iGEM 2020 — Team SCU-WestChina

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# Team:SCU-WestChina/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:SCU-WestChina/Engineering

Collaborations

[![](https://static.igem.org/mediawiki/2020/thumb/8/87/T--SCU-WestChina--planD.png/798px-T--SCU-WestChina--planD.png)](https://2020.igem.org/Team%3ASCU-WestChina)

* [Home](https://2020.igem.org/Team%3ASCU-WestChina)
* [Project](https://2020.igem.org/Team%3ASCU-WestChina/Description)
  + [Description](https://2020.igem.org/Team%3ASCU-WestChina/Description)
  + [Design](https://2020.igem.org/Team%3ASCU-WestChina/design)
  + [Engineering](https://2020.igem.org/Team%3ASCU-WestChina/Engineering)
  + [Model](https://2020.igem.org/Team%3ASCU-WestChina/Model)
  + [Entrepreneurship](https://2020.igem.org/Team%3ASCU-WestChina/Entrepreneurship)
  + [Safety](https://2020.igem.org/Team%3ASCU-WestChina/safety)
  + [Implementation](https://2020.igem.org/Team%3ASCU-WestChina/Implementation)
* [HP](https://2020.igem.org/Team%3ASCU-WestChina/HP%20Overview)
  + [Human\_Practices](https://2020.igem.org/Team%3ASCU-WestChina/Human_Practices)
  + [Experts](https://2020.igem.org/Team%3ASCU-WestChina/experts)
  + [Enterprise](https://2020.igem.org/Team%3ASCU-WestChina/enterprise)
  + [Education](https://2020.igem.org/Team%3ASCU-WestChina/Education)
  + [Collaborations](https://2020.igem.org/Team%3ASCU-WestChina/Collaborations)
* [Parts](https://2020.igem.org/Team%3ASCU-WestChina/parts)
  + [Parts](https://2020.igem.org/Team%3ASCU-WestChina/parts)
  + [Contribution](https://2020.igem.org/Team%3ASCU-WestChina/Contribution)
* [Team](https://2020.igem.org/Team%3ASCU-WestChina/team)
  + [Team](https://2020.igem.org/Team%3ASCU-WestChina/team)
  + [Attributions](https://2020.igem.org/Team%3ASCU-WestChina/Attributions)
  + [Partnership](https://2020.igem.org/Team%3ASCU-WestChina/Partnership)
* [Notebook](https://2020.igem.org/Team%3ASCU-WestChina/note)
  + [Note](https://2020.igem.org/Team%3ASCU-WestChina/note)
  + [Protocols](https://2020.igem.org/Team%3ASCU-WestChina/protocols)
* [Awards](https://2020.igem.org/Team%3ASCU-WestChina/awards)
  + [Awards](https://2020.igem.org/Team%3ASCU-WestChina/awards)
  + [Criteria](https://2020.igem.org/Team%3ASCU-WestChina/criteria)

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## Categories

# Engineering

##### **Click the button in the upper right corner to open the directory menu**

#### In our project, we want to create and invent a highly targeted drug to overcome the shortcomings of current IPF treatment drugs, such as poor efficacy and strong side effects, in order to precisely target pulmonary fibrosis tissue, reverse the fibrosis process, and thus make IPF patients return to health.

To do this, we need a comprehensive understanding of the pathogenesis and important signaling pathways of IPF.
Through literature review and brainstorming, we first clarified that TGF-related signaling pathways play a vital role
in the development of fibrosis, and are very likely to be a breakthrough point in the treatment of IPF. We made our
goal clear. We need to block the cell signaling pathway that induces pulmonary fibrosis to avoid the increase of
fibroblasts and thereby inhibit the further deepening of pulmonary fibrosis.

We understand that fusion proteins are very suitable for this type of treatment. With reference to existing research
on the treatment of fibrotic diseases, we have designed a variety of fusion proteins targeting lung fibrosis regions
to block fibrosis signaling pathways. These proteins are made up of three main parts:

(1) The head is the fibrosis treatment-related domain. Here we consider using IPF truncated receptors to capture and
inactivate TGF-β;

(2) The tail is the domain which can recognise and target nidus to enhance the targeting of drugs;

(3) The rigid linker stabilizes the protein structure and separate two functional domains.

