iGEM 2024 — Team uma-malaga

{% extends "layout.html" %}
{% block title %}NOTEBOOK{% endblock %}
{% block lead %}*“Not only should you have a to-do list, but it must become your best friend.”*
- Jim Kwik{% endblock %}
{% block page\_content %}

[2023 September](#23-sep)

[2023 October](#23-oct)

[2023 November](#23-nov)

[2023 December](#23-dec)

[2024 January](#24-jan)

[2024 February](#24-feb)

[2024 March](#24-mar)

[2024 April](#24-apr)

[2024 May](#24-may)

[2024 June](#24-jun)

[2024 July](#24-jul)

[2024 August](#24-aug)

[2024 September](#24-sep)

[2024 October](#24-oct)

![](https://static.igem.wiki/teams/5466/static/notebook/start.png)

## September 2023

### 12th

**⬤ Attendance to Sonia Blanco's presentation at IBIMA-Plataforma BIONAND**

![](https://static.igem.wiki/teams/5466/static/notebook/10-09-23.jpeg)

On September 12th, some members of the UMA-MALAGA team had the opportunity to attend the lecture of the Doctor in Audiovisual Communication, Sonia Blanco, about how to disseminate science and use social networks successfully. In this talk, they were able to learn tools on how to take advantage of the booming potential of networks when disseminating science information faithfully, effectively and responsibly.

### 18th

**⬤ Brainstorming for project planning**

The members brainstormed about which problem to address. After various proposals, it was decided that the topic could be related to biomedicine and several interesting ideas were proposed that would mature over the weeks.

### 30th

**⬤ Team Building - Internal Communication**

![](https://static.igem.wiki/teams/5466/static/notebook/30-09-23.png)

On Saturday, we attended a workshop proposed by the networking instructor, Antonia. We learned the beneficts and the impact of a correct internal communication between team members, as well as assertiveness when it comes to conflict resolution and the importance of identifying and valuing the strenght of each one within the different areas.

## October 2023

### 1st

**⬤ Maturation of ideas**

Gradually, the most attractive ideas were selected. Ideas as diverse as a healing and sun protection cream, *Aspergillus* detection in hospitals, methods to prevent coeliacs from reacting to gluten and to avoid problems caused by aflatoxins. Everyone was very active in coming up with new ideas!

### 7th

**⬤ Laboratory Safety Introduction**

Together with the team instructors, all team members were taugh about basic principles of working with microorganisms and GMOs and the safety measures necessary to work in an experimental environment. We learned how to grow, treat and dispose of microorganisms.

### 12th

**⬤ Team Building - Escape Room**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-02.jpg)

The members of the team went to an Escape Room because we considered it a very good way to see how we work as a team in stressful situations. We could also have a good time together and get to know each other better. Of course, althought only one team was able to win the game, we were all winners in terms of camaraderie.

### 13th

**⬤ Team Building - How to speak in public and make amazing presentations**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-03.png)

Networking instructor Antonia conducted a second workshop at the Faculty of Science where we learned basic notions and tools to overcome the fears of public speaking and have a good presence in front of an audience.

### 20th

**⬤ The approach of a possible final idea**

We selected the idea that we thought was the most interesting and technically feasible. The goal of our project would be to tackle the problems caused by aflatoxins, for which the whole team searched for literature and information on previous projects in iGEM before going on with this adventure.

### 25th

**⬤ Demo Day of Link by UMA**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-04.png)

On October 25th, an event called Demo-Day was organized at the Rayo Verde building of Link by UMA, which was attended by our team member Lucía Campos and PI Dr. Manuel Bernal. They made a brief presentation explaining the evolution of our ideas and presenting the iGEM competition.

### 26th

**⬤ Get-together "A germ in Synthetic Biology"**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-05.jpg)

The members of the UMA-MALAGA team, Lucía Campos and Ángel Navarro, gave an informative talk about what is understood by synthetic biology and its application. This workshop was possible thanks to the support and trust provided by UMA Divulga.

## November 2023

### 6th

**⬤ ⬤ UMA-MALAGA team meeting**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-09.jpg)

The team met at the Faculty of Science of the University of Málaga to plan the start of the project, after accepting the idea of developing a probiotic with the ability to capture AFB1. During the meeting, we discussed several key points to analyse how the selected problem could impact the population and to which sectors our solution could be relevant. To facilitate this assessment, we decided to create a paper outline that would allow us to visualise all the important aspects of the project in a clear and orderly manner.

### 7th

**⬤ Logo creation training**

Two of our colleague, Ángel Navarro and Lucía Aguilar, held an online meeting with Mónica Sanz, photographer and in charge of the image of many large companies. In this meeting, we learned the most important keys on how to design a logo and how to use the righ tools for it; the most important points to take into account when creating the name of the projects, and, of course, how to get a quality corporate image.

