iGEM 2020 — Team NJU-China

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

# Team:NJU-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)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

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

# Team:NJU-China/Engineering

Title

[![logo](https://static.igem.org/mediawiki/2020/4/41/T--NJU-China--team_logo.jpg)](https://2020.igem.org/Team%3ANJU-China)

* [Home](https://2020.igem.org/Team%3ANJU-China)
* Project
  + [Description](https://2020.igem.org/Team%3ANJU-China/Description)
  + [Design](https://2020.igem.org/Team%3ANJU-China/Design)
  + [Results](https://2020.igem.org/Team%3ANJU-China/Results)
  + [Model](https://2020.igem.org/Team%3ANJU-China/Model)
  + [Implementation](https://2020.igem.org/Team%3ANJU-China/Implementation)
  + [Proof of Concept](https://2020.igem.org/Team%3ANJU-China/Proof_Of_Concept)
  + [Engineering](https://2020.igem.org/Team%3ANJU-China/Engineering)
  + [Contribution](https://2020.igem.org/Team%3ANJU-China/Contribution)
  + [Safety](https://2020.igem.org/Team%3ANJU-China/Safety)
* Parts
  + [Parts](https://2020.igem.org/Team%3ANJU-China/Parts)
* Human Practices
  + [Overview](https://2020.igem.org/Team%3ANJU-China/Human_Practices)
  + [Collaborations](https://2020.igem.org/Team%3ANJU-China/Collaborations)
  + [Education](https://2020.igem.org/Team%3ANJU-China/Education)
  + [Partnership](https://2020.igem.org/Team%3ANJU-China/Partnership)
  + [Art](https://2020.igem.org/Team%3ANJU-China/Art)
* Notebook
  + [Calendar](https://2020.igem.org/Team%3ANJU-China/Calendar)
  + [Protocol](https://2020.igem.org/Team%3ANJU-China/Protocol)
* Team
  + [Members](https://2020.igem.org/Team%3ANJU-China/Team)
  + [Attributions](https://2020.igem.org/Team%3ANJU-China/Attributions)
* Achievements
  + [Achievements](https://2020.igem.org/Team%3ANJU-China/Achievements)

### ENGINEERING

[PD-L1 siRNA overexpressed in HEK293T cell and exosome](#1)
[CD47 siRNA overexpressed in HEK293T cell and exosome](#2)
[Composite part](#3)
[KIBRA promotes exosomes release](#4)

This year we developed an alternative strategy to silence the Immune checkpoint therapy  by
employing exosome-mediated siRNA delivery. Silencing of immune checkpoints will allow tumor cells to
be attacked by the immune system.At the same time, we used iRGD-Lamp2b to achieve exosome targeting.

Particularly, we reprogrammed cells to simultaneously express siRNA and Lamp2b. Lamp2b is an
exosomal membrane protein, in fusion with a tumor-penetrating internalizing RGD (iRGD) peptide
(CRGDKGPDC), and then produce the tumor-targeting exosomes as  siRNA delivery system.

With this system, we can target tumor cells and deliver siRNA.

![](https://static.igem.org/mediawiki/2020/7/7d/T--NJU-China--eng1.png)

Figure 1. Basic parts and Composite parts

## PD-L1 siRNA overexpressed in HEK293T cell and exosome

Tumor cells evade immune system surveillance by overexpressing PD-L1. Therefore, we combined the
fusion expression of iRGD and Lamp2b to characterize the exosome targeting. At the same time, siRNA
targeting PD-L1 is produced to reduce mRNA expression. This allows the immune system to avoid the
effects of PD-L1 and attack tumor cells.

Lamp2b is an exosomal membrane protein, in fusion with a tumor-penetrating internalizing RGD (iRGD)
peptide (CRGDKGPDC), and then produce the tumor-targeting exosomes as siRNA delivery system.

We detected the high expression of iRGD-Lamp2b after transfection by RT-qPCR, which proved that our
parts are effective and provided assistance for the production of targeted exosomes (Figure1.).

![](https://static.igem.org/mediawiki/2020/9/98/T--NJU-China--en1.png)

Figure 2. iRGD-Lamp2b is overexpressed in HEK293T cell

We detected the correct expression of PD-L1 siRNA through RT-qPCR to ensure that
sufficient targeted siRNA could be produced in cell. CMV-iRGD-siRP is this part.
CMV-iRGD-siRP+C+K
is a Composite part.

![](https://static.igem.org/mediawiki/2020/8/84/T--NJU-China--t.png)

Figure 3. PD-L1 siRNA is overexpressed is HEK293T cell

At the same time, We detected siRNA in exosomes produced by HEK293T cells, confirmed
that siRNA could be properly wrapped into exosomes, and prepared for its role (Figure.2).