There are many choices for the peptide sequence of the head and tail. We need to read the literature and do
experiments to determine the optimal protein combination. Through literature review, we finally determined four
candidates for the head TGF-β truncated receptor: the domains are M7824, P144, TMED10 and an unnamed peptide; We used
HL5 as a rigid linker due to its good stability. At the same time, after extensively collecting and comparing the
collagen types of various fibrotic diseases (including scars, liver fibrosis, pulmonary fibrosis) and the
corresponding targeting methods, we selected six targeted probes. The picture below shows all possible combinations：

![](https://static.igem.org/mediawiki/2020/thumb/5/58/T--SCU-WestChina--Engineering1.png/1200px-T--SCU-WestChina--Engineering1.png)

In dry lab, we use the Z-DOCK model to obtain the parameters of four truncated receptors and perform Z-score
normalization on these parameters to compare receptors’ bind ability to TGF-β. The following result shows M7824 and
Unnamed have strong binding ability to TGF-β. Considering that M7824 is already a relatively mature clinical targeted
probe, we finally determined its choice as the head.

![](https://static.igem.org/mediawiki/2020/thumb/f/fd/T--SCU-WestChina--Engineering2.png/1200px-T--SCU-WestChina--Engineering2.png)

For probe targeting type I collagen, we used Z-DOCK and I-TASSER for molecular docking and stability experiments and
select three types of collagen-binding targeting probes with KD values of 0.01, 0.1, 10 as the candidates. These
probes need to be verified in wet experiments.

In wet lab, we realize the microcellular expression and prokaryotic expression of four fusion proteins (M7824-CILY,
M7824-SILY, M7824-Type4, M7824-Type4(reversed)). Firstly, we need a quick and easy way to rapidly produce a certain
amount of proteins for preliminary molecular verification test and cell verification test. We use cell-free protein
synthesis which is used firstly in iGEM competition. We designed special primers for template reconstruction and make
experiment to recover the glue with correct size of strip and then concentrate. The SDS-PAGE result is shown below to
verify their successful expression:

![](https://static.igem.org/mediawiki/2020/thumb/a/ae/T--SCU-WestChina--Engineering3.png/914px-T--SCU-WestChina--Engineering3.png)

Going further, we used *E.Coli (BL21)* with DH5-a and *E.Coli (BL21)* with pET28a to amplificate the
plasmids inserted with our sequence. The direct SDS-PAGE and SDS-PAGE after purification results are shown below to
verify their successful expression:

![](https://static.igem.org/mediawiki/2020/thumb/0/01/T--SCU-WestChina--Engineering4.jpg/1200px-T--SCU-WestChina--Engineering4.jpg.png)

Then, we need to build a cell model to simulate the lesion tissue of IPF. We successfully establish a pulmonary
fibrosis model with HFL-1 (treated by TGF-β1). After that, we

We successfully establish a pulmonary fibrosis model with HFL-1 (treated by TGF-β1) then set nine groups

and apply RT-q PCR to determine whether their epithelial-mesenchymal transition (EMT) process was blocked with the
level of collagen I.

![](https://static.igem.org/mediawiki/2020/thumb/b/bb/T--SCU-WestChina--Engineering5.png/1200px-T--SCU-WestChina--Engineering5.png)

![](https://static.igem.org/mediawiki/2020/thumb/8/85/T--SCU-WestChina--Engineering6.png/1200px-T--SCU-WestChina--Engineering6.png)

![](https://static.igem.org/mediawiki/2020/thumb/2/2c/T--SCU-WestChina--Engineering7.png/1200px-T--SCU-WestChina--Engineering7.png)

It can be obviously refered from the cell morphology that, the HEL-1 cells differentiated into myofibroblasts in the
TGE-beta-treated group while this process was depressed in the drug-treated group.

To get more evidence to prove our cell model and drug treatment success, we used RT-qPCR to get more evidence in gene
expression degree.

We can see that it occurs expression degree of collagen I differences between three groups though the difference is
not that ovious. But we are making more drug concentration comparisons to detect the differences more obviously, and
we believe that these experiments will provide guidance for later SPR experiments and animal experiments in the
future.