### 8th

**⬤ First presentation of ideas for the project**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-10.png)

After several working meeting, we presented a barinstorming to the PI's responsible for UMA-MALAGA where we showed the intial organization that we expected to implement, sectors we wanted to address and the logo with which we wanted to be associated.

### 11th

**⬤ First Dry Lab meeting**

The Dry Lab team met for the first time to discuss the more technical aspects of the project. They discussed how to capture AFB1, synthetic receptors, information on *Lactococcus lactis*, among others.

### 16th

**⬤ Talk at Torre Atalaya high school**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-11.png)

On the 16th, we gave our first talk to students of Torre Atalaya high school where we showed AflaxOFF in a dynamic and fun way. It was our first public talk, how thrilling!

### 20th

**⬤ Meeting with Tecnalia**

We visited the applied research and technological development centre Tecnalia. We explained the ideas we had about our project and asked them for feedback to improve and optimise our product sales.

### 24th

**⬤ Meeting with Torcuato**

The UMA-MALAGA team met with Torcuato Fernández García, who had worked as a consultant in different medical and pharmaceutical companies. We presented our work to him and he helped us to focus the project on different areas, without pigeonholing us in one. We discussed the purpose of our project applied to different sectors such as food, biotechnology, medical, veterinary and even bioremediation.

### 25th

**⬤ Meeting with advisors**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-12.jpg)

First meeting with the advisors, where we introduced them to our project concepts and the techniques we plan to use to detect aflatoxins. During the meeting, we received feedback on the errors present in our approach, as well as constructive criticism that helped us to identify areas for improvement.

### 30th

**⬤ First brainstorming in Dry Lab**

We started to develop, from the bibliography found, the genetic circuit, with still several options on the table. We started to work with a more engineering view, breaking down the functions our probiotic has to do in terms of *input module* and *output module*, and their relevant submodules. We also learned how to use Benchling to facilitate the recording of the sequences and the construction of the circuits.

## December 2023

### 5th

**⬤ Presentation of progress to the rest of the team.**

Colleagues from Human Practice and Dry Lab updated team members on their progress. In Dry Lab the information regarding genetic circuits, and in Human the advances in the search for funding.

### 11th

**⬤ ⬤ First Day - Informative session about UMA-MALAGA for possible new members**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-13.png)

The UMA-MALAGA team held an informative day at the University of Málaga in aim of attracting different profiles of various educational branches in order to raise the opportunity to participate in the AflaxOFF project. Thanks to this, we got people from computer engineering, health engineering, communication and law degrees interested in participating in the adventure that is iGEM.

### 15th

**⬤ Lecture on Artificial Intelligence with Sonia Blanco “Artificial Intelligence, your ally or your enemy in the classroom?”**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-14.png)

On December 15, members of the UMA-MALAGA team attended another magnificent lecture given by Dr. Sonia Blanco about the use of artificial intelligence in education. This talk inspired us to make changes in our project.

### 18th

**⬤ ⬤ Second "New Members" Day**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-15.png)

On this day, we organized activities to see the potential of our new members. We carried out activities and games where we promoted internal communication, team building and, above all, the values of the UMA-MALAGA team.

### 23rd

**⬤ Team Building - Christmas dinner**

With the Christmas spirit in the air, the members of the AflaOFF team decided to organise a more festive dinner to get to know each other better outside of the iGEM work environment. We had a great time!

## January 2024

### 12th

**⬤ Team Building - Dinner with new members**

The new members have arrived! To integrate them into the work dynamic and the team, we organised a general meeting in which we explained our working method and the areas in which they could collaborate to push the project forward. We know that integration can be exhausting, so, after the meeting, we decided to go for a typical Málaga dinner, a campero, which allowed us to get to know each other better in a more relaxed atmosphere.

### 13th

**⬤ The genetic circuit is finished**

The gene designs were finalised, and the idea of using *Lactococcus lactis* was abandoned as there was no literature on synthetic receptors in this species, a key point of our project.

### 25th

**⬤ Presentation of the genetic circuit to the PI's**

We showed our design to Carlos, one of our PI's, who helped us to revise them to avoid small errors in order to send them to synthesis. Together with the circuits, we presented the associated bibliography to demonstrate their robustness at a theoretical level.

### 30th

**⬤ Gene synthesis**

Our designed parts were ordered, finally materialising in a period of effort. In addition, it was decided to use the collection of pSEVAs as vectors.

## February 2024

### 1st

**⬤ General meeting**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-16.jpg)

In this meeting, a general tour of the social networks of UMA-MALAGA was made, focusing on the purpose of each one and the use of LinkedIn. The email signature of all members were formalised, the work areas were delimited and doubts of all members were solved. In addition, we discussed the approach we want to give to Instagram and X posts.

