iGEM 2020 — Team NOVA_LxPortugal

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# Team:NOVA LxPortugal/Notebook

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[![pinemato fight logo](https://static.igem.org/mediawiki/2020/e/ec/T--NOVA_LxPortugal--logo.jpg)](https://2020.igem.org/Team%3ANOVA_LxPortugal)

[Home](https://2020.igem.org/Team%3ANOVA_LxPortugal)

Project

[Project Description](https://2020.igem.org/Team%3ANOVA_LxPortugal/Description)
[Implementation](https://2020.igem.org/Team%3ANOVA_LxPortugal/Implementation)
[Design](https://2020.igem.org/Team%3ANOVA_LxPortugal/Design)
[Sustainability](https://2020.igem.org/Team%3ANOVA_LxPortugal/Sustainability)
[Notebook](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)

Lab

[Experiments](https://2020.igem.org/Team%3ANOVA_LxPortugal/Experiments)
[Project Modeling](https://2020.igem.org/Team%3ANOVA_LxPortugal/Model)
[Results](https://2020.igem.org/Team%3ANOVA_LxPortugal/Results)
[Engineering Success](https://2020.igem.org/Team%3ANOVA_LxPortugal/Engineering)

[Safety](https://2020.igem.org/Team%3ANOVA_LxPortugal/Safety)
[Human Practices](https://2020.igem.org/Team%3ANOVA_LxPortugal/Human_Practices)
[Parts](https://2020.igem.org/Team%3ANOVA_LxPortugal/Parts)
[Contribution](https://2020.igem.org/Team%3ANOVA_LxPortugal/Contribution)
[Collaborations](https://2020.igem.org/Team%3ANOVA_LxPortugal/Collaborations)

Team

[Team Members](https://2020.igem.org/Team%3ANOVA_LxPortugal/Team)
[Attributions](https://2020.igem.org/Team%3ANOVA_LxPortugal/Attributions)

[Science Communication](https://2020.igem.org/Team%3ANOVA_LxPortugal/Education)

# Notebook

[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### March](#march)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### April](#april)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### May](#may)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### June](#june)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### July](#july)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### August](#august)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### September](#september)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### October](#october)

# March

---

* iGEM kick-off session at ITQB NOVA

![first session schedule](https://static.igem.org/mediawiki/2020/0/0b/T--Nova_LxPortugal--notebook1.png)

This was the very first time we were together and showed interest in participating in iGEM! It was a sneak peak on what to expect for the next few months. We had the chance to listen to Fran Quero talking about the spanish participation in iGEM over the years and showing us what are the most challenging activities for a team, and Nemanja gave us the global perspective of what it means to be an iGEMmer. We have also simulated a mini iGEM by working in groups in some crazy momentary ideas. It was a really funny activity and all of us got excited to be part of the iGEM world.

![first session photo](https://static.igem.org/mediawiki/2020/f/ff/T--Nova_LxPortugal--notebook2.png)

* Week 1 (March 30 to April 5)

A week after the kick off session, the whole country went to confinement, so we will only get together online from now on (at least until further notice). This week we officially created the NOVA\_LxPortugal team with all the members that showed interest after the kick-off session, the first ever Portuguese team on iGEM. We are all so excited to be participating in this competition!
The team gathers nine students: seven are from the Master in Biochemistry for Health and the remaining two are from the Bachelor in Cellular and Molecular Biology.
Being the first year we are participating in iGEM is hard enough on a team, but if you add a world pandemic to the equation, it promisses to be a challenge (to say the least). But we are Fighters so… challenge accepted!

![team ready to rumble](https://static.igem.org/mediawiki/2020/6/65/T--Nova_LxPortugal--notebook3.png)

Around this time we also created our iGEM slack account. We could not be together so this was the way we found to communicate.

![team slack](https://static.igem.org/mediawiki/2020/1/12/T--Nova_LxPortugal--notebookpi.png)

# April

---

* Week 2 (April 6 to 12)

First online meeting where we talked about iGEM and what we were supposed to do as a team for this project. In this first meeting we had our co-PI Sofia Ferreira who gave examples of synthetic biology case-studies and started the initial brainstorming of ideas.

* Week 3 (April 13 to 19) and Week 4 (April 20 to 26)

The ideas are starting to flow. In this weekly meeting the team has come up with five potential projects for iGEM:

+ Control of *Carpobrotus ebulis*
+ Hyaluronic acid production
+ Control of *Xylella fastidiosa* infections
+ Strictosidine production
+ Control and prevention of pine wilt disease (PWD).

* Week 4 (April 20 to 26)

In our weekly meeting, each working group presented their ideas, and we all discussed their potential and relevance for the society. All the ideas seemed potential contenders. It is hard to only pick a theme, so many problems that can be attenuated or even solved with synthetic biology!

# May

---

* Week 5 (April 27 to May 3)

This week we were all focused on the synthetic biology component of the projects and how we could use molecular biology strategies to implement these ideas.

* Week 6 (May 4 to 10)

This week we said goodbye to three of our projects: Control of the invasive plant Carpobrotus edulis, Control of Xylella fastidiosa infections in olive trees, Strictosidine production in *E. coli*, the precursor of several anti-cancer compounds. Don´t get us wrong, they were nice projects, and we are putting a pin on them, but they were just not special enough for iGEM at this stage.
Due to the COVID-19 pandemic, we were told we would have to wait until September to start our lab work and still even then it might not be possible.

* Week 7 (May 11 to 17)

We are down to only two projects, Hyaluronic acid production to fight osteoarthritis and the control of pine wilt disease (PWD) by an engineered bacteria. The two sub-teams are now more focused on finding molecular strategies, designing modelling programs in Optflux and the impact of our project in Portugal. Ultimately, only one project can be chosen to represent the Portuguese team on iGEM and be elected as the ultimate survivor, let’s see what the future holds.

