iGEM 2023 — Team navarrabg


{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block banner\_name %}engineering{% endblock %}
{% block page\_content %}

## Goals

---

The project's main aim is to create specialized plants, known as biosensors, that can identify specific substances. For this initial test run, we've chosen to focus on detecting carbon dioxide (CO2).

Another goal is to develop new designs that enhance the effectiveness of the ones you've been using. To achieve this, we're incorporating different marker genes, which are like labels, with advantages that are better than what you get from GFP (a common marker), and a system that causes the signal from the marker protein to disappear when the stimulus is no longer present. This makes the whole process smarter and more efficient.

This year we are working with the 4 selected promoters, and we are following the project by researching genes for the codificant region of the transcriptional unit.

1. For this task we have developed three lines of investigation to make the project viable CO2 inducible promoters with the Gateway Cloning Method.
2. **Adding visible reporter genes** (EYGFPuV and RUBY) to our construction with the Gateway Cloning Method.
3. **Adding protein degradation sequences** (PEST sequence) to our construction with the Golden Braid Technology.

The biological engineering in each part is not always the same, due to the complexity of the task we have used two, the **Gateway Cloning Method** and the **Golden Braid Golden Method**.

Genetically modify *Arabidopsis thaliana* using CO2-responsive promoters from last year, employing the Gateway method.

## Design

---

#### Gateway method

The Gateway Cloning Method is a powerful and flexible molecular biology method advanced through Invitrogen for the efficient and unique cloning of DNA fragments into plasmid vectors. It simplifies the manner of moving genes or DNA fragments among unique plasmid vectors without the need for traditional restriction enzyme-based totally cloning. The Gateway Cloning Method is broadly utilized in molecular biology and Synbio research and has turned out to be a trendy technique in many laboratories.

Here's an overview of how the Gateway Cloning Method works:

**Create Entry Clones**: In this step, you begin by using amplifying your DNA fragment of hobby (e.g., a gene) using PCR. You then introduce DNA recombination websites referred to as "attB websites" to the PCR product. These attB websites are recognized with the aid of special recombination enzymes.

**BP Recombination**: After developing Entry Clones, you perform a BP (Gateway BP) recombination response. This response includes blending the Entry Clone (containing your DNA fragment with attB sites) with a plasmid vector containing attP sites. The BP Clonase enzyme recognizes those websites and allows recombination. This affects the transfer of your DNA fragment into the destination vector, which is referred to as a "Donor Vector." The resulting Donor Vector now incorporates your DNA fragment flanked with the aid of attL websites. It is called the BP reaction because the B-s are changed with the P-s

**LR Recombination**: In the next step, you perform an LR (Gateway LR) recombination reaction. You mix the Donor Vector (containing your DNA fragment) with a destination vector that includes attR websites. The LR Clonase enzyme acknowledges these sites and catalyses recombination. This step transfers your DNA fragment from the Donor Vector to the Destination Vector, resulting for your preferred expression construct.

**Transform Host Cells**: After the LR recombination, the resulting Destination Vector, which now contains your DNA fragment beneath the manipulate of a promoter of your choice, can be transformed into a suitable host organism, in our case we introduce it inside *Agrobacterium tumefaciens* for plant transformation.

The Gateway Cloning Method offers several benefits, such as excessive cloning performance, no want for restrict enzymes or ligase, and the capacity to hastily assemble complex expression constructs with a couple of components. It is especially useful for excessive-throughput and modular cloning initiatives, wherein you need to transport DNA fragments among exceptional vectors effectively.

In our case, we have used this method twice, more specifically in the two first lines of research.

In the first line, we introduced our **promoters inside the plasmid** **with the GFP and the terminator**. Afterward, the construction was introduced in *Agrobacterium tumefaciens* with no capability of forming tumours in plants. Next, we submerge the solution of the modified bacteria in the ovules of *Arabidopsis thaliana*, so it passes the modification to the next filial generation.

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/engineering-1.jpg)

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/engineering-2.jpg)

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/engineering-3.jpg)

In the case of the next line of investigation, we did it in two rounds. First, we **introduced the promoter and the** **reporter** in two different pDONRS, going from a linear sequence to a plasmid one. Then, in the second round we fused both plasmids using the LR CLonase.

So, our final constructions are plasmids with the promoters, the visible reporters (RUBY and eYGFPuV) and the terminator, TNos.

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/engineering-4.jpg)

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/engineering-5.jpg)

#### Goldenbraid method

The GoldenBraid (GB) technique is a modular and versatile DNA meeting technique utilized in synthetic biology for the development of genetic circuits and multigene constructs. Developed at the Centre for Research in Agricultural Genomics (CRAG) in Spain, the GoldenBraid technique presents researchers with an effective tool for assembling and manipulating DNA parts, inclusive of genes, promoters, and different genetic factors, to create complex organic structures.

Its technique is based in:

**Type IIs Restriction Enzymes**: GoldenBraid is based on Type IIs restrict enzymes, which cut DNA sequences outside in their popularity site. This characteristic is essential for unique and scarless DNA meeting. Common Type IIs enzymes used in GoldenBraid consist of BsaI and BsmBI.

**Standardized DNA Parts**: DNA components used in GoldenBraid are usually flanked via Type IIs enzyme popularity sites and are standardized in terms of length and structure. These elements can encompass promoters, coding sequences, terminators, and other genetic elements.