### 5th

**⬤ Colonia Santa Inés Day Care Center Workshop**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-17.jpg)

On the morning of 5th February, we had the opportunity to give a workshop at the Colonia de Santa Inés day centre, where people with different types of cognitive disorders live. Here, we were able to show them in a playful and entertaining way what synthetic biology is, the real applicability it has and the work we do. In addition, after giving the talk, we raised the possibility that, in the future, we could join forces with this and other day centres to seek the inclusion of these people in our work.

**⬤ Meeting with the Mayor of Málaga and with the President of the 11th District Council**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-20.jpg)

The Mayor of our city, Francisco de la Torre, and the President of the 11th District Council, Borja Vivas, were also going to visit the facilities of this day centre, and thanks to the fact that they liked the workshop we had given that same morning, they decided to invite us to visit and formally present our work to both of them. Thus, we had a conversation in which both were receptive to know our initiative and to strengthen the links we had with the City Council of Málaga.

### 7th

**⬤ General meeting: Day of reflection**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-21.jpg)

Today we had our first general meeting after welcoming the new team members. We worked in groups to further elaborate on various aspects of the aflatoxin's problem, such as its scope, regulations and ethical implications. We shared ideas on how to approach the next feedback loop and discussed Special Prizes that the team could focus on. We also added some new features to our team’s website.

### 8th

**⬤ UPA meeting**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-22.jpg)

In the meeting with the Unión de Pequeños Agricultores y Ganaderos, Ángel Navarro, Adrián Gómez and Lucía Aguilar presented the project. They placed great emphasis on the problem of aflatoxins in the agricultural sector and on the methods of action and prevention that are active today to solve this problem. The conclusion was that there are no simple and cheap methods that farmers and ranchers can use routinely. In addition, it is the cooperatives that are responsible for analysing the samples they received and taking the necessary measures. Finally, a series of contacts were obtained to contrast this information.

### 10th

**⬤ Talk at ASA (High Ability Association)**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-23.jpg)

We gave a talk to the kids of the High Abilities Association of Málaga about the UMA-MALAGA team, the Grand Jamboree competition and the possible outlets for students in science, which allowed the attendees to ask questions about their possible participation in future editions of UMA-MALAGA and gave us feedback with their opinion of our idea, AflaxOFF.

### 13th

**⬤ Meeting with Cordis**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-24.png)

In the meeting with Cordis Software Solutions, Adrián and Lucía Aguilar met with Jaime Mahillo, to whom they presented the AflaxOFF project and the different areas of work. They discussed the work done by our software engineering colleagues to assess the feasibility of it. We got advice on the importance of organisation in developing good software and the time usually spent on it.

**⬤ Meeting with Toomba**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-30.png)

On February 13th, our colleagues Álvaro and Lucía Aguilar had the opportunity to meet with Patrick Berends, Raúl Domínguez and Jordi Van der Ven; all of them belong to the company Cordis Software Solutions. We presented our project, from the most experimental to the most theoretical part. In addition, the possibility of support and intervention in the IT part of the project was valued, where it was agreed to develop a more in-depth presentation of the objectives we had and the deadline of the work.

### 15th

**⬤ Meeting with Promega in Madrid, attended by professors Carlos and Manuel together with Lucía Campos and Alonso**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-26.jpg)

Today we were lucky enough to visit the offices of the biotech company Promega in Madrid. There we had a meeting with Gijs Jochems, director of Promega Biotech Ibérica, and with several of their marketing specialists, molecular biology PhDs and functional analysts (among many others). We presented the AflaxOFF project and received feedback on how to address aflatoxin issues and on future proposals for iGEM. In addition, we were provided with help and solutions for the future in case we had any doubts when we started working in wetlab.

### 16th

**⬤ Talk at IES Jardines de Puerta Oscura High school**

On 16th February, we visited IES Jardines de Puerta Oscura to give an informative talk on synthetic biology and to present the UMA-MALAGA initiative, as well as our new project, AflaxOFF.

**⬤ Structuring our own website**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-27.png)

On the 16th of February, work started on the WordPress of the AflaxOFF website. A gallery was made for each team of UMA-MALAGA showing the members and the projects of previous years.

### 19th

**⬤ Meeting with CEM**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-28.jpg)

Our colleagues attended the meeting with Felix Infante, technician of the Confederation of Entrepreneurs of Málaga. In this meeting, our project was presented and we discussed different proposals for improvement.First, the entrepreneurial nature of the initiative and the possibility of enhancing its business projection in the future.Secondly, the feasibility of such an ambitious project, especially under the possibility of creating software in parallel to the laboratory work, which simplifies the dynamics in this area.

### 22nd

**⬤ Meeting with ASP Ozono**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-29.jpg)

ASP Ozono is a company dedicated to the disinfection of spaces and food products by different methods, among which they are specialised in the use of ozone. After presenting our project to them, we received a very satisfactory response to the approach. They congratulated us for our work and encouraged us to orient it towards the agricultural field to improve food quality.