![first project](https://static.igem.org/mediawiki/2020/a/a3/T--Nova_LxPortugal--notebook4.png)
![second project](https://static.igem.org/mediawiki/2020/1/1c/T--Nova_LxPortugal--notebook5.png)

Team Hyaluronic acid was constituted by Daniela, Fabiana, Beatriz, Joana and Madalena, while the PWD team had João, Carolina, Tiago, and Isabel.

* Week 8 (May 18 to 24)

Up to this moment, we all have been participating in several tasks. However, this week we started to work on different working groups for both projects:

![connect](https://static.igem.org/mediawiki/2020/9/93/T--Nova_LxPortugal--connect-icon.png)

Team human practices: Isabel, João - This week we focused on finding sponsors and potential stakeholders for both our projects.

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

Team Dry lab: Carolina and Beatriz - We are designing optflux models for the production of hyaluronic acid and spectinabillin. Unfortunately neither of our models are producing the compound. We still have a lot of work ahead of us.

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

Team wet Lab: João, Isabel, Tiago - Searched previous studies on the subjects to address whether our project could be innovative in that field.

Madalena, Daniela, Fabiana and Joana at this point were just helping everyone with their tasks.

* Week 9 (May 25 to 31)

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

Finally, we were able to finalize our Hyaluronic acid optflux model, so we are starting the optimizations. The spectinabilin model is still not completely functional so our computational team is working on it.

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

This week we have been focusing on finding the better way to administer both our compounds and how that might change the way we approach the problem. Also, we started to think more about the outline of the experiments and how the restriction of time in the lab due to COVID might affect our experimental work.

# June

---

* Week 10 (June 1 to 7)

After a very fruitful discussion, we all agreed that this was the right moment to choose only one and that was… drumroll please… production of spectinabillin by a genetically engineered *Pseudomonas putida* to fight the PWD.

You must be wondering where the idea of finding a strategy to control and prevent PWD came from. It all started because João, our student leader, lives in Setúbal, a beautiful city in Portugal known for its vast forest, which harbours many species of trees, including *Pinus Pinaster*. João had noticed that in Setúbal the maritime pines had become more and more affected by the pine wilt disease, so when he heard about iGEM he thought it was the perfect opportunity to find a way to prevent the spreading of the disease. Ultimately we thought this project was the most promising because, not only was it relevant to our society but it was also related to many of the sustainable development goals.

To achieve this ambitious goal, we are going to develop a *Pseudomonas putida* model with the spectinabilin production pathway to determine the optimum genotype for our desired phenotype: maximum spectinabilin production while maintaining the maximum growth rate possible. After the in silico validation of the spectinabilin production in *P. putida*, we will implement the heterologous production pathway of spectinabilin by expressing the respective genes in *P. putida* under the control of an alpha-pinene’s inducible promoter. The results obtained from the computational analysis will support the further development of our engineered bacteria by manipulating its metabolism in order to maximize the flux production of spectinabilin.

* Week 11 (June 8 to 14)

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

We were able to conclude the spectinabilin-producing *P. putida* genome-scale metabolic model and started the simulations with different media. We now know that we can use *P. putida* to produce our nematicidal compound: spectinabilin! It seems very promising to us!

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

We started to plan how we can insert the same genes we have added to the *P. putida* model in vivo. A lot of genes are needed to produce spectinabilin, so it is not an easy task! We are analysing which genes/proteins are already validated for these reactions and thinking about molecular biology strategies to insert them in our bacteria.

* Week 12 (June 15 to 21)

![talk](https://static.igem.org/mediawiki/2020/5/54/T--Nova_LxPortugal--talk-icon.png)

We welcomed Rita, our Science Communication instructor, to the team.
Team communication: Daniela, Madalena, Fabiana, Rita and Joana. This week we started the discussion about our team name. We had tons of ideas, from PineNematoControl to NematoFighters, we thought about it all. Ultimately, we voted on the different names and we decided to call ourselves… PineNematoFight. Nice, right?

* Week 13 (June 22 to 28)

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

Continuation of the simulations in optflux, however the yields are still very low. Modifications on the model are being done.

![connect](https://static.igem.org/mediawiki/2020/9/93/T--Nova_LxPortugal--connect-icon.png)

First contact with ICNF (Conservation of Nature and Forests Institute).

# July

---

* Week 14 (June 29 to July 5)

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

We started the optimizations of our model, but we have noticed some problems in obtaining nice yields of spectinabilin. Must try different simulation and optimization methods and parameters!

* Week 15 (July 6 to 12)

![talk](https://static.igem.org/mediawiki/2020/5/54/T--Nova_LxPortugal--talk-icon.png)

First reach out to the Science Communication and Image Office at ITQB-NOVA for the collaboration in the designing of our team Logo.

* Week 16 (July 13 to 19)

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

Since we are having some troubles with the model in terms of optimization results, we decided to search for another pre-existing model to add the spectinabilin and (S)-methylmalonyl-CoA pathways.

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

This week we outlined some experiments that we intended to do to help validate our project:

+ We intend to induce the expression of our nematicidal agent, spectabilin, in the presence of α-pinene. To do this, we are going to express our genes under the control of an α-pinene inducible promoter. To test if our promoter works, we will first associate this promoter to the green fluorescent protein, GFP, using it as a reporter gene for the activity of our promoter by analysing the fluorescence emitted by the bacteria transformed with this construct. This is a rapid and visual experiment we pretend to validate our promoter.
+ Test the toxicity of spectabilin in the nematodes by in vitro experiments.
+ If we are able we would also like to make a proof of concept by joining our engineered bacteria with the nematode in an in vitro experiment and induce the production of spectabilin by α-pinene.

