iGEM 2022 — Team sesame-shenzhen

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CONTENTS

[I. Preparation](#a1)
[II. Plans and procedures](#a1)
[III. Experimental Records](#a1)

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I. Preparation

During the preparation stage we have listed down the materials we will need for the experiment, including extraction kits, enzymes and carriers, apparatus and consumables. All kits we used for the experiemnt are products of TIANGEN company.

![](https://static.igem.wiki/teams/4299/wiki/note/lb1.jpg)
![](https://static.igem.wiki/teams/4299/wiki/note/lb2.jpg)
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II. Plans and procedures

We planned to finish our laboratory work within 5 weeks, including the time needed for repeating the experiment in case there is an error. We have listed down tasks that are required to be finished for every week and also wrote step-by-step experiment protocols.

![](https://static.igem.wiki/teams/4299/wiki/note/lb4.jpg)
![](https://static.igem.wiki/teams/4299/wiki/note/lb5.jpg)
![](https://static.igem.wiki/teams/4299/wiki/note/lb6.jpg)
![](https://static.igem.wiki/teams/4299/wiki/note/lb7.jpg)
![](https://static.igem.wiki/teams/4299/wiki/note/lb8.jpg)

III. Experimental Records

The picture below is a brief outline for our experiment. It also includes real photo record taken in the lab and explanation about those photos' significance.

![](https://static.igem.wiki/teams/4299/wiki/note/lb9.jpg)

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CONTENTS

[Generation 1](#a1)
[Design:](#b1)
[Build:](#b2)
[Test:](#b3)
[Learn:](#b4)
[Generation 2](#a2)
[Design:](#b5)
[Build:](#b6)
[Test:](#b7)
[Learn:](#b8)

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![](https://static.igem.wiki/teams/4299/wiki/engi/eng-1.png)

Incorporating the engineering thought process is central to every design, including synthetic biology. This process is repeated until a biological system with the desired function is identified. This incremental and iterative approach enables engineering despite our (often largely) incomplete understanding of many biological processes. This process is essential to successfully incorporate and implement our project. Thus we followed the process and iterated 2 repeats of the engineering cycle.

The first generation/repeat of the cycle, we produced a simple E.coli Nissle 1917 introduced with Urate Oxidase.

Generation 1

Design:

During the first generation of our bacteria, our sole purpose was to introduce urate oxidase into a e.coli.

Build:

To build the model of our bacteria, we inserted the Uricase plasmid into DH5alpha e.coli for testing. (More details can be found at lab notes)

Test:

We verified the ability of the production and catalytic effect of the enzyme under intentinal conditions. The final results were satisfactory and proved the effectiveness of our bacteria.

We constructed E.coli 1917 with only urate oxidase as the experimental group, and transferred an empty plasmid into another strand as the control group.

![](https://static.igem.wiki/teams/4299/wiki/engi/eng-3.png)

Final results proved the functionality of the urate oxidase enzyme.

More testing was also performed on other factors influencing the efficacy of Urate Oxidase, such as the temperature and pH. More details can be found at the Proof of concepts page.

Learn:

We learned that only introducing an enzyme may not be enough to ensure a safe implementation in the human body (please see Integrated Human practices - CCIC to see details) as there is a risk of GM contamination.

Generation 2

Design:

We redesigned our genetic circuit and introduced a dynamic regulation system detecting uric acid concentration, linking the expression of uricase and the suicide protein together.

![](https://static.igem.wiki/teams/4299/wiki/engi/eng-4.png)

More details on how this system works can be found at the project description page.

Build:

We constructed the bacteria.

Test:

We introduced both systems, the Uric acid promoter and the catabolic enzyme, into E.coli nissle 1917, and simulated the conditions inside the gut.

![](https://static.igem.wiki/teams/4299/wiki/engi/eng-5.png)

Experimental conditions:

* pH of 7
* The temperature of 37 degrees Celsius
* Hypoxia (hypoxic bag used)
* Add bile salts
* Pancreatic juice
* Liquid LB medium
* Adjust the uric acid concentration to 2000 μM.

Experimental Process:

Co-culture the bacteria with the above materials.

Co-incubate for 48 hours, incubate at 37°C on a shaker at 180 rpm

Test for uric acid concentration

![](https://static.igem.wiki/teams/4299/wiki/engi/eng-6.png)

The final degradation rate of the control group was about 10%, and the experimental group was about 45%, proving the functionality of our system.

Learn:

We could have implemented a dynamic population control system based on signaling molecules when we attended the conference with 嘉亿生物, Jiayi research institute.

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