### 23rd

**⬤ Team building in Juan Antonio's Dojo**

Today we participated in a self-defence workshop given by our colleague Juan Antonio, a black belt in the martial art known as Shorinji Kempo. Although the workshop was of an individual nature, it fostered communication between us and allowed us to connect on a more personal level.

**⬤ Sponsorship of Publikit**

Our first sponsorship came from Publikit, who prepared t-shirts for the AflaxOFF team. After contacting Jesús and telling him about iGEM and the AflaxOFF initiative, he showed immediate interest in supporting us. In return for his generous contribution, we made some posts on his social networks showing the t-shirts and thanking Publikit for his support.

### 28th

**⬤ Human Practices meeting**

This meeting was dedicated to reflection and the distribution of tasks of Integrated Human Practices. After the day of reflection on February 7, we considered how we could obtain information from society about the problem, how to make our project ethical and responsible, and how to approach Special Prizes. All this reflection materialized in several tasks.

### 29th

**⬤ Collection of aflatoxin data from hospitals**

We contacted 18 different public and private hospitals in Málaga, as well as the General Organization of Toxicology to obtain information on the incidence of aflatoxins in our region. This allowed us to consider the possibility of carrying out our own study for the team, which would allow us to know precisely the scope of the project.

## March 2024

### 5th

**⬤ General meeting**

In this meeting we discussed how to start a crowdfunding compaign to obtain funding for the project. We discussed the content that should be included in the texts to request its opening and we evaluated which would be the most efficient channel for its dissemination in society. In addition, we designed a series of videos to share on our social networks in order to promote the crowdfunding in advance.

### 7th

**⬤ Update of the website of the team**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-31.png)

With the idea of subsequently shaping the contents of the website, we began to set up the entire team's website. To do this, the explanatory texts for each section were uploaded in general and the initial presentation video showing the team and highlighting its objectives.

### 11th

**⬤ Wet Lab Team debut**

This week, the Wet Lab team started working to get used to the basic safety measures and protocols applied in Synthetic Biology. The 6 Wet Lab team members were divided into pairs to ensure an appropriate learning environment for all the members (there is no room for 6 people pipetting at the same time.) In addition, working in pairs allows one person to perform an experiment while the other ensures that correct technique and safety procedures are followed.

### 17th

**⬤ Opening of a Crowdfunding**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-31.png)

With a view to financing the team’s participation in the competition, we developed during the previous month the content shown on the crowdfunding platform GOTEO. Through it, we managed to raise the money necessary for the attendance of two more members for the competition, as well as funds for the development of material for informative events and for the competition itself.

### 18th

**⬤ First Steps in the Lab**

With the growth media (LB, 2YT) and competent *Escherichia coli* cells obtained last week, we decided to try to get a first stock of SEVA plasmids. We started to keep track of our experiments in our laboratory notebook and performed our first transformations.

### 20th-22nd

⬤ Attendance at Foro Transfiere

![](https://static.igem.wiki/teams/5466/static/notebook/nb-34.jpg)

We attended a meeting at European level on science, technology and innovation where we had the opportunity to talk and share information with various agents such as employers or technology and research centers as well as organizing bodies such as the Junta de Andalucía. Thanks to the synergies established, we obtained advice for the development of our project, taking into account socio-economic and commercial factors, we applied everything we had learnt to our business plan.

### 25th

**⬤ Troubleshooting Plasmid verification Problems**

We are having a few problems recovering plasmid DNA from our precultures, getting some low concentrations or purity of extracted DNA, leading to failure in DNA digestion verification. We are working very hard to improve our technique and hope to get better results soon.

### 26th

**⬤ First contact with IBIMA Plataforma BIONAND**

We contacted IBIMA Plataforma BIONAND with the objective of exploring a possible collaboration, given the importance of the sector they work in. The interaction proved to be successful as a couple of months later we had the opportunity to visit their facilities. During the visit, we were able to continue the conversations and advance our collaborative discussions.

### 27th

**⬤ Defining the aesthetic of the website**

With the aim of offering the public a serious and quality presentation letter, on 27 March, we defined and implemented a characteristic style already defined for the project's presentation web page. Once that was done, we homogenised the rest of the pages that made up the UMA-MALAGA website following the previous criteria in order to offer a better visual experience.

### 29th

**⬤ First SEVA stock**

After solving the problems with Minipreps and digestions, we managed to get our first stock of pSEVA 181 with a red fluorescent protein-expression device as a dropout. Now we are ready to transform new DNA parts into this plasmid!

## April 2024

### 2nd

**⬤ Meeting with the Vice Dean Carmen del Rocío Monedero Morales**

The social networking team met with the Vice-Dean of the Faculty of Communication Sciences, Carmen del Rocío Monedero Morales, in which an agreement was reached for the use of the facilities, specifically the photography set and the radio room.