![talk](https://static.igem.org/mediawiki/2020/5/54/T--Nova_LxPortugal--talk-icon.png)

We thought we needed a nice logo for our awesome project, so we asked Luís Morgado from the Science Communication and Image Office at ITQB NOVA to help us with this task. He kindly designed a batch of logos. We all had very different opinions, so it took a while to reach an agreement. Finally, we temporarily settled on this version:

![old logo](https://static.igem.org/mediawiki/2020/3/38/T--Nova_LxPortugal--old_logo.png)

* Week 17 (July 20 to 26)

![talk](https://static.igem.org/mediawiki/2020/5/54/T--Nova_LxPortugal--talk-icon.png)

Lucas Boldrini, a former member of the São Carlos team from Brazil in 2019, started mentoring us. We had our first meeting with Lucas in which we showed progress and achievements we accomplished so far in the spectinabilin production project. We also discussed Luca’s experience on iGEM and what we could do to be a successful team in this competition. He gave us several tips and told us the must-do’s in an iGEM project.

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

The search for our model continues.

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

Searching for alpha-pinene inducible promoters, so our pathway is only activated in the presence of this compound.

# August

---

* Week 18 (July 27 to August 2)

![connect](https://static.igem.org/mediawiki/2020/9/93/T--Nova_LxPortugal--connect-icon.png)

Gathering of information about the pine wilt disease that was kindly provided by ICNF.

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

The search for the model comes to an end. The model iJN1462 was chosen since it was the most recent and complete model of *P.putida*, we are now adding the heterologous reactions to the model.

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

We outlined an experiment to test if our compound of interest, spectabilin, is safe for the environment in particular for the plants. So, we will test the effect of this compound on the germination of seeds of *Arabidopsis thaliana*.

* Week 19 (August 3 to 9)

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

The reactions of spectinabilin formation pathway and (S)-methylmalonyl-CoA production pathway were added.

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

We ordered the genes of the spectinabilin and methylmalonyl-CoA production pathways. We also ordered the α-pinene inducible promoters together with GFP.

![video](https://static.igem.org/mediawiki/2020/e/e8/T--Nova_LxPortugal--video-icon.png)

This week Fabiana, Madalena, Beatriz and Daniela started to plan the promotional video. We had too many ideas so choosing one was very hard.

* Week 20 (August 10 to 16)

![connect](https://static.igem.org/mediawiki/2020/9/93/T--Nova_LxPortugal--connect-icon.png)

Response from ICNF with statistics about the pine wilt disease in Portugal. Resipinus was officially associated with our project.

![talk](https://static.igem.org/mediawiki/2020/5/54/T--Nova_LxPortugal--talk-icon.png)

Start of our collaboration with the Groningen team. Daniela, Beatriz and João worked hard and were able to design a great presentation about soil biodiversity and conservation.

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

Once again our model shows some problems in the production of spectinabilin, since the stoichiometry of some equations was not balanced.

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

The reagents needed to perform the experimental activities planned were ordered. There is a possibility of getting a new sponsor that will provide us some lab material, looking forward to that.

* Week 21 (August 17 to 23)

![talk](https://static.igem.org/mediawiki/2020/5/54/T--Nova_LxPortugal--talk-icon.png)

We finally heard back from Luís, who sent us a new set of proposals with eight more logos, from which we chose the winner, after careful consideration, online voting through Slack and final decision at our weekly meeting. Let's take a moment to appreciate all the hard work that went into designing our logo:

![logo](https://static.igem.org/mediawiki/2020/e/ec/T--NOVA_LxPortugal--logo.jpg)

![talk](https://static.igem.org/mediawiki/2020/5/54/T--Nova_LxPortugal--talk-icon.png)

Creation of our Instagram and Twitter accounts. We are so excited to start sharing our experience with everyone!

![talk](https://static.igem.org/mediawiki/2020/5/54/T--Nova_LxPortugal--talk-icon.png)

Webinar about soil biodiversity in collaboration with the Groningen team. Tiago did a great job representing our team in the presentation.

![thankgroningen](https://static.igem.org/mediawiki/2020/b/b1/T--Nova_LxPortugal--notebook6.png)

![talk](https://static.igem.org/mediawiki/2020/5/54/T--Nova_LxPortugal--talk-icon.png)

Participation in the iGEM Global Meet-Up’s Poster Session. Daniela designed our poster by hand and Madelena made it come to life on the computer . This event was a great way to meet other teams and share iGEM experiences.

![poster](https://static.igem.org/mediawiki/2020/1/12/T--Nova_LxPortugal--notebook7.png)

* Week 22 (August 24 to 30)

![video](https://static.igem.org/mediawiki/2020/e/e8/T--Nova_LxPortugal--video-icon.png)

Start of the discussion about the promotional video. This week Fabiana, Daniela, Beatriz, Madalena and Rita came up with ideas for our video.

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

Finalization of the *Pseudomonas putida* optflux model (We finally fixed the model and were able to produce spectinabillin for the first time, yeah!). Beginning the simulation and optimization process in Optflux.

![optimizationsongoing](https://static.igem.org/mediawiki/2020/2/2c/T--Nova_LxPortugal--notebook8.png)

A lot of simulations are being performed every day!

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

Started planning the experimental work by searching and adapting protocols. We also started to book the necessary lab equipment to perform the planned experiments.

![talk](https://static.igem.org/mediawiki/2020/5/54/T--Nova_LxPortugal--talk-icon.png)

Update our instagram with information about Synthetic Biology, since several of our followers were unaware of what it means.

![talkposter](https://static.igem.org/mediawiki/2020/9/97/T--Nova_LxPortugal--notebook9.png)

# September

---

* Week 23 (August 31 to September 6)

![talk](https://static.igem.org/mediawiki/2020/5/54/T--Nova_LxPortugal--talk-icon.png)

Madalena and Beatriz prepared a short presentation video and a small presentation of our project to present at the pitching session of the Paris Global Meetup. This day was great, not only did we win first place at the pitching session but we also got to meet our fellow iGEMmers again.

![shortpresentation](https://static.igem.org/mediawiki/2020/b/b3/T--Nova_LxPortugal--notebook10.png)

![connect](https://static.igem.org/mediawiki/2020/9/93/T--Nova_LxPortugal--connect-icon.png)

PINUS centre confirmed their interest in our work saying our solution could be of great interest and offered support for our project.

