iGEM 2023 — Team tu-eindhoven

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# **Notebook**

# **Notebook**

An overview of what we did in the lab each week to develop cELPro can be found here

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### Week 20

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### Week 21

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### Week 22

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### Week 23

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### Week 24

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### Week 25

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### Week 26

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### Week 27

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### Week 28

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### Week 29

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### Week 30

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### Week 31

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### Week 32

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### Week 33

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### Week 34

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### Week 35

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### Week 36

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### Week 37

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### Week 38

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### Week 39

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# **Engineering**

# **Engineering**

Here we go through two iterations of the iGEM engineering cylce, implementing all four stages of the cycle

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## Engineering success according to the iGEM cycle

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Engineering

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Cycle 1: Hydrogel

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Cycle 2: Therapeutic

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Learn

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Test

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Build

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Build

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Design

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# **Experiments**

# **Experiments**

Find our protocolbook with our biolab experiments here, designed to guide you through experiments with ease and accuracy

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Working in the lab during your iGEM journey can be both exciting and challenging. Ensuring you have all the necessary protocols in place can significantly improve your research experience. That's why we've put together an extensive bundle of Biolab protocols, made to support your bacterial laboratory experiments. Our Biolab protocols have been carefully designed to guide you through each experimental phase during iGEM with ease and accuracy.

You can download our protocolbook *[here](https://static.igem.wiki/teams/4905/wiki/biolab-protocols.pdf)*.

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In an attempt to impact the health sector and improve the quality of life of patients suffering from inflammatory bowel disease (IBD), cELPro was designed. As is described in more detail in the  [*Project Description*](https://2023.igem.wiki/tu-eindhoven/description), the purpose of this therapy is to make treatment of IBD more affordable, more specific, and less invasive. To this end, we created genetically engineered bacteria, which were inhibited from dividing due to in situ hydrogelation from block Elastin-Like Polypeptides (ELPs). In addition, they were still able to produce a target protein such as a therapeutic protein, IL-10, for the treatment of IBD or mNeonGreen, a model protein. This platform could be less infectious while still retaining the ability to produce drugs for the treatment.

# Achievements

![Image 1](https://static.igem.wiki/teams/4905/wiki/z2-a120-z2-10-g.jpeg)

We successfully **cloned** the genetic sequences encoding the self-assembling ELPs and  **expressed**  them for the designated  **hydrogel**  at different weight-to-volume percentages.

![Image 2](https://static.igem.wiki/teams/4905/wiki/od600-over-time-square.png)

We managed to form this hydrogel **inside the bacteria** which made them  **more resistant to ampicillin**  and furthermore  **inhibited the division**  of these cells, making them more controllable.

![Image 3](https://static.igem.wiki/teams/4905/wiki/achievement-frap.png)

Finally, we were able to express an  **ELP-mNeonGreen fusion**  protein inside the bacteria after formation of the hydrogel, indicating that the bacteria are still  **functional after intracellular gelation**.

**Scroll down** below to find a more elaborate reporting and discussion of the results from the different stages of the project.

## Cloning and Expression of ELPs

To make our bacteria more controllable we wanted to prevent them from dividing. We designed a sequence of ELPs that are able to form a hydrogel within the cells, and potentially inhibit bacteria division. We cloned these sequences into the appropriate vectors, expressed the proteins and subsequently characterized them.

**[Click me for results!](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27result-cloning%27%29%20%7D%7D)**

## Hydrogel

In this section, we show that we were able to form hydrogels of our constructs. We then test the properties and utilities of these gels with a variety of experiments. These include testing the diffusion rates, viability tests of the gelated bacteria and microscopy.

**[Click me for results!](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27result-hydrogel%27%29%20%7D%7D)**

## Therapeutic

As a proof of concept, we decided to express a therapeutic for the treatment of IBD. After speaking to several experts and stakeholders, and doing our own literature research, we found the cytokine IL-10 to be the best candidate for this purpose. Apart from expressing the protein, we tried to perform an immunoassay on it in order to quantify the expression levels.

**[Click me for results!](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27result-therapeutic%27%29%20%7D%7D)**

## Outlook

Our project has known its successes and failures. Both mean that there is still enough work that could be done to improve the platform. Here, we provide an overview of what we think the right next steps are for the project. We think that, with the right optimization, there is a lot of potential for the platform.

**[Click me for results!](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27result-outlook%27%29%20%7D%7D)**

If the boxes to the result pages do not appear, please click the links here: ***[Cloning](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27result-cloning%27%29%20%7D%7D)**

**[Hydrogel](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27result-hydrogel%27%29%20%7D%7D)**

**[Therapeutic](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27result-therapeutic%27%29%20%7D%7D)**

**[Outlook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27result-outlook%27%29%20%7D%7D)**

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