iGEM 2022 — Team lzu-hs-pro-b


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{% block title %}Engineering Success{% endblock %}
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![](https://static.igem.wiki/teams/4301/wiki/covers/engi.png)

[Our initial design](#a1)

[Design Stage](#b1)

[Build Stage](#b2)

[Test Stage](#b3)

[Visible light examination](#b4)

[Range](#b5)

[Time](#b6)

[Learn stage](#a2)

[Our amplification system](#a3)

[Design Stage](#b7)

[Build Stage](#b8)

[Test Stage](#b9)

> Our initial design

Design Stage

![](https://static.igem.wiki/teams/4301/wiki/engi/img1.png)

> Learn stage

When analyzing the outcomes of our experiment， we noticed that the sensitivity of the mere sense system and the luminous system is too low. It was found that the signal was hardly large enough for the actual application. Therefore, we also need to devise another way to amplify the signal of uric acid and render larger results to be detected.

We noticed the importance of enhancing the visibility of our system, ie, amplifying the signals released by RFP protein. The natural HRP regulatory network contained the activating proteins HrpR and HrpS. The two would form a hypersensitive high-order co-composite HrpRS. This has been shown to amplify the input transcriptional signal.

> Our amplification system

Design Stage

  In this way, we considered the architecture of transduced signal amplifier based on the HRP regulation system in order to achieve this demand. Promoter LacUV5, genes downstream of which will be transcribed in response to IPTG activation. HrpR and hrpS are the genes encoding the activator protein PhrpL, a promoter induced by hypersensitive high-order co-complex HrpRS. The ribosome binding site was B0030, and the terminator was B0015. Red fluorescent protein was used as our reporter protein.

![](https://static.igem.wiki/teams/4301/wiki/engi/img5.png)

Build Stage

We obtained the gene fragments we designed through DNA synthesis.

-First, the plasmid was extracted for PCR amplification. Then double digestion of PCR fragments and plasmids, nucleic acid electrophoresis, and gel recovery experiment were carried out.

-The PCR fragments and plasmids digested successfully were recovered. The target gene and plasmid were linked to transforming the linked product.

-After the screening, the plasmid was successfully constructed.

-Prepare receptor cells, and transform the engineering vector into the expression host E.coli BL21.

Test Stage

For the experimental results, it was evidence presented that the perceptive rate of uric acid greatly increased after we added the amplification system into our experiment. We have discovered and concluded the general rule that the amplification system we designed and added was able to amplify the signal by a factor of four, which enabled us to better sense and detect the amount of uric acid in our bodies.

![](https://static.igem.wiki/teams/4301/wiki/engi/img6.png)

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