![](https://static.igem.org/mediawiki/2020/2/28/T--NJU-China--en3.png)

Figure 4. PD-L1 siRNA is overexpressed in exosome

![](https://static.igem.org/mediawiki/2020/2/2a/T--NJU-China--en4.png)

Figure 5. Absolute quantification of PD-L1 siRNA

According to the absolute quantification of PD-L1 siRNA, we found that the amount of siRNA in
HEK293T cells is 3.946E-4pM, the amount of siRNA in exosome is 0.4370E-4pM. The exosome siRNA is
equivalent to 11.07% of the siRNA concentration in the cell.

## CD47 siRNA overexpressed in HEK293T cell and exosome

Tumor cells escape macrophage phagocytosis by overexpressing CD47. Therefore, we
combined the fusion expression of iRGD and Lamp2b to characterize the exosome targeting.At the same
time, siRNA targeting CD47 was produced, and mRNA expression was targeted to be reduced.This allows
macrophages to avoid the effects of CD47 and attack tumor cells.

Then, we detected the correct expression of CD47 siRNA. CMV-iRGD-siRC is this part.
CMV-iRGD-siRP+C+K is a Composite part.

![](https://static.igem.org/mediawiki/2020/8/8a/T--NJU-China--en5.png)

Figure 6. CD47 siRNA is overexpressed is HEK293T cell

![](https://static.igem.org/mediawiki/2020/c/c1/T--NJU-China--en6.png)

Figure 7. CD47 siRNA is overexpressed in exosome

![](https://static.igem.org/mediawiki/2020/d/df/T--NJU-China--en7.png)

Figure 8. Absolute quantification of CD47 siRNA

At the same time, We detected siRNA in exosomes produced by HEK293T cells, confirmed
that siRNA could be properly wrapped into exosomes, and prepared for its role (Figure.2).

According to the absolute quantification of CD47 siRNA, we found that the amount of
siRNA in HEK293T cells is 5.710E-4 pM, the amount of siRNA in exosome is 0.2544E-09 pM. The exosome
siRNA is equivalent to 4.45% of the siRNA concentration in the cell.

## Composite part

After the normal expression of siRNA of KRAS,PD-L1 and CD47 in HEK293T cells and
exosomes was verified, the composite Part of three siRNA fusion was designed and each siRNA was
separately detected.

![](https://static.igem.org/mediawiki/2020/e/ea/T--NJU-China--eng5.png)

Figure 9. siRNA of Composite Part was overexpressed in HEK293T cells

![](https://static.igem.org/mediawiki/2020/d/d1/T--NJU-China--eng6.png)

Figure 10. siRNA of Composite Part was overexpressed in exosome

The above experimental results showed that the three types of composite siRNA were
strongly expressed in HEK293T cells and exosomes. This opens up the possibility of combining the
three siRNA as a treatment.

![](https://static.igem.org/mediawiki/2020/2/27/T--NJU-China--eng7.png)

Figure 11. Absolute quantitative of siRNA

After passing the absolute quantification, siRNA concentration in the Composite Part
was measured as shown in the table below.

|  | Intracellular siRNA Amount (10-4 pM) | Exosome siRNA Amount (10-4 pM) |
| --- | --- | --- |
| PD-L1 siRNA | 3.818 | 0.1627 |
| KRAS siRNA | 272.9 | 16.14 |
| CD47 siRNA | 49.55 | 2.544 |

As can be seen from the table, the amount of siRNA in exosomes was about 5% of the
amount in cells, which laid a foundation and prepared for siRNA to play its role through exosomes.

## KIBRA promotes exosomes release

At the same time, in order to promote exosome release and thus enhance the efficacy of
exosome therapy, we chose KIBRA as an exosome biological generator. By avoiding ubiquitination of
Rab27A, KIBRA stabilizes Rab27A and promotes exosome release.

The expression of each mRNA was confirmed by RT-qPCR after transfected to HEK293T
cells.

![](https://static.igem.org/mediawiki/2020/2/2a/T--NJU-China--engi1.png)

Figure 12. KIBRA mRNA relative expression in HEK293T cell (vs GADPH)

To further verify the expression of KIBRA at protein level, we used Western Blotting
experiment to prove that KIBRA was indeed overexpressed in HEK293T cells.

![](https://static.igem.org/mediawiki/2020/c/c9/T--NJU-China--engi2.png)

Figure 13. Western Blotting result shows that KIBRA is overexpressed in
HEK293T cell.

In order to further characterize the ability of KIBRA to promote exosome release, NTA
analysis was selected to characterize the diameter and number of exosomes produced by cells after
transfection with KIBRA Part. The results showed that KIBRA significantly promoted exosome release
compared with NC, while the diameter of exosomes did not change significantly. All this shows that
KIBRA can play a good role as an exosome biological generator.

