iGEM 2021 — Team LZU-CHINA

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:LZU-CHINA/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:LZU-CHINA/Engineering

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Look! Our engineering~

(°▽°)

![](https://static.igem.org/mediawiki/2021/thumb/4/42/T--LZU-CHINA--logo-new.png/320px-T--LZU-CHINA--logo-new.png)

* [Home](https://2021.igem.org/Team%3ALZU-CHINA)
* [Project](https://2021.igem.org/Team%3ALZU-CHINA/Project)
  + [Description](https://2021.igem.org/Team%3ALZU-CHINA/Description)
  + [Design](https://2021.igem.org/Team%3ALZU-CHINA/Design)
  + [Proof of concept](https://2021.igem.org/Team%3ALZU-CHINA/Proof_Of_Concept)
  + [Model](https://2021.igem.org/Team%3ALZU-CHINA/Model)
  + [Future](https://2021.igem.org/Team%3ALZU-CHINA/Future)
  + [Engineering](https://2021.igem.org/Team%3ALZU-CHINA/Engineering)
  + [Implementation](https://2021.igem.org/Team%3ALZU-CHINA/Implementation)
* [HP](https://2021.igem.org/Team%3ALZU-CHINA/Human_Practices)
  + [Integrated Human Practices](https://2021.igem.org/Team%3ALZU-CHINA/Human_Practices)
  + [Education&Communication](https://2021.igem.org/Team%3ALZU-CHINA/Communication)
* [Team](https://2021.igem.org/Team%3ALZU-CHINA/Team)
  + [Members](https://2021.igem.org/Team%3ALZU-CHINA/Team)
  + [Attributions](https://2021.igem.org/Team%3ALZU-CHINA/Attributions)
  + [Partnership](https://2021.igem.org/Team%3ALZU-CHINA/Partnership)
  + [Collaborations](https://2021.igem.org/Team%3ALZU-CHINA/Collaborations)
  + [Contribution](https://2021.igem.org/Team%3ALZU-CHINA/Contribution)
* [Parts](https://2021.igem.org/Team%3ALZU-CHINA/Parts)
  + [Parts](https://2021.igem.org/Team%3ALZU-CHINA/Parts)
  + [Improvement](https://2021.igem.org/Team%3ALZU-CHINA/Improvement)
* [Safety](https://2021.igem.org/Team%3ALZU-CHINA/Safety)

![](https://static.igem.org/mediawiki/2021/2/2d/T--LZU-CHINA--enginnering.png)

**[Part.1](#E1)**
**[Part.2](#E2)**
**[Part.3](#E3)**
**[Part.4](#E4)**

---

## 1. Constructed HEK293T-hACE2 stable cell line

To construct SARS-CoV-2 pseudovirus’ target cell, first we constructed the
transfer plasmid pLENTI-ACE2-PURO in virto. We then introduced it with packaging
plasmids into HEK293T cells. Then we screened HEK293T-hACE2 stable cell line using
purinomycin, but we have difficulty in screening. To screen the stable cell line
easier, we designed a part [BBa\_K3760004](http://parts.igem.org/Part%3ABBa_K3760004)
which contains T2A and mRFP to replace
ACE2 coding sequence in pLENTI-ACE2-PURO.

![](https://static.igem.org/mediawiki/2021/b/b4/T--LZU-CHINA--3-6Engineering_Fig1.png)

Fig.1. (a)pLENTI-ACE2-PURO; (b)The Western Blot of ACE2

---

## 2. Constructed the SARS-CoV-2 pseudovirus

We transfected pCMV14-3X-Flag-SARS-CoV-2 S, pLenti-CMV-GFP-Puro (658-5),
psPAX2 into HEK293T cells and packaged the pseudovirus. Then We harvested
the virus suspension and stored at -80°C. We also used Western Blot
to make sure the Spike protein was expressed.

![](https://static.igem.org/mediawiki/2021/4/43/T--LZU-CHINA--3-6Engineering_Fig2.png)

Fig.2. The Western Blot result of Spilke protein

---

## 3. Constructed and characterized a new part：**BBa\_K3760001** 6x His tag-*Rfx*Cas13d

### 3.1 Cloning

We ordered *Rfx*Cas13d gene from Addgene, then we cloned the gene into pET-28a.
Additionally, we attached a 6 x His tag to 5' end of the gene. We transformed the
ligation product into *E.coli* Rosetta (DE3) and confirmed the cloning by colony-PCR
and sequencing.