![video](https://static.igem.org/mediawiki/2020/e/e8/T--Nova_LxPortugal--video-icon.png)

The storyboard for our promotional video is completed, so we are starting to shoot.

* Week 24 (September 7 to 13)

![video](https://static.igem.org/mediawiki/2020/e/e8/T--Nova_LxPortugal--video-icon.png)

We have decided to shoot our video in Portuguese because it is always more meaningful when we speak in our mother language. Madalena was our movie star and Fabiana our amazing camerawoman. No one would say it was the first time they were doing this! We had a blast recording the video.

![talk](https://static.igem.org/mediawiki/2020/5/54/T--Nova_LxPortugal--talk-icon.png)

Our team Leader João Costa gave a talk about the iGEM competition to the new master students. We really want that next year more Portuguese teams participate in iGEM so we are trying to spread the word.

![joaotalk](https://static.igem.org/mediawiki/2020/b/ba/T--Nova_LxPortugal--notebook11.png)

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

We performed optimizations to maximize the production of spectinabilin. In these optimizations, we tried to find reactions that could be under/over expressed to maximize spectinabilin production

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

Gathering all the last details needed to fill the final safety form.

* Week 25 (September 14 to 20)

![talk](https://static.igem.org/mediawiki/2020/5/54/T--Nova_LxPortugal--talk-icon.png)

This week we got together with our Iberian brothers and we co-hosted the Iberian Meeting with the UPF\_Barcelona team. It was really nice to present our project to our iberian fellows and watch their presentation. We had fun playing some games in the end inspired by the iGEM Global meetup’ spirit.

![iberian meetup](https://static.igem.org/mediawiki/2020/e/e6/T--Nova_LxPortugal--notebook12.png)

We really did try to spell iGEM 2020, well… A for effort!

![spell igem](https://static.igem.org/mediawiki/2020/6/63/T--Nova_LxPortugal--notebook13.png)

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

We have continued performing optimizations, but this time we are looking for reactions knockouts. We are fighters, so knockouts are our expertise!

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

In silico design of the DNA constructs from the building of plasmid constructs to the assembly of all the genes in the pathways needed for the production of spectinabilin and more.
Still waiting for the arrival of some DNA parts and cells to start the experimental work.

* Week 26 (September 21 to 27)

![talk](https://static.igem.org/mediawiki/2020/5/54/T--Nova_LxPortugal--talk-icon.png)

Biotechnology and Forest Scientific Meeting organized by the NOVA\_LxPortugal team. In this meeting we learned the different chemotypes and infection profiles of *Pinus pinaster* present in the Portuguese forest, the treatments that are being developed and we got to know more about ongoing projects to increase our knowledge about pine wilt disease. We also discussed the utilization of alpha-pinene as an inducer and the possibility of testing spectinabilin’s toxicity in pine trees.

![forest meeting](https://static.igem.org/mediawiki/2020/4/4f/T--Nova_LxPortugal--notebook14.png)

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

In the optimizations, we switched the reactions for the genes, meaning, we performed under/overexpression and knockout of the genes present in the model instead of the reactions.

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

Due to delay in the arrival of some products and material we weren’t able to start the experiments yet. Continued with the design of the experiments in silico.
We got our new sponsor NZYTech!

![video](https://static.igem.org/mediawiki/2020/e/e8/T--Nova_LxPortugal--video-icon.png)

The promotion video was successfully submitted. Honestly, we think it is great! We are so proud of our two minute video (one can almost forget the hard work it took). One video is done, one to go….

# October

---

* Week 27 (September 28 to October 4)

![video](https://static.igem.org/mediawiki/2020/e/e8/T--Nova_LxPortugal--video-icon.png)

The Presentation video is on the making. If a two minute video is hard try a 20 minute one… We are optimistic but also a bit worried, we have never had to make such a long video. We have some great ideas though, so it is looking good.

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

Finally, we started the wet-lab work by assessing if spectinabilin has a toxic effect on *Arabidopsis thaliana* germination and growth. We prepared the growth medium with different spectinabilin concentrations and planted the seeds.
 Finally, *Pseudomonas Putida* strain arrived!

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

We continued the optimizations and started to analyse some of the results obtained, looking good!

THE MONTH OF WIKI FREEZE IS HERE! At this point, all the team members are writing for the wiki.How did time go so fast?

* Week 28 (October 5 to 11)

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

Analysis of the last week results on the impact of spectinabillin in *Arabidopsis thaliana* germination and growth.
 Finalization of the promoter sequences. Just a few weeks to finish everything but so much to do!

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

We finished our optimizations and compared the results. The four best results included: 5 knockouts, 3 underexpressions, 4 underexpression and 5 underexpressions. The knockout result had a bigger production of spectinabilin. Let’s knockout some genes!

* Week 29 (October 12 to 18)

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

Everything is being prepared for the experiments we will perform next week Unfortunately, we won’t be able to finish it all before the wiki freeze. Next week will be rough.

![connect](https://static.igem.org/mediawiki/2020/9/93/T--Nova_LxPortugal--connect-icon.png)

Forestis showed interest in our project and gave us more information about the PWD in Portugal.

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

We proceed with the analysis of the best result.

* Week 30 (October 19 to 25)

![lab](https://static.igem.org/mediawiki/2020/6/6b/T--Nova_LxPortugal--lab-icon.png)

We constructed a plasmid with GFP under the control of two different alpha-pinene inducible promoters and transformed in an *Escherichia coli* strain. The plasmids were extracted and the sequences were confirmed by sequencing.

![pc](https://static.igem.org/mediawiki/2020/a/af/T--NOVA_LxPortugal--pc-icon.png)

The wiki pages were all concluded. It was exhausting but totally worth it!
The poster is on the making.