![](https://static.igem.org/mediawiki/2020/1/11/T--NJU-China--res7.png)

Figure 14.(A) Averaged FTLA Concentration / Size for Exosome
(B) Intensity / Size graph for Exosome

Contact Us

School of Life Science, 163 Xianlin Road, Qixia District, Nanjing University, Jiangsu Province, China

181850128@smail.nju.edu.cn

Navigation

[Home](https://2020.igem.org/Team%3ANJU-China)
[Team](https://2020.igem.org/Team%3ANJU-China/Team)
[Projects](https://2020.igem.org/Team%3ANJU-China/Description)
[HP](https://2020.igem.org/Team%3ANJU-China/Human_Practices)
[Model](https://2020.igem.org/Team%3ANJU-China/Model)

![校徽](https://static.igem.org/mediawiki/2020/9/90/T--NJU-China--xiao_hui.jpg)

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# Team:NJU-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

* [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:NJU-China/Results

Title

[![logo](https://static.igem.org/mediawiki/2020/4/41/T--NJU-China--team_logo.jpg)](https://2020.igem.org/Team%3ANJU-China)

* [Home](https://2020.igem.org/Team%3ANJU-China)
* Project
  + [Description](https://2020.igem.org/Team%3ANJU-China/Description)
  + [Design](https://2020.igem.org/Team%3ANJU-China/Design)
  + [Results](https://2020.igem.org/Team%3ANJU-China/Results)
  + [Model](https://2020.igem.org/Team%3ANJU-China/Model)
  + [Implementation](https://2020.igem.org/Team%3ANJU-China/Implementation)
  + [Proof of Concept](https://2020.igem.org/Team%3ANJU-China/Proof_Of_Concept)
  + [Engineering](https://2020.igem.org/Team%3ANJU-China/Engineering)
  + [Contribution](https://2020.igem.org/Team%3ANJU-China/Contribution)
  + [Safety](https://2020.igem.org/Team%3ANJU-China/Safety)
* Parts
  + [Parts](https://2020.igem.org/Team%3ANJU-China/Parts)
* Human Practices
  + [Overview](https://2020.igem.org/Team%3ANJU-China/Human_Practices)
  + [Collaborations](https://2020.igem.org/Team%3ANJU-China/Collaborations)
  + [Education](https://2020.igem.org/Team%3ANJU-China/Education)
  + [Partnership](https://2020.igem.org/Team%3ANJU-China/Partnership)
  + [Art](https://2020.igem.org/Team%3ANJU-China/Art)
* Notebook
  + [Calendar](https://2020.igem.org/Team%3ANJU-China/Calendar)
  + [Protocol](https://2020.igem.org/Team%3ANJU-China/Protocol)
* Team
  + [Members](https://2020.igem.org/Team%3ANJU-China/Team)
  + [Attributions](https://2020.igem.org/Team%3ANJU-China/Attributions)
* Achievements
  + [Achievements](https://2020.igem.org/Team%3ANJU-China/Achievements)

### RESULTS

[Targeting Part](#1)
[RNAi Part](#2)
[Exosome booster part](#3)

In the Design section, we introduced the conception of the whole project. In order to realize our
idea, we rationally designed hierarchical genetic circuits that allows the verification of each
parts and the free combination of different functional modules. Then we confirm the expression and
function of each part in vitro and in vivo, followed by the confirmation of each system. After that,
we will package the whole system with into a lentivirus delivery system, of which the pathogenic
genes have been deleted. The lentivirus can effectively infect human cells and be reprogrammed into
a exosome production unit by continuous expression of the circuits. Eventually, we will test the
therapeutic effect of our strategy in KRAS mutation driven lung cancer mice model.

The design of each module is shown below.

![](https://static.igem.org/mediawiki/2020/d/db/T--NJU-China--res1.png)

According to our plan, this year we finished the verification of RNAi part, Exosome
Booster part and Targeting part. Next year, we will test the in vivo efficacy and therapeutic effect
of our strategy.

## Targeting Part

For the design of the targeting part, a sequence encoding a tissue-targeting tag was fused to the
N-terminus of Lamp2b (a canonical exosome membrane protein). Then the fused protein was inserted
downstream of the CMV promoter. Upon translation, this tag should be anchored to the exosome surface
via Lamp2b, thus guiding the delivery of the exosomal cargo to the desired tissue. In our project,
we chose the well-known homing peptide iRGD (CRGDKGPDC) as the targeting tag, which will penetrate
normal tissues and guide the exosomes binding to integrins specifically expressed on tumor
vasculature. We confirmed the expression of iRGD-Lamp2b in HEK293T cells by RT-qPCR, which proved
that our parts are effective. The targeting of iRGD-Lamp2b to lung cancer cells has been fully
studied, so we have sufficient reasons to believe that it can achieve a good target.

![](https://static.igem.org/mediawiki/2020/f/fd/T--NJU-China--Result7.png)

Contact Us

School of Life Science, 163 Xianlin Road, Qixia District, Nanjing University, Jiangsu Province, China

181850128@smail.nju.edu.cn

Navigation

[Home](https://2020.igem.org/Team%3ANJU-China)
[Projects](https://2020.igem.org/Team%3ANJU-China/Team.html)
[HP](https://2020.igem.org/Team%3ANJU-China/Human_Practices)
[Model](https://2020.igem.org/Team%3ANJU-China/Model)

![校徽](https://static.igem.org/mediawiki/2020/9/90/T--NJU-China--xiao_hui.jpg)