Team:NOVA LxPortugal/Notebook
March
iGEM kick-off session at ITQB NOVA
Week 1 (March 30 to April 5)
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.
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!
Around this time we also created our iGEM slack account. We could not be together so this was the way we found to communicate.
April
Week 2 (April 6 to 12)
Week 3 (April 13 to 19) and Week 4 (April 20 to 26)
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)
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.
The ideas are starting to flow. In this weekly meeting the team has come up with five potential projects for iGEM:
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)
Week 6 (May 4 to 10)
Week 7 (May 11 to 17)
Week 8 (May 18 to 24)
Week 9 (May 25 to 31)
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.
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.
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.
Team Hyaluronic acid was constituted by Daniela, Fabiana, Beatriz, Joana and Madalena, while the PWD team had João, Carolina, Tiago, and Isabel.
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:
Team human practices: Isabel, João - This week we focused on finding sponsors and potential stakeholders for both our projects.
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.
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.
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.
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)
Week 11 (June 8 to 14)
Week 12 (June 15 to 21)
Week 13 (June 22 to 28)
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.
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!
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.
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?
Continuation of the simulations in optflux, however the yields are still very low. Modifications on the model are being done.
First contact with ICNF (Conservation of Nature and Forests Institute).
July
Week 14 (June 29 to July 5)
Week 15 (July 6 to 12)
Week 16 (July 13 to 19)
- 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.
Week 17 (July 20 to 26)
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!
First reach out to the Science Communication and Image Office at ITQB-NOVA for the collaboration in the designing of our team Logo.
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.
This week we outlined some experiments that we intended to do to help validate our project:
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:
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.
The search for our model continues.
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)
Week 19 (August 3 to 9)
Week 20 (August 10 to 16)
Week 21 (August 17 to 23)
Week 22 (August 24 to 30)
Gathering of information about the pine wilt disease that was kindly provided by ICNF.
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.
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.
The reactions of spectinabilin formation pathway and (S)-methylmalonyl-CoA production pathway were added.
We ordered the genes of the spectinabilin and methylmalonyl-CoA production pathways. We also ordered the α-pinene inducible promoters together with GFP.
This week Fabiana, Madalena, Beatriz and Daniela started to plan the promotional video. We had too many ideas so choosing one was very hard.
Response from ICNF with statistics about the pine wilt disease in Portugal. Resipinus was officially associated with our project.
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.
Once again our model shows some problems in the production of spectinabilin, since the stoichiometry of some equations was not balanced.
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.
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:
Creation of our Instagram and Twitter accounts. We are so excited to start sharing our experience with everyone!
Webinar about soil biodiversity in collaboration with the Groningen team. Tiago did a great job representing our team in the presentation.
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.
Start of the discussion about the promotional video. This week Fabiana, Daniela, Beatriz, Madalena and Rita came up with ideas for our video.
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.
A lot of simulations are being performed every day!
Started planning the experimental work by searching and adapting protocols. We also started to book the necessary lab equipment to perform the planned experiments.
Update our instagram with information about Synthetic Biology, since several of our followers were unaware of what it means.
September
Week 23 (August 31 to September 6)
Week 24 (September 7 to 13)
Week 25 (September 14 to 20)
Week 26 (September 21 to 27)
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.
PINUS centre confirmed their interest in our work saying our solution could be of great interest and offered support for our project.
The storyboard for our promotional video is completed, so we are starting to shoot.
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.
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.
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
Gathering all the last details needed to fill the final safety form.
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.
We really did try to spell iGEM 2020, well… A for effort!
We have continued performing optimizations, but this time we are looking for reactions knockouts. We are fighters, so knockouts are our expertise!
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.
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.
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.
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!
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)
Week 28 (October 5 to 11)
Week 29 (October 12 to 18)
Week 30 (October 19 to 25)
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.
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!
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?
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!
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!
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.
Forestis showed interest in our project and gave us more information about the PWD in Portugal.
We proceed with the analysis of the best result.
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.
The wiki pages were all concluded. It was exhausting but totally worth it! The poster is on the making.