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# Team:NOVA LxPortugal/Engineering

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# ENGINEERING SUCESS

[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### Engineering Sucess](#engineering_1)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### Research](#engineering_2)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### Imagine](#engineering_3)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### Design](#engineering_4)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### Test](#engineering_5)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### Learn](#engineering_6)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### Improve](#engineering_7)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### References](#engineering_8)

# Engineering Sucess

Due to the COVID-19 pandemic, the access to the laboratories and to the necessary material tend to be more restricted. So the planning of our experiments was limited by the regulation implemented at Instituto de Tecnologia Química e Biológica António Xavier (ITQB-NOVA) and some of the planned experiments had to be postponed.

#### RESEARCH

Our project started by reviewing the literature to infer which species of bacteria are present in *Pinus pinaster* microbiota that are capable of metabolizing alpha-pinene1. This monoterpene tends to be overproduced in stress responses2, including the nematode infection of *Bursaphelenchus xylophilus*, so we designed a synthetic biology strategy dependent on the presence of this compound. Two bacteria were chosen for the analyses: *Pseudomonas putida* and *Serratia marcescens*. Both species have the ability to metabolize alpha-pinene1,3, so both possess regulatory mechanisms as a response to the presence of this compound. Nevertheless, the species *Pseudomonas putida* was selected to further proceed with the project since this microorganism is easier to acquire and work in the laboratory. In this phase of the project, we started to investigate which promoters are associated with the enzymes constituting the alpha-pinene degradation pathway. The main goal was to activate the production of a nematicidal compound as a response mechanism to a high concentration of alpha-pinene. After some research, we selected spectinabilin as our nematicidal compound since (i) it has been proven that it has a significant nematicidal effect4, (ii) the biosynthetic pathway for its production is fully disclosed ([here](https://biocyc.org/META/new-image?type=PATHWAY&object=PWY-8034)) and (iii) its prokaryotic origin.

#### IMAGINE

The utilization of alpha-pinene as an inductor was debated in the Biotechnology and Forest Scientific Meeting organized and hosted by us with the other scientists and researchers in this area. Alpha-pinene tends to be produced as a stress response in several situations, including infection by the nematode and tends to be present in both *Pinus pinaster* and *Pinus pinea*, with the last one not being as affected by Pine Wilt Disease (PWD). Furthermore, *Pinus pinea* tends to produce a lower quantity of alpha-pinene than *Pinus pinaster*. So, the utilization of this metabolite as an inductor must be considered if applied to other species. Probably, alternative compounds associated with the response of other species to the nematode infection must be researched to find other possible pathway triggers. Another important topic discussed in our meeting, was the existence of different chemotypes of *Pinus pinaster* that could lead to variations on the quantity of alpha-pinene produced in the tree. Nevertheless, despite all the different chemotypes and the consequent variations in the amount of alpha-pinene released, it is still observed an increase in its concentration after the infection and continues to be one of the most characteristic compounds associated with the PWD.
After considering all the challenges that have arisen from our debate, we concluded that the project was still feasible for combating the PWD in the Portuguese territory and we proceeded with alpha-pinene as our chosen compound to induce the expression system of the nematicidal compound.

#### DESIGN

After selecting the inductor, we had to select the respective inducible promoters. After some research in the literature and databases, we have selected two different promoters to regulate the expression of the genes constituting the spectinabilin biosynthetic pathway. We have focused on the promoters involved in the alpha-pinene metabolism in *Pseudomonas* strains1. Particularly, only a few genes belonging to this pathway were already annotated and well-characterized. We have selected two possible options: one promoter regulating the expression of the alpha-pinene oxide lyase gene from *Pseudomonas rhodesiae* CIP107491 and one putative promoter from the gene enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (fadB) from *P. putida KT2440*. The sequence of the first promoter was already annotated as a promoter region in NCBI. The last promoter was not yet annotated, so its potential role as a promoter was inferred by using the online bioinformatic tool BPROM from [softberry](http://www.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb). This tool allows recognizing potential transcription start positions of bacterial genes and it was suggested by other iGEMmers in the iGEM 2020 Global Slack (Thank you!).
Additionally, for this project we also used a terminator sequence that is suited for *P. putida*. This terminator with 158 bp, called T1, was already proven in previous research to efficiently terminate transcription in *P. putida*, *Escherichia coli* and other microorganisms5.

#### TEST

To validate the selected alpha-pinene inducible promoters, we constructed plasmids where the expression of the green fluorescent protein (GFP) is under the control of the previously chosen promoters. To do this, we designed two composite parts, each one containing three individual parts that were registered in the iGEM’s Part's Registry page. Our individual parts correspond to the enhanced green fluorescent protein (EGFP) (BBa\_K3675002), the terminator (BBa\_K3675003) and our promoters (BBa\_K3675000 and BBa\_K3675001). The composite parts were designed with one of the promoters, followed by the EGFP and ending with the terminator as depicted in Figure 2. The first composite part was the one with the promoter regulating the expression of the gene encoding the alpha-pinene oxide lyase (BBa\_K3675004) and the second composite contains the promoter from fadB gene (BBa\_K3675005). Both parts were cloned into the plasmid pSEVA234, a vector compatible with the selected expression strain *Pseudomonas putida* KT2440. Two different molecular biology strategies were designed using *SnapGene* to clone each promoter: one eliminating the original operon lac and trc promoter from the plasmid pSEVA234, and another one cloning the DNA constructs directly in the original multiple cloning site of the plasmid. For the first strategy, the restriction enzymes used were *Pac*I and *Kpn*I, while for the last design, *EcoR*I and *Kpn*I were the selected enzymes. The resulting plasmid maps are presented in Figure 1.

![Plasmids](https://static.igem.org/mediawiki/2020/7/7e/T--NOVA_LxPortugal--engineering.png)

Figure 1- Maps of the constructed plasmids. Schemes designed in SnapGene

The constructed vectors were finished, but we were not able to transform them into *P. putida*and continue the experiment for validation of the selected promoters. Since our promoters are regulated by alpha-pinene, we assume that by supplementing this compound to the medium, the transcription of GFP would be activated with the consequent emission of fluorescence, easily visible under a UV light or measured by a fluorimeter. The same experiment without supplementing alpha-pinene would work as the negative control. The schematic representation of this experiment is present in Figure 2.