**Entry Vectors**: GoldenBraid uses Entry Vectors, which are circular plasmids that hold person DNA elements. These Entry Vectors have standardized cloning websites and are designed to accept DNA fragments generated through Type IIs restrict enzyme digestion.

In our project we used the GB tech in the third line of investigation (PEST DNA sequences) to research protein degradation. This method is based on developing small plasmids known as PUPD2 with the different subunits of our transcriptional unit and fusing them using the enzymes BSal and DNA Ligase.

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/engineering-6.jpg)

Our final plasmid will have the name pDGB1 a1 p35S:YGFP-PEST:Tnos, in this case we have used only eYGFPuV. Giving as a result the following plasmid.

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/engineering-7.jpg)

## Practical demostration

---

Our structures have been effectively assembled using the described methods.

Subsequently, these constructs were introduced into *Arabidopsis thaliana*, the plant designated for modification. This plant has shown a correct response to the transformation, resulting in varying levels of GFP expression depending on the promoter used. Additionally, the genetically modified organisms (GMOs) containing the RUBY marker have also proven successful, causing the plant to exhibit a red coloration in response to the promoter. In summary, the entire experiment was a triumph, as we achieved the anticipated results successfully.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

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

---

* [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)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block banner\_name %}protocols{% endblock %}
{% block page\_content %}

## Selection of CO2 plant biosensors

---

#### Arabidopsis thaliana with CO2 inducible promoters

After carrying out the permanent transformation of Arabidopsis thaliana plants, with different CO2 inducible promoters and the GFP as reporter gene, we obtained several independent lines of each construct.

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/results-1.jpg)

We exposed these plants to high and ambient CO2 (control plants) for 6 and 24 hours and measured the expression of GFP in both CO2 conditions.

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/results-2.jpg)

In order to select the best line of each construction, to be used as CO2 biosensors, we have made two sequential experiments with their corresponding GFP quantification measurements:

* In the first experiment, we test several the lines for each of the CO2 inducible promoters and measured the activation of the promoter in high CO2 by quantifying GFP content.
* Based on the results obtained in the first experiment, we selected the best line for each construction that respond to high CO2 concentration and measured the activation of the promoter in high CO2 by quantifying GFP content.

The CO2 inducible promoters selected to our proof of concept were ADH1, ATPS, CYS4 y G3PDH.

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/results-3.jpg)

To quantify the GFP properly and to obtain comparative data, the first thing we did was to prepare a calibration curve for GFP determination (Figure 1) and a calibration curve for total protein determination (Figure 2) in leaf samples.

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/results-4.jpg)

**Figure 1.** GFP calibration curve

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/results-5.jpg)

**Figure 2.** Total protein calibration curve

In the first experiment we extracted the proteins of the leaves of several the lines for each of the CO2 inducible promoters and determined their protein content and GFP expression levels. As we can see in Figure 3 not all the obtained lines were induced in high CO2.

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/results-10.jpg)

**Figure 3.** GFP (ng/mg protein) content of different lines of Arabidopsis plants grown at ambient and high CO2 concentrations.

Based in these results, we decided to carry out the second measuremet with the following line for each promoter:

* Line 2 for ADH1
* Line 7 for ATPS
* Line 9 for CYS4
* Line 1 for G3PDH

For determining the reliability of the obtained results and confirming that the selected lines are good candidates for being use as CO2 biosensor plants we decide to repeat the experiment. As observed in figure 4, all selected lines responded to high CO2. Even more, we observed that 6 hours of exposure was enough for sensing the presence of “toxic” concentrations of CO2.

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/results-11.jpg)

**Figure 4.** GFP content of different lines of Arabidopsis plants grown at ambient and high CO2 concentrations. Values are the mean of two repetitions.

## Parts

---

#### Fusing visible reporter genes to selected promoters

One of our lines of work consists of fusing visible reporter genes (eYGFPuv and RUBY) to the promoters that we have previously selected. We have done this with the MultiSite Gateway cloning method (see “Protocols”)

First of all, we must produce entry clones with the promoters, eYGFPuv and RUBY into the pDONRs vectors. We did this first step in a BP reaction (using BP clonase).

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/results-6.jpg)

Once the Entry clones were ready, we had to transfer to a secondary plasmid, the Destination vector (R4pGWB501 plasmid).

We did LR reactions (using LR clonase) to fuse, on the one hand, the selected promoters with the eYGFPuv and on the other, the promoters with the RUBY cassette. Thus we have obtained the following final plasmids:

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/results-7.jpg)

#### Adding protein degradation sequence (PEST sequence)

In this line of work, we have used the Golden Braid cloning method (see “Protocols”). Using a domestication protocol to adapt basic DNA parts to the GB grammar, we introduce our PCR products, in four pUPD2, as we see in the image.

These four pUPD2 correspond to: the constitutive 35S promoter, the visible marker that we have used for this construction, which is eYGFPuv, the PEST degradation sequence and the NOS terminator.

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/results-8.jpg)

![](https://static.igem.wiki/teams/5011/wiki/web-img/document-img/results-9.jpg)

In order to create a new transcriptional unit, we need to join all parts using a Multipartite GB assembly reaction. We used the pDGB1α plasmid as receptor.

Our final plasmid contains a constitutive promoter (p35S), that in the future would be substituted with an inducible promoter, the reporter gene eYGFPuv, the degradation sequences (PEST sequence), and the terminator NOS. To select the future modified plants, we will add the kanamycin resistance gene.

This plasmid will be used as a proof of concept to see how the PEST sequence used in protein degradation works.

{% endblock %}