### 3rd

**⬤ Possible collaboration with Toomba**

After a first approach to the company TOOMBA, it was agreed that an explanatory document would be presented showing the possible software to be developed jointly. However, after several attempts at compatibility, no agreement could be reached.

### 4th

**⬤ Cloning of basic DNA parts**

We started cloning basic parts (Level 0) such as constitutive and inducible promoters, RBS with different strengths, reporters and terminators into our pSEVA 181 in order to get a toolkit for further Wet Lab experiments.

### 5th

**⬤ Contact with Unicaja Foundation**

We saw in the Unicaja Foundation, which belongs to the Spanish bank of Málaga origin Unicaja, the perfect opportunity to publicize the AflaxOFF project in society having as support an entity of such renown and given to support a wide variety of scientific projects. So, that day we spoke for the first time with Miguel Gil, director of Social Activities of the foundation, who was very interested in collaboration and with whom we kept in touch during the following months.

### 10th

**⬤ Chassis change**

In reviewing the literature, we encountered a problem. There were no papers confirming that aflatoxin could pass through the bacterial cell wall, which undermined our idea of using a receptor. There was only evidence to suggest that it could damage the cell wall. In view of this, we decided to change the chassis. We opted for yeast, whose papers did show that aflatoxin could pass through its cell wall. As a result, we immediately started the new development.

### 11th

**⬤ Presentation of the project to Fernando Leguina Roig, from the Department of Education and Employment Promotion of the City Council of Málaga**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-35.jpg)

On April 11th, our colleagues Ángel Navarro and Lucía Aguilar met with Fernando Leguina to present our project and preserve the legacy of the UMA-MALAGA discipline. After a very satisfactory presentation, they discussed topics such as the official presentation of the team, or the possibility of establishing synergies thanks to his support.

### 19th

**⬤ Blend Hub Meeting**

The team met in the T1 classroom of the Faculty of Science for a call with Juan José Valderrama, Innovation Director of the company Blend Hub. The project was presented to him and he was shown different ways in which his company could collaborate. He identified some of the failures we had when it came to exposing a company. After giving us his feedback, he invited us to make a new presentation following his advice and with a defined deadline, which he would later revisit, and give us his opinion again.

**⬤ New yeast genetic circuit**

It was a challenge to go from designing a genetic circuit in prokaryotes to eukaryotes, so we decided to keep the same approach we designed for bacteria but adapt it to yeast. We presented our research and provisional circuits to our colleagues.

### 22nd

**⬤ First Integrated Human Practices loop meeting**

We caught up with the work done in Human Practices. On one hand, the task of communication with companies and personnel directly related to aflatoxins (loops) was addressed. On the other hand, the focus of the Inclusivity Special Prize was finally decided.

### 26th

**⬤ ⬤ Presentation model advised by Blend Hub**

Thanks to the collaboration of Juan José Valderrama, Innovation Director of the company Bledhub, we received the necessary advice to make an appropriate presentation for companies, which would allow us to understand the different mechanisms we use and their objectives. This was very useful for us to apply it later to the rest of the meetings we held with important entities.

**⬤ University fundraising party**

In order to raise funds in a more informal way, the UMA-MALAGA team organised a university party, offering music, drinks and a fun atmosphere. As part of our commitment to promoting science among young people, we also organised a trivia game in which we tested the attendees' knowledge of synthetic biology.

### 30th

**⬤ Application for European Researchers’ Night**

Since we want to bring synthetic biology research closer to as many people as possible, we considered that taking the opportunity to participate in the European Researchers' Night was something we could not miss. Therefore, we met online to plan the activity that we would organise for this event, and we concluded that a micro-meeting in which we would talk about the socio-health aspects of our project could be the most interesting. After sending it to the central service of scientific dissemination of the University of Málaga, they gave us the go-ahead, so we continue our work for September.

## May 2024

### 6th

**⬤ Second Integrated Human Practices loop meeting**

We shared the progress of the Human Practices work and agreed to attend different companies in person to learn about the aflatoxin problems first hand.

### 8th

**⬤ First photoshoot for UMA-MALAGA members**

The first photo session for UMA-MALAGA members took place in the photo lab of the Faculty of Communication Sciences.

### 9th

**⬤ Visit to BIOLES cooperative**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-36.jpg)

This company is responsible for the packaging of organic production, as well as its distribution for sale. Their main products are dried fruits, such as nuts, legumes and cereals, although they have also worked with food spices. In the meeting with Carlos Aragón, co-founder of the company, we were able to learn about real cases of mycotoxin contamination in food that reach the market, and how to act in those situations. We also learned about how quality control works, who is responsible for carrying it out, and the traceability of food that may be contaminated.Undoubtedly, it was a very productive and rewarding meeting.

### 11th

**⬤ Team building and conflict resolution workshop by Nogal**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-37.jpg)

To ensure the efficiency and comfort of all team members, it is important to establish a joint work dynamic, learn how to manage conflicts and have enough confidence to communicate your personal situation to the rest of your colleagues. That is why we organized a workshop in which we addressed these points through various reflections and dynamics.