![Promoters](https://static.igem.org/mediawiki/2020/0/0c/T--NOVA_LxPortugal--PromoterAssay.png)

Figure 2- Construction of a plasmid with the potential alpha-pinene inducible promoter(s) controlling the expression of the Green fluorescence protein (GFP) and a terminator sequence. The validation of the selected promoter(s) will be performed by observing/measuring variations on fluorescence after adding alpha-pinene to the culture.

#### LEARN

If the results of this experiment validate the use of alpha-pinene to induce gene expression with the chosen promoter(s), several experiments can follow. First, it is important to understand if the DNA construct(s) can be improved by measuring the emitted UV light or by running SDS-PAGE to evaluate the GFP levels of expression. Different plasmids and combinations of DNA parts can also be tested to improve the GFP expression levels. The minimal concentration of alpha-pinene to induce gene expression must be quantified to validate if it is sufficient to induce the gene expression in pine trees. We also intend to proceed with our main goal by expressing the spectinabilin biosynthetic pathway under the control of the validated promoter(s). By doing so, the spectinabilin production is only activated in the presence of alpha-pinene after the tree is infected by the nematode. Finally, we intend to co-culture our engineered *Pseudomonas putida* expressing GFP with nematodes to better understand the association behaviour between the two organisms. By adding alpha-pinene to the medium and consequently activating GFP expression, we would be able to detect the bacteria inside the nematode.

#### IMPROVE

If the promoters fail to induce the gene expression in the presence of alpha-pinene, we have planned different strategies to address this problem. The first option would be to create genetic variability in the sequences of previously selected promoters by using techniques like random or site directed mutagenesis. For instance, we can design degenerate primers for regions that more likely affect the gene expression to create genetic diversity. The best mutants can be rapidly identified by measuring the intensity of the emitted UV light with a 96-well plate in a fluorescence microplate reader. Several rounds of optimizations can be performed until achieving the optimum GFP level of expression. In parallel, we can sequence the best performing promoters to identify the respective punctual mutations and implement a more rational strategy with site directed mutagenesis. In alternative, we can also test other promoters from different strains of *Pseudomonas*, or from other bacteria like *Serratia marcescens*, as mentioned earlier. Finally, we can broaden the research spectrum of potential promoters to other bacteria species from the pine microbiota, capable of metabolizing alpha-pinene. If all these strategies fail, we can use other compounds associated with the PWD to activate the gene expression.

##### References

1. Tudroszen NJ, Kelly DP, Millis NF. alpha-Pinene metabolism by Pseudomonas putida. Biochem J. 1977;168(2):315-318. doi:10.1042/bj1680315.
2. Li, Y. et al. Comparative Transcriptome Analysis of the Pinewood Nematode Bursaphelenchus xylophilus Reveals the Molecular Mechanism Underlying Its Defense Response to Host-Derived α-pinene. Int. J. Mol. Sci. 2019; 20, 911.
3. Wright SJ, Caunt P, Carter D, Baker PB. Microbial oxidation of alpha-pinene by Serratia marcescens. Appl Microbiol Biotechnol. 1986; 23:224–227. doi:10.1007/BF00261919.
4. Liu MJ, Hwang BS, Jin CZ, Li WJ, Park DJ, Seo ST, Kim CJ. Screening, isolation and evaluation of a nematicidal compound from actinomycetes against the pine wood nematode, Bursaphelenchus xylophilus. Pest Manag Sci. 2019;75(6):1585-1593. doi: 10.1002/ps.5272.
5. Vanesa Amarelle, Ananda Sanches-Medeiros, Rafael Silva-Rocha, and María-Eugenia Guazzaroni. ACS Synthetic Biology. 2019;8 (4), 647-654. doi: 10.1021/acssynbio.8b00507.

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# Team:NOVA LxPortugal/Experiments

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# EXPERIMENTS

[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### Experiments](#experiment_1)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### Protocols](#experiment_2)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### References](#experiment_3)

# Experiments

*Arabidopsis thaliana* germination assays

To evaluate the effect of spectinabilin on seed germination, we inoculated *Arabidopsis thaliana* seeds in three different types of plates: Murashige and Skoog (MS) medium; MS medium with DMSO and MS medium supplemented with spectinabilin. The seeds inoculated in MS medium plates represent a positive control of germination.

Since spectinabilin was solubilized and stored in DMSO, we wanted to assess its effect on germination of the seeds to avoid incorrect attribution of secondary effects of DMSO to spectinabilin. Therefore, four plates with 10, 20, 100, 200 and 400 µL of DMSO were inoculated.

According to Min-Jiao et al.1, the LC50 value (concentration that kills 50% of the test subjects) of spectinabilin against *Bursaphelenchus. xylophilus* is 0.84 µg/mL. Therefore, five different concentrations below and above the LC50 value were tested: 0.05; 0.1; 0.5; 1 and 2 µg/mL.

After the inoculation of the *A. thaliana* seeds in the different tested conditions, all the plates were incubated at 4 °C in a dark chamber for 3 days. This step is crucial to overcome seed dormancy. It is known that temperature and maternal plant photoperiod during seed development plays a relevant role in setting dormancy level and, consequently, germination timing2,3. Hence, this step allowed seed synchronization and triggering of the germination-related processes.

The following step consisted in an incubation at 20 °C with a 12 h/12 h photoperiod enabling both photosynthesis and chemosynthesis, which contribute to seed germination and growth. One week later, we evaluated the germination rate and phenotype present in the three types of media.