![](https://static.igem.org/mediawiki/2020/thumb/9/92/T--SCU-WestChina--Engineering8.png/1200px-T--SCU-WestChina--Engineering8.png)

Here are the five steps from research to testing. Design2&3 are the next steps for learning and improvement.

## Collagen degeneration in fibrosis focus

Taking into account that the above treatment module can only delay the condition of IPF, but cannot reverse the
lesion area. We considered designing a plan to restore normal function of the tissue in the lesion area. To this end,
we focused on myofibroblasts. We hope to design drugs to properly degrade collagen to improve tissue compliance and
restore normal tissue function. We knew that plasmin has been used in the treatment of thrombosis and ss a proteolytic
enzyme, plasmin is also promising in applications in terminal pulmonary fibrotic diseases. In consideration of
controlling side effects, we did not choose plasminogen activating peptides with enzymatic activity, but chose
peptides with carboxyl terminal lysines. Among the peptides with carboxyl terminal lysines, the α2-antiplasmin
C-terminal 26 peptide was selected. We chose a relatively mild activating peptide with dual effects and a highly
selective collagen targeting peptide to control the scope of effect by enriching the drug in the lesion. We hope to
reverse the lesion tissue of IPF patients by designing the above-mentioned fusion protein for administration.

## Drug Delivery System Design

Through investigations in the hospital, we learned about the huge side effects of traditional IPF treatment drugs
(such as nintedanib and pirfenidone), and we realized that the side effects of our fusion protein drugs are also a
potential hidden danger. Although the targeted nature of the drug can limit side effects to a large extent, the harm
to the body when the drug is metabolized in the body is still inevitable. In this regard, doctors suggest that we
should consider to explore the inhibitory effects of different administration methods on side effects, and use
liposomes to make sustained-release drug formulations to increase the utilization of drugs and reduce side effects. In
response, we designed a pharmacokinetic experiment. By establishing a PBPK model, we can theoretically simulate the
metabolism of drugs in the body.

We analyzed the metabolism of peptide drugs in animal models and humans in the two treatment modules, and used the
physiological pharmacokinetic (PBPK) model prediction software GastroPlusTM to predict all peak concentration-time
(Cp-time) curves. We focus on the metabolism of peptide drugs through intravenous administration and pulmonary
administration, as well as the influence of the presence or absence of liposomes on drug metabolism. We use the second
treatment module--the activation peptide (AP26 and SAK) involved in the degradation of collagen in fibrotic lesions as
the research object.

We first established a PBPK model for intravenous administration, and the results are as follows:

![](https://static.igem.org/mediawiki/2020/thumb/3/33/T--SCU-WestChina--Engineering9.png/795px-T--SCU-WestChina--Engineering9.png)

Furthermore, we established a PBPK model for pulmonary administration as a comparison. The PK curve is as follows:

![](https://static.igem.org/mediawiki/2020/thumb/b/bc/T--SCU-WestChina--Engineering10.png/870px-T--SCU-WestChina--Engineering10.png)

Our results prove that after lung administration, the deposition of the drug in the lungs is significantly increased
and the bioavailability is higher, suggesting that lung administration is a better choice than intravenous
administration.

In order to predict the effect of liposome delivery methods on drug metabolism, we established a release curve model
of sustained-release preparations based on transdermal administration and predicted the PK curve. The simulation
results show that the exposure of AP26 and SAK sustained-release preparations administered percutaneously in rats and
humans is significantly increased, and their bioavailability basically reaches 100%.

The simulation results of pharmacokinetics gave us a lot of encouragement, and the results characterized the
advantages of the drug delivery methods in our design, helped us understand the metabolism of drugs in the body and
helped us to screen better drugs. After discussing with the Professor Zhenlei Wang, we knew that we can design in
vitro experiments and animal experiments to optimize the parameter settings of the pharmacokinetic model, so that we
can understand the metabolism of the drug in the body more accurately.

## Categories

[![](https://static.igem.org/mediawiki/2020/thumb/9/9f/T--SCU-WestChina--footer1.png/800px-T--SCU-WestChina--footer1.png)](http://sklb.scu.edu.cn/)

## Contact us

1 Keyuan 4th Road, Gaopeng Avenue,Chengdu, Sichuan 610041,China

* Phone:  +86 028 85164088
* Email: SCuWestChina@163.com

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# Team:SCU-WestChina/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:SCU-WestChina/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)