### 13th

**⬤ New team member: Jimena from Translation and Interpreting**

Given the team's need for someone with a solid knowledge of foreign languages and a strong desire to work, we valued and decided that Jimena García from the Translation and Interpreting degree would become part of the team.

### 14th

**⬤ Visit to DCOOP**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-38.jpg)

As part of our meeting with different sectors to learn about the aflatoxin problem first hand, we visited the cooperative DCOOP, a world producer of olive oil. We were welcomed by the head of R&D and the head of the laboratory to explain their relationship with aflatoxins, the quality controls their products go through, show us their facilities and give us feedback on what our project could contribute and what we could improve.

### 16th

**⬤ Fill in the Safety Form**

The team met to answer the questions on the Safety Form. The meeting generated discussion on the issues and implications that our project could have.

**⬤ Second photoshoot of the UMA-MALAGA members**

The second photography session for UMA-MALAGA members was held in the photography laboratory of the Faculty of Communication Sciences.

**⬤ Meeting with the Diputación Provincial de Málaga**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-39.jpg)

In the meeting with the Diputación Provincial de Málaga, our colleagues, Ángel Navarro and Lucía Aguilar, had the opportunity to present our project to…, and to talk about our values as a team and our short and long term goals.

### 20th

**⬤ Third loop integrated Human Practices meeting**

After having visited several companies in person, we met to share all the relevant information we had gathered over the last month about the aflatoxin problems in society, and how it affects the primary, secondary and tertiary sectors in particular.

### 21st

**⬤ Contact with Canal Sur**

Given that in previous years Canal Sur had given us coverage on the television of our autonomous community, we again proposed to them to make a report to appear in the programme Tesis. We didn’t think they were going to answer us so quickly, the next day the recording was going to take place!

### 22nd

**⬤ TV Report for Canal Sur**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-40.jpg)

Today, we welcomed Paula Peralta from Cedecom to record a report about our AflaxOFF project. It was a great opportunity for the team to communicate their ideas and raise a wide local audience. Don’t miss out Lucía A and Daniel, coming soon on Canal Sur in the programme Tesis!

### 24th

**⬤ Visit to IBIMA plataforma BIONAND**

The team was invited to visit the Institute of Biomedical Research of Malaga, where different researchers briefly presented their current lines of research, we visited the different laboratories of the centre and we had the great opportunity to talk with them during a break. As a result of this visit, the collaboration with this entity to which we are so grateful arose.

### 25th

**⬤ Visit to Sabor en Rama**

Another of the companies that took the time to help us and provide us with information about how aflatoxin B1 affected their business was Sabor en Rama. We met their manager, who gave us information about the protocols they have to follow for the correct maintenance of the facilities, in order to prevent their product from being contaminated by aflatoxin B1. He also promised that, when we needed it, we could visit him again and he would give us more information. Without a doubt, it was a very rewarding experience to get to know this company closely!

### 26th

**⬤ Meeting for the Special Prize Inclusivity approach**

After a reflection on the different social groups that may have difficulties in accessing science and inspired by the talk we have previously given in a Mental Health Day Center, we decided that working with people with conditions such as intellectual disability could be a benefit to society and the inclusion of differences in science. Thus we agreed to participate in Inclusivity’s Special Prize.

### 28th

**⬤ Final Constructs Presentation**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-42.jpg)

This time, the Dry Lab team put aside the computers to capture all the ideas on the chalkboard. Genetic constructs were presented to our PI Carlos, including the final target design, as well as other transcriptional units for further validation of our parts. We discussed our synthesis strategy in order to take full advantage of the special offers provided by the official iGEM’s sponsors and decided on a top-down approach to test our designs.

### 30th

**⬤ General meeting**

Dry Lab informed the rest of the team on the final yeast constructs, and explained the new functions it has compared to our original design in bacteria. Since the design followed the same idea as the first one, Human Practice had almost no need to adapt their work, only to introduce these new functions when talking to companies or organisations.

## June 2024

### 20th

**⬤ Project description**

After collecting bibliographic information and specifying the main points of our project, we wrote the preliminary project description. This meant the opening of our Wiki and the first content that we uploaded. We are so excited!

### 21st

**⬤ Essential wiki preparations**

Before starting to work fully on the wiki, basic preparatory work was carried out, including an analysis of how the wiki works and the repository rules associated with the wiki on the IGEM servers. In addition, the ‘Description’ tab contains a description of our project, marking the rest of the pages as *under construction*.

## July 2024

### 3rd

**⬤ Distribution Kit Plates Test**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-43.jpg)

The Distribution Kit Plates are finally in our laboratory and we have tried transforming some plasmids today to check that the DNA has arrived in good condition after its long journey from Asimov’s laboratories. We can't wait to test the Open Yeast Collection (OYC)!