Promoters insertion into cloning vector pSEVA234

The final aim of our project is to build a plasmid that activates the expression of the spectinabilin production genes in the presence of alpha-pinene. So, our first experiment is to find alpha-pinene inducible promoters and validate their induction by the compound. To achieve this, we selected two different promoters: the alpha-pinene oxide lyase promoter (P1), an enzyme from the alpha-pinene degradation pathway known to be overexpressed in the presence of this compound from *Pseudomonas rhodesiae* CIP107491 - and the P2, a promoter associated with the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, an enzyme from the alpha-pinene catabolic pathway from *Pseudomonas putida*. To validate the efficiency of the promoters inducibility by alpha-pinene, two DNA constructs containing the Green Fluorescent Protein (GFP) under the control of each promoter and a terminator sequence downstream were built (P1\_GFP\_T and P2\_GFP\_T) and cloned into the plasmid pSEVA234, which is compatible with the *P. putida* strain. The goal was to infer if the supplementation of alpha-pinene could induce the expression of GFP and consequently fluorescence could be observed.

Two different molecular biology strategies were designed to clone each promoter: one eliminating the original operon lac and trc promoter from the plasmid pSEVA234, and another one cloning the DNA constructs directly in the original multiple cloning site of the plasmid. For the first strategy, the restriction enzymes used were *Pac*I and *Kpn*I, while for the last design, *EcoR*I and *Kpn*I were the selected enzymes. In Figure 1, a scheme of the constructed plasmids is shown.

![Plasmid](https://static.igem.org/mediawiki/2020/0/0b/T--NOVA_LxPortugal--Plasmid_2.png)

Figure 1 - Scheme of the constructed plasmids

The first step in this assay was to amplify both DNA inserts with the two different promoters (P1 and P2) and the terminator by Polymerase Chain Reaction (PCR). This technique allows the generation of copies of a specific DNA sequence in vitro, relying on a thermostable DNA polymerase, Taq polymerase. In a PCR reaction, it is necessary to add forward and reverse primers (short sequences of nucleotides flanking the target DNA sequence) that bind to the DNA template and can be extended by Taq polymerase with the addition of nucleotides.

The PCR protocol includes three main steps as represented in Figure 2: **denaturation** (at 98°C), in which the high temperature will denature DNA strands providing single-stranded DNA template; **annealing** (depending on the primers melting temperature), by lowering the reaction temperature the primer bind to their complementary sequences on the DNA template; **extension** (at 72°C), the step where Taq polymerase synthesizes new DNA strands through primers extension4.

![PCR](https://static.igem.org/mediawiki/2020/e/ea/T--NOVA_LxPortugal--PCRII.png)

Figure 2 - Scheme representing the main steps in Polymerase Chain Reaction main steps scheme. Created in BioRender

The genes used in this study were amplified by polymerase chain reaction (PCR) using Phusion High-Fidelity DNA Polymerase (Thermo Scientific) in a LifeECO Thermal Cycler (Bioer Technology). All DNA constructs and primers were purchased from IDT. DNA fragments were purified using DNA Clean and Concentrator DNA Kit from Zymo Research. The success of the PCR was checked by running an agarose gel electrophoresis. In Table 1, the sequence of the primers used in this project are summarized.

Table 1 - List of primers used in this study.

| Primer | Sequence (the restriction cloning sites are in italic) | Restriction enzyme |
| **P1\_GFP\_fw\_PacI** | cccccc*TTAATTAA*cgatcgcccatgggc | *Pac*I |
 **P2\_GFP\_fw\_PacI** | cccccc*TTAATTAA*ggctggggctgcaac | *Pac*I | **P1\_GFP\_fw\_EcoRI** | cccccc*GAATTC*cgatcgcccatgggc | *EcoR*I | **P2\_GFP\_fw\_EcoRI** | cccccc*GAATTC*ggctggggctgcaac | *EcoR*I | **P12\_GFP\_rev\_KpnI** | cccccc*GGTACC*agcccccagtcgatg | *Kpn*I |

Plasmids were extracted using NZYMiniprep kit (Nzytech). All digestions were performed using the appropriate FastDigest® restriction endonucleases (Thermo Scientific). Ligations were performed with T4 DNA Ligase (Thermo Scientific) and transformed by heat-shock in chemically competent *E. coli* DH5-alpha cells. The success of the ligation was checked through Colony PCR using DreamTaq (Thermo Scientific) and further confirmed by sequencing (StabVida). Protocols were performed in accordance with manufacturer’s instructions.

# Protocols

*Arabidopsis thaliana* germination assays

Murashige and Skoog (MS) medium

- 1x MS macro salts

- 10 g/L sucrose

- 0.5 g/L 2-(N-morpholino)ethanesulfonic acid (MES)

- 8 g/L agar

- Adjust pH to 5.7

MS medium + Dimethyl sulfoxide (DMSO)

- Four plates of MS medium with DMSO were made by adding 10, 20, 100, 200 and 400 µL of DMSO

MS medium + Spectinabilin

- Five plates of MS medium were supplemented with 0.05, 0.1, 0.5, 1 and 2 µg/mL of Spectinabilin

*Arabidopsis* seeds plate inoculation

1. Place enough *Arabidopsis thaliana* seeds in 1.5 mL Eppendorfs to cover the bottom
2. Add 200 µL of Ethanol 70%
3. Vortex each Eppendorf for 20 to 30 seconds
4. Remove the ethanol with caution to avoid seeds loss
5. Add 200-250 µL of sterile H2Odd
6. Vortex each Eppendorf for 10 to 20 seconds, followed by a spin-down to allow the seeds to seed
7. Remove the water
8. Repeat steps 5-7 three more times
9. Add 200 µL of sterile H2Odd
10. Using a pipette, place 10 seeds evenly over a line in the medium plate
11. Repeat step 10 until each plate has approximately 40 isolated seeds
12. Incubate the plates at 4°C, protected from light for 3 to 4 days to overcome seeds dormancy and to synchronize germination
13. Change incubation conditions to 20°C with 12h/12h photoperiod for 1 week