![](https://static.igem.org/mediawiki/2021/e/e9/T--LZU-CHINA--3-6Engineering_Fig3.png)

Fig.3. Colony PCR result of 6xHis *Rfx*Cas13d in *E.coli* Rosetta.
Note: Lane 3 is the positive clone

### 3.2 *Rfx*Cas13d protein expression and purification

We expressed the protein in *E.coli* Rosetta (DE3).
Expressed it at 16°C in LB medium under the condition of 1 mM IPTG.
And as the sequence contains 6×His tags,
we purified the protein through Ni-NTA agarose.

![](https://static.igem.org/mediawiki/2021/9/91/T--LZU-CHINA--3-6Engineering_Fig4.png)

Fig.4. The *Rfx*Cas13d purification result. 1.empty vector supernatant 2.empty vector precipitation 3.uninduced supernatant 4.uninduced precipitation 6.Inducted supernatant 7.Inducted precipitation 1 8.Inducted supernatant 9.Inducted precipitation

### 3.3 Characterization: Test the cleavage activity of *Rfx*Cas13d protein

After we got the expressed and purified *Rfx*Cas13d protein, we needed to
verify its activity to make sure our CRISPR-Cas13d system can target the
coding RNA of ACE2. We tested the kinetic curve of the protein to see the
dynamic change of the flourescence. We can clearly see that after adding
the target, the activity of the protein is greatly improved, which enables
us to tell whether the coding RNA of ACE2 was splited. After 100 mins of
detecting, the fluorescence signal gradually became stable.

We analysed it on the model page, click [>>Model<<](https://2021.igem.org/Team%3ALZU-CHINA/Model) to see more details.

![](https://static.igem.org/mediawiki/2021/7/7e/T--LZU-CHINA--3-6Engineering_Fig6.png)

Fig.5. Cleavage activity of *Rfx*Cas13d in virto.(a)The fluorescence signals of target RNA(A,B,C) and control(D,E,F,G) were detected on real-time PCR.(b) The cleavage activity of *Rfx*Cas13d were tested with ssRNA reporter. Control: Without target RNA. Error bars represent the means ± SD from three replicates (n = 3)

---

## 4. Constructed the lentivirus carried CRISPR-Cas13d system to knock down ACE2

We constructed the plasmid pLenti-sgRNAs-*Rfx*Cas13d-PURO to express *Rfx*Cas13d and sgRNAs which is
desgined to knock down the expression of ACE2 in HEK293T-ACE2 cells. This vector and two
packaging plasmids were co-transfected into cells.

![](https://static.igem.org/mediawiki/2021/3/35/T--LZU-CHINA--321.png)

Fig.6. pLenti-sgRNAs-*Rfx*Cas13d-PURO

Thorugh RT-PCR, we detected that the expression level of ACE2 has degraded significantly.
In the simulated infection of SARS-CoV-2 pseudovirus, we used flow cytometry (LSRFortessa, BD, USA)
to measure the fluorescence intensity of all samples. The results exhibited that the knockdown of ACE2
receptor decreased the infection level of pseudovirus in cells. This system can prevent HEK293T-ACE2
cells from the infection of SARS-CoV-2 pseudovirus.

![](https://static.igem.org/mediawiki/2021/6/66/T--LZU-CHINA--2-3_POC_Fig20.png)

Fig.7. Analysis of CRISPR-Cas13d system targeting ACE2 on the infection of SARS-CoV-2 pseudovirus

![](https://static.igem.org/mediawiki/2021/8/89/T--LZU-CHINA--logo_lzu.png)
Lanzhou University

![](https://static.igem.org/mediawiki/2021/5/59/T--LZU-CHINA--1_home_snapgene_logo.png)
SnapGene

![](https://static.igem.org/mediawiki/2021/b/b6/T--LZU-CHINA--logo_shengkeyuan.png)
School of Life Science , LZU

![](https://static.igem.org/mediawiki/2021/3/34/T--LZU-CHINA--Fig9.png)
addgene

Contact Us

E-mail: 1981846330@qq.com

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

Copyright ©2021 iGEM LZU-CHINA Team All rights reserved.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# 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.

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

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:LZU-CHINA/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.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)