**⬤ Video game Brainstorm**

On the 3rd of July we started a new challenge with the aim of participating in the Special Prize for Education. We started to develop our educational video game, focusing on brainstorming about the possible design of the story, the characters that could appear and the path that will guide players through the experience.

### 4th

**⬤ ⬤ Wet Lab & Dry Lab Update**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-44.jpg)

The last few changes in genetic constructs and assembly strategy were communicated to the Wet Lab members. Once everything was clear, we proposed a workflow for the following weeks and coordinated to get the lab running again.

### 5th

**⬤ First idea for the characters**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-45.jpg)

Initial sketches of the characters for the educational video game are presented, each designed to represent a specific area of the work team. Each character reflects the characteristics and skills associated with their respective role, creating a connection between the dynamics of the team and the narrative of the game.

### 6th

**⬤ First script and final characters style**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-46.jpg)

At the same time as working on the character sketches, an initial draft of the script of the video game is generated, in which the structure and theme of the game is specified.
After a meeting with the members of the team, the colour of the characters is decided and the final sketches and designs are obtained.

### 9th

**⬤ ⬤ Madrid European MeetUp Deliverables**

Getting ready for the iGEM European MeetUp organized by Madrid-UCM. Today we finished designing our first Team Poster to present our project in Madrid and practised our speech. Late-night bus today for Ángel and Daniel in order to make it to Madrid. We are very excited to meet the other teams and projects and exchange ideas with all the participants!

### 10th

**⬤ First day at the European MeetUP in Madrid**

After a long overnight bus ride, we met in the morning with iGEM team members from Madrid, Maastricht, Stockholm, Paris and Leuven. On this first day we were able to enjoy several talks, snacks and free time. We really enjoyed the company and the knowledge we got from the other teams.

### 11th

**⬤ Second day at the European MeetUp in Madrid**

This was the most important day for our project. We brought with us the poster that we had designed a couple days of before, and in front of a jury of PI's from the UCM team, as well as iGEM ambassadors, we defended in 3 minutes the key ideas of the project. Then, it was our turn to defend the project in more detail through a 10 minute presentation, where we explained certain concepts in more detail and showed how multidisciplinary our team is. After both presentations, the jury evaluated the work we had done and gave us a lot of feedback. Once the hardest part was over, we went to lunch…and to get to know the University and Madrid!

### 12th

**⬤ First transformations from the iGEM Distribution Kit Plates**

After testing the Kit Plates, we started working with the DNA Parts needed for the assembly of a plasmid for *Saccharomyces cerevisiae* using the OYC.

### 15th

**⬤ General meeting**

Once the exam period was over and after attending the MeetUP organized by the team of the Complutense University of Madrid, we saw the need to organize a face to face meeting in which we could share everything we had learned during the event, distribute the work in order to attend the CEEBI, and we could properly establish the work for the rest of July and part of August. We also discussed in detail some aspects of the Wiki fundamental to organize our work and taking into account all members’ opinions.

### 16th

**⬤ First verified stock**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-47.png)

Today, we obtained our first DNA and glycerol stock from a bacterium transformed with an OYC plasmid. The miniprep DNA was confirmed by digestion with BsaI and electrophoresis. It is great to see the DNA bands with the expected size!

**Elaboration of the final script of the video game**

Once the structure of the video game and the parts to be shown were defined, the team started working on the collaborative writing of the final script. In several sessions, the narrative, characters and dialogue were discussed, ensuring that the story was coherent and engaging, aligned with the overall vision of the project.

### 17th

**⬤ Inclusivity Special Prize: Contact with centers**

We initiated contact with the Mental Health Centers via e-mail and telephone. They all seemed so happy with our proposal, and we were so excited too!

### 19th

**⬤ Participation in the III Congreso Estatal de Estudiantes de Biociencias (CEEBI)**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-48.jpg)

We attended the third edition of the State Congress of Biosciences Students (CEEBI), where we were offered the opportunity to perform several activities: First, a Pechakucha; a short talk in which we gave the attendees a few hints about iGEM, synthetic biology and our project. After that, the business forum began, where we had a stand dedicated to the dissemination of information about the institution and community of iGEM and about our project. Finally, we organized a workshop where we could explore an extended explanation about synthetic biology and our project in a playful and interactive way.

### 20th

**⬤ Start of the development of the video game**

The implementation of the different scenes of the video game begins, while, in parallel, the mini-games that will be integrated in later phases are designed.

### 21st

**⬤ Notebook writing**

All the members meet to clarify the format of the notebook and start writing it. We worked in groups to speed up the process and have it finished as soon as possible, and we agreed to record all the events directly in a common document. Thus, we started to compile all the information in a definitive way to be able to transfer it to the team Wiki.

### 24th

**⬤ Starting with Wiki work**

The members responsible for software development carry out extensive research on visually appealing wiki designs, collecting examples of good and bad practices (*must do* and *>must not*). We then select the most attractive approaches according to our own criteria.

