iGEM 2023 — Team squirrel-shanghai

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# Notebook

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## Part 1

## Part 2

## Part 3

## Part 4

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# Engineering Success

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Our project consists of three parts: metal ion response, metal ion binding, and the E. coli induced death. When
designing and testing these parts, we thought about a large number of practical and theoretical factors and
applied the DBTL cycle into our practical exercises.

The first is the metal ion response module. The process involved two DBTL cycles in which the mathematical
modeling gave us ample learning. We first constructed the plasmid for cadmium ion response.

**Why is this design necessary?**

Because we aim to develop a strain of Escherichia coli with heavy metal detection and recovery capabilities,
it is necessary for our strain to be able to respond to external heavy metal ions in order to achieve rapid
detection of heavy metal ion levels in agricultural land.

![](https://static.igem.wiki/teams/4698/wiki/engineering-success/img0.png)

![](https://static.igem.wiki/teams/4698/wiki/engineering-success/img1.png)

Gel Electrophoresis Results

![](https://static.igem.wiki/teams/4698/wiki/engineering-success/img2.png)

Verification of Bacterial PCR Gel Electrophoresis Results

We detected the intensity of YFP by microplate reader, which is induced by the concentration of cadmium ions in
the culture medium, so the fluorescence level of YFP can reflect the function of our metal detection module. Our
experimental results showed that the fluorescence intensity of YFP did increase in the presence of cadmium ions,
demonstrating that our system worked.

![](https://static.igem.wiki/teams/4698/wiki/engineering-success/img3.png)

Here, we implemented the first DBTL, i.e., we designed the cadmium ion-metal response module, constructed the
plasmid, tested it, and evaluated its effect.

Although it worked, we felt that this was not efficient enough. Then, we introduced mathematical modeling and
performed the second DBTL module.

Through reviewing a lot of literature, we learned that T7 RNA polymerase is expressed with high intensity, so we
hypothesized that we could use it to amplify the signal.

![](https://static.igem.wiki/teams/4698/wiki/engineering-success/img4.png)

As the picture above shown, the left side is the preset result without T7 polymerase for amplification, the right
side is the theoretical simulation result with T7 polymerase for signal amplification, and the vertical
coordinate is the YFP production. We could find that indeed more protein is produced per unit time, which
indicates that the strategy is feasible. Detailed results are shown in our modeling section. Therefore, we
designed and constructed the plasmid

![](https://static.igem.wiki/teams/4698/wiki/engineering-success/img5.png)

**The results of the microplate reader are as follows:**

![](https://static.igem.wiki/teams/4698/wiki/engineering-success/img6.png)

Obviously, the enhancement of fluorescence signal is higher after adding cadmium ions, indicating that our
design succeeded. Meanwhile, our modeling results also showed that the amplification effect of T7 will be
significantly
enhanced if the translation rate is appropriately increased, which also provides a direction for our future
improvement.

In conclusion, we applied two DBTL cycles in this part and realized our purpose.

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# Results

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