Promoters insertion into cloning vector pSEVA234

1. Amplification of P1 and P2 via Polymerase Chain Reaction (PCR)

**P1**: promoter 1 – GFP – terminator (BBa\_K3675004)

**P2**: promoter 2 – GFP – terminator (BBa\_K3675005)

2. Purify both inserts with the DNA Clean and Concentrator Zymo Research Kit
3. Digest both PCR inserts and plasmid pSEVA234 with the *Pac*I/*Kpn*I and *EcoR*I/*Kpn*I
4. Purify inserts and plasmid with the DNA Clean and Concentrator Zymo Research Kit
5. Ligation of inserts with the plasmid using T4 Ligase
6. Transform 5 μL of the ligation reaction into chemically competent *E.coli* DH5α cells by heat shock
7. Plate the cells in LB-agar medium supplemented with kanamycin and grow overnight at 37 °C
8. Colony PCR with appropriate primers to find successful clones
9. Grow overnight positive colonies in LB+Kanamycin
10. Plasmid extraction with NZYMiniprep kit and send for sequencing to confirm if the sequence is correct

##### References

1. Liu, M.‐J., Hwang, B.‐S., Jin, C.‐Z., Li, W.‐J., Park, D.‐J., Seo, S.‐T. and Kim, C.‐J. (2019), Screening, isolation and evaluation of a nematicidal compound from actinomycetes against the pine wood nematode, Bursaphelenchus xylophilus. Pest. Manag. Sci., 75: 1585-1593. doi:10.1002/ps.5272
2. Galloway LF, Etterson JR. (2007), Transgenerational plasticity is adaptive in the wild. Science 318: 1134–1136. doi: 10.1126/science.1148766
3. Burghardt, L.T., Edwards, B.R. and Donohue, K. (2016), Multiple paths to similar germination behavior in Arabidopsis thaliana. New Phytol, 209: 1301-1312. doi:10.1111/nph.13685
4. Garibyan L, Avashia N. Polymerase chain reaction. J Invest Dermatol. 2013;133(3):1-4. doi:10.1038/jid.2013.1

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# Team:NOVA LxPortugal/Results

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# RESULTS

[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### Computational results](#results_1)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### Germination rates](#results_2)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### Phenotype analysis](#results_3)
[![pine tree figure](https://static.igem.org/mediawiki/2020/c/ce/T--Nova_LxPortugal--pinefigure.png)

##### Promoters insertion into cloning vector](#results_4)

# Computational results

The results obtained from the *in silico* analysis are presented in the [Project Modeling](https://2020.igem.org/Team%3ANOVA_LxPortugal/Model) section.

# *Arabidopsis thaliana* germination assays

* ### Germination rate

The germination rates are shown in Table 1. These values were calculated as the quotient of the germinated seeds and the total number of seeds per plate.

Table 1 - Germination rates calculated for the three types of plates: Murashige and Skoog (MS), MS with Dimethyl sulfoxide (DMSO) and MS supplemented with spectinabilin.

|  | Culture medium | | | | | | | | | | |
| MS | MS + DMSO (µL) | | | | | MS + Spectinabilin (µg/mL) | | | | |
| 10 | 20 | 100 | 200 | 400 | 0.05 | 0.1 | 0.5 | 1 | 2 |
| Germination rate | 40/40 | 40/40 | 38/40 | 40/40 | 38/40 | 39/40 | 39/40 | 38/40 | 38/40 | 39/40 | 39/40 |

In Figure 1, it is shown a plate layout with both germinated and non-germinated seeds. Here, it is possible to observe that germinated seeds are easily identified by its green dicotyledonous. Also, we can observe a thin root growing out of the seed involucre with a closer look.

![germinated seeds](https://static.igem.org/mediawiki/2020/d/d5/T--Nova_LxPortugal--results1.png)

Figure 1 - Plate of MS medium with 400 µL of DMSO. (Right) Approximately 40 seeds were inoculated evenly in which only one non-germinated seed highlighted in red was observed. (Left) Germinated seeds macroscopic identification of dicotyledonous and root.

Overall, both DMSO and spectinabilin are responsible for a slight decrease in the germination rate, although this effect is not sufficient to compromise the germination and growth viability of *Arabidopsis thaliana*.

* ### Phenotype analysis

The phenotype analysis was performed through the observation of all seeds in a microscope. In Figure 2, the differences identified in roots of seeds inoculated in MS medium supplemented with DMSO or spectinabilin are depicted and compared with the MS medium.

![microscopic analysis of phenotypes](https://static.igem.org/mediawiki/2020/a/a4/T--Nova_LxPortugal--results2.png)

Figure 2 - Microscopic analysis of possible phenotypes triggered by the three different culture mediums.

Most seed roots which germinated in MS medium exhibit a curly and thick appearance. However, in the medium supplemented with DMSO or spectinabilin two distinct root forms are observed: one similar to the ones described previously and another one characterized by a thin and long filament appearance. Since spectinabilin is diluted in DMSO, it is not possible to conclude if the modified phenotype is a response only to the presence of DMSO or if spectinabilin also contributes to this aspect.

* ### Promoters insertion into cloning vector pSEVA234

Both promoter constructions were amplified by PCR. To confirm the success of the PCR, we run an agarose gel electrophoresis. As shown in Figure 3, a band of the expected size is visible for both inserts which confirms both inserts were amplified by PCR.

![PCR amplified products](https://static.igem.org/mediawiki/2020/8/8b/T--Nova_LxPortugal--results_3_correct.jpg)

Figure 3: Agarose gel electrophoresis (1% agarose) of PCR amplified products using species-specific PCR primer sets. Lane 1: 1kB plus DNA size marker (Thermo); Lane 2: Promoter 1-GFP-terminator *PacI*; Lane 3: Promoter 1-GFP-terminator *EcoRI*; Lane 4: Promoter 2-GFP-terminator *PacI*; Lane 4: Promoter 2-GFP-terminator *EcoRI*;

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