification mechanism

The picture shows the DNA electrophoresis photo. Apoptosis caused by activation of casp3, which is randomly cleaved between nucleosomes and degraded into 200 bp or an integral multiple thereof. Since the nuclear membrane is intact during DNA extraction, there is almost no DNA in the extract of normal cells, and the DNA in the apoptotic cell of apoptotic cells is extracted, and the DNA molecules of 200 bp in length or their integral multiples are electrophoresis. Strips.

![](https://static.igem.org/mediawiki/2019/7/7e/T--LZU-CHINA--res33.png)

#### Fluorescence microscopy for cell death

Different cells can produce different degrees of apoptosis under different conditions. Under the fluorescence microscope, the normal cells stained with Hochest33258 showed a uniform blue color, while the apoptotic cells showed dense dense stained particles with a cluster-like distribution.

![](https://static.igem.org/mediawiki/2019/a/a4/T--LZU-CHINA--res34.png)

#### Flow cytometry to detect the degree of apoptosis

![](https://static.igem.org/mediawiki/2019/e/ed/T--LZU-CHINA--res35.png)
![](https://static.igem.org/mediawiki/2019/d/d9/T--LZU-CHINA--res36.png)
![](https://static.igem.org/mediawiki/2019/4/4c/T--LZU-CHINA--res37.png)

Fig A: blank control, under high oxygen concentration, sw1990 cell status is normal cells

Fig B: blank control, under low oxygen concentration, sw1990 cell status is normal cells

Fig C: Negative control, at high oxygen concentration, after binding to empty exosomes, the sw1990 cell status is normal cells.

Fig D: Negative control, at low oxygen concentration, after binding to empty exosomes, the sw1990 cell status is normal cells.

Fig E: In the experimental group, after high oxygen concentration, after binding to the exosomes after transformation, the state of sw1990 cells was more early apoptosis.

Fig F: In the experimental group, after the combined oxygen concentration in the medium, the sw1990 cell status was more early apoptosis.

Fig G: In the experimental group, after low-oxygen concentration, after binding to the exosomes after transformation, the sw1990 cell status was more late apoptosis.

## Delivery System

---

Exosomes (Fig. A), sw1990 (Fig. B), under confocal microscopy, it can be seen that the PKH26-labeled exosome showing red fluorescence is mainly located in the cytoplasm of sw1990, distributed in the perinuclear region, and most of the cells are visible. To the red fluorescent signal (Fig.C)

![](https://static.igem.org/mediawiki/2019/4/48/T--LZU-CHINA--res21.png)

NTA analysis refers to nanoparticle tracking analysis. The experimental results showed that the number of particles of HEK293T cells overexpressing exosome booster at 100 nm and 250 nm was significantly higher than that of control cells (about 23 times that of control cells). This suggests that exosome potentiators can increase the number of exosomes secreted by 293T cells.

![](https://static.igem.org/mediawiki/2019/9/9c/T--LZU-CHINA--res22.png)

## Basic Progress

---

![](https://static.igem.org/mediawiki/2019/d/d9/T--LZU-CHINA--res43.png)
![](https://static.igem.org/mediawiki/2019/2/2e/T--LZU-CHINA--res42.png)

Fig.A: AND logic gate. The reporter was used at a concentration of 150 ng, the fluorescence value was measured at 515 nm, and the ordinate was the normalized fluorescence intensity value. The and logic gate pathways show that when TEVP and TVMVP are present alone, the fluorescence value at 515 nm is very low, and when both are present, the fluorescence value is high, indicating that both proteins are required to function in the pathway.

Fig.B: OR logic gate. The reporter was used at a concentration of 150 ng, the fluorescence value was measured at 515 nm, and the ordinate was the normalized fluorescence intensity value. The or logic gate pathway shows that when TEVP and TVMVP are present alone, the fluorescence value at 515 nm is high, and when both are present, the fluorescence value is very low, indicating that two proteins are required to function in the pathway alone.

Fig C: NOR logic gate. The reporter was used at a concentration of 150 ng, and the fluorescence value was measured at 515 nm. The norm gate showed that the ordinate was the normalized fluorescence intensity value. When TEVP and TVMVP are present, the fluorescence value at 515 nm is very low, and when both are absent, the fluorescence value is high, indicating that it is necessary to have two proteins at the same time in the pathway to function.

![](https://static.igem.org/mediawiki/2019/e/ee/T--LZU-CHINA--xiaohui.png)Lanzhou University

![](https://static.igem.org/mediawiki/2019/c/c8/T--LZU-CHINA--jiaowuchu.png)Academic Affair Office of LZU

![](https://static.igem.org/mediawiki/2019/4/41/T--LZU-CHINA--shengming.png)School of Life Science , LZU

![](https://static.igem.org/mediawiki/2019/d/df/T--LZU-CHINA--yiyuan.png)The First Hospital of LZU

Contact Us

E-mail：1746921258@qq.com 752786236@qq.com

Address：No. 222, Tianshui South Road, chengguan district, Lanzhou City, Gansu Province,730000, P. R. China.

Copyright ©2019 iGEM LZU-CHINA Team All rights reserved.