iGEM 2022 — Team navarra-bg


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{% block title %}Engineering Success{% endblock %}
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## Project's objective

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Our objective is to use plants as CO2 sensors. In order to achieve this, we had to search for inducible promoters to high concentrations of CO2. We have then tested them by constructing a plasmid with the **Gateway Cloning Method**

## Design

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Our constructions had the following parts: a high CO2 inducible promoter, the GPF marker gene, and a terminator. To test the selected promoters, we created 11 constructions with this structure using the Gateway cloning method.

Once we had the constructions we electroporated them in  *Agrobacterium tumefaciens* to infiltrate Nicotiana. When the plants were transformed we exposed them to high levels of CO2 to compare GFP expression with transformed plants at ambient CO2.

![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/engineering/judith.jpg)

Preparing the infiltration solution with the transformed  *Agrobacterium tumefaciens*

## Cloning method

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Gateway is a **clonation method** developed by Invitrogen and is the method we have used to construct our plasmids. Briefly explaining, the gateway method is divided into two steps.

In the first step, we introduce our selected **promoter** in the **pDONR plasmid** by the recombination of the attB1/attB2 and attP1/attP sites. This way, the promoter substitutes the ccdB gene, a lethal gene that inhibits helicases and as a result, we have our pDONR plasmid with our promoter.

In the next step, we transfer our **promoter** into the **final plasmid** which already has the GFP and the terminator. In this second reaction, the recombination is between attL1/attL2 and attR1/attR2 sites to substitute ccdB of the **final plasmid** by our promoter. As a result, we have our **complete transcriptional unit**, promoter, GFP, and terminator in the final plasmid (see “Parts”).

## Practical demostration

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The infiltration process consists of introducing transformed *Agrobacterium tumefaciens*, a bacteria that naturally infects wounded plants and transfers **part of its DNA** to the plant cell, into our *Nicotiana benthamiana* with a syringe. The bacteria are introduced between the two layers of the **epidermis** of the leaves. For the process to be efficient we had to **infiltrate** in leaves that were between 3 and 5 cm in diameter, which normally corresponds to young plants with tender leaves.

![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/engineering/yassin.jpg)

Selecting the plants for infiltration

After a few days we divided our transformed plants in two groups: one was **exposed to high concentrations of CO2** (1,000 ppm) for 24 h and the other one was left at **ambient CO2** levels (400 ppm) as control.

Finally we analyzed the samples on a **confocal microscope**, to see **GFP expression**(See “Protocols” section to see all the explanations of the process).

Our **goal** was to identify plants that expressed more GFP under high CO2 conditions than under control conditions. Throughout the entire project, we have been **improving different steps**, such as the time between the moment of infiltration and the GFP analysis, plant growth conditions, time CO2 exposure…

## Success

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From the eleven tested promoters, we have encountered significant differences between exposed and control plants in three cases. These promoters are: **Citrate synthase 4 (CSY4)**, **Glyceraldehyde-3-phosphate dehydrogenase (G3PDH)** and **Adenosine Triosephospate Synthase (ATPS)**. For more data go to the “Results” section.

![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/engineering/results.jpg)

Photo of the results we see on the confocal microscope

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## What should this page contain?

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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).

## Inspirations

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* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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{% block title %}Results{% endblock %}
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Our results show that the best promoters for our objective are **Citrate synthase 4 (CSY4)**, **Glyceraldehyde-3-phosphate dehydrogenase (G3PDH)** and **Adenosine Triosephosphate Synthase (ATPS)**. But let us explain the data in more detail.

### G3PDH

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Visual exam

| Ambient CO2 | Higher CO2 |
| --- | --- |
| ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/1.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/2.jpg) |

Quantitative exam

| Ambient CO2 | Higher CO2 |
| --- | --- |
| ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/3.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/4.jpg) |
| ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/5.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/6.jpg) |

Both on the visual examination and in the quantification graph we can see that the intensity is much higher in the plants exposed to higher CO2 than in the ones on ambient CO2. The first reaches an intensity of 80 units while the exposed to higher CO2 has a peak of 150 units of intensity.

### CSY4

---

Visual exam

| Ambient CO2 | Higher CO2 |
| --- | --- |
| ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/7.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/8.jpg) |

Quantitative exam

| Ambient CO2 | Higher CO2 |
| --- | --- |
| ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/9.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/10.jpg) |
| ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/11.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/12.jpg) |

Both on the visual examination and in the quantification graph we can see that the intensity is much higher in the plants exposed to higher CO2 than in the ones on ambient CO2. The first reaches an intensity of 25 units while the exposed to higher CO2 has a peak of 250 units of intensity.

### ATPS

---

Visual exam

| Ambient CO2 | Higher CO2 |
| --- | --- |
| ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/13.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/14.jpg) |

Quantitative exam

| Ambient CO2 | Higher CO2 |
| --- | --- |
| ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/15.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/16.jpg) |
| ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/17.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/18.jpg) |

Both on the visual examination and in the quantification graph we can see that the intensity is much higher in the plants exposed to higher CO2 than in the ones on ambient CO2. The first reaches an intensity of 150 units while the exposed to higher CO2 has a peak of 250 units of intensity.

### Hypothesis

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We have found a relationship between the function of the promoters and the results of our three most successful samples.

* Stimulated leaf nocturnal respiration is a response that happens when plants are exposed to high CO2 concentration. **GA3PDH** and **CSY4** are enzymes involved in this process. This is a way to obtain energy from the metabolites formed by a higher rate of photosynthesis.
* Respiratory chain activation is needed to maintain both the carbon and energy balance in leaves during growth at high CO2. In this process **ATPase** plays an important role.

So we hope the next Navarra Biogalaxy team would continue our work by doing further studies with these three promoters.

### Discarded promoters

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We have classified a sample as discarded when the **difference between exposed and non-exposed was similar**. This is because the absence of changes indicates that the promoter is not activated when exposed to high CO2 concentrations and, therefore, cannot be used to generate plant biomarkers.

In the next table we show the results of the promoters that had **similar expression** in ambient and high CO2 concentration.

| Promoter | Ambient CO2 | Higher CO2 |
| --- | --- | --- |
| ADH1 | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/19.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/20.jpg) |
| CI76 | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/21.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/22.jpg) |
| PEPCI | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/23.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/24.jpg) |
| ALDH2 | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/25.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/26.jpg) |
| CIII14 | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/27.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/28.jpg) |
| HB1 | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/29.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/30.jpg) |
| HB2 | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/31.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/32.jpg) |
| ACO3 | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/33.jpg) | ![](https://static.igem.wiki/teams/4374/wiki/web/web-images/document-img/results/34.jpg) |

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