## August 2024

### 2nd

**⬤ First Golden Gate Assembly**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-51.jpg)

We have successfully assembled our first fragment through Golden Gate. We constructed a transcriptional unit for constitutive expression of green fluorescent protein (GFP), which will be a valuable tool for future cloning experiments. Our next goal will be to perform the protocol with a larger number of fragments to assemble our shuttle plasmids.

### 5th

**⬤ Meeting with Sergio Ragel from Cervezas Victoria**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-52.jpg)

That morning we visited the facilities of Cervezas Victoria, where we spoke with Sergio Ragel, head of External Relations of the company. Since Cervezas Victoria is a Málaga brand and they work with barley, food that is easily contaminated by AFB1, we arranged a visit to their quality control center to obtain data for the Human Practice work. They also consolidated their position as sponsors of the team, offering to provide beer, bottled water and free access to their facilities for future events.

### 5th

**⬤ Keeping on with the lab work!**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-53.jpg)

Even though we are in August and the heat strikes, the lab work continues at a great pace! Today we decided to try out a new technique: colony PCR. These are days of intense work and sometimes it can be hard for our team members to keep the neurons working...Ángel even got confused and claimed that A was paired with G. He should rest until tomorrow.

### 12th

**⬤ First workshop for Inclusivity Special Prize: CEPER**

During the morning of the 12th, all the work that Nogal had designed began to take shape in that first visit to the CEPER center for people with intellectual disabilities. This first session consisted of getting to know the needs and motivations of these people in a playful way, through games and questions and doing experiments to stimulate their creativity and participation. We wrote down a lot of ideas they were telling us and, together with what we learn in more intellectual disabilities centers, we will do more sessions that we hope they enjoy a lot and get them involved.

**⬤ Visit to Piensos Campillo**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-54.jpg)

That day we also had to continue with the work of Integrated Human Practices. We managed to talk to a company that manufactures cattle feed, one of the areas more affected by AFB1 contamination. There, they showed us the preventive measures they apply, as well as the complete feed preparation process. We also visited the quality control laboratory and were given samples of feed and aflatoxin B1 sequestrant, which will be very useful for comparative experiments with the control techniques already used.

### 13th

**⬤ Start of wiki development**

All the documentation collected throughout the project starts to be translated into final documents. The essential elements of the website are implemented, such as the navigation bar, the side menu, the footer and the team page.

### 14th

**⬤ Day of Reflection**

On this day, the team met to discuss again certain issues concerning our project, for example, we reflected and sought information about the future projection of our product, whether it fosters inequalities, the associated risks... In addition, we considered whether our project had followed the established ethics. It was a very productive day that also helped us to write in the wiki.

### 15th

**⬤ General Meeting to see the video games progress**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-55.png)

On Thursday 15, the current state of the game was presented to the team in order to get feedback on possible areas for improvement. This will allow to correct potential bugs and ensure a satisfactory user experience, while facilitating the learning of key concepts through dialogues and mini-games.

**⬤ Meeting with Carlos from the iGEM team at Pompeu Fabra University in Barcelona**

Our colleagues met online with Carlos, a member of the iGEM team at Pompeu Fabra University in Barcelona. In this meeting, besides getting to know each other and presenting our projects, we discussed a possible collaboration between both teams in the educational and informative field. Both teams are applying for the special prize of education, so we have both developed a project that we will present in schools, high schools and universities; reaching from Málaga to Barcelona, in both directions. We are looking forward to take our work to the classroom with UPF-BARCELONA!

### 26th

**⬤ First workshop for Inclusivity Special Prize: Aidis**

With the same methodology, we tried to get to know the interest of the people in this center, as well as explaining our project in a visual and practical way.

### 29th

**⬤ Recording voices for the Promotion video**

The dialogue that would later appear in the promotion video was recorded by team members Lucía campos and Álvaro Acedo

### 30th

**⬤ First workshop for Inclusivity Special Prize**

Following the methodology previously used, we discover the interest of this center and explained our project to them in a practical and visual way.

**⬤ ProGnosis sponsorhip**

After visiting Piensos Campillo earlier this month, we decided to talk to ProGnosis, the company that provided them the test to detect AFB1 in their samples. After talking to Jesús Romero, Iberian Sales Manager at the company, they decided to sponsor us and provide us with two different ELISA kits to analyse the capability of our engineered yeast to capture the toxin. We are so thankful for it!

## 2024 September

### 3rd

**⬤ First workshop for Inclusivity Special Prize: APROINLA**

As we did in the other workshops, we try to explained to them our project in an entertaining way.

### 6th

**⬤ Presentation Video**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-56.jpg)

On Friday, September 6, we were able to share with the world the AflaxOFF presentation video. In it we show the problem we are facing and the solution which we have developed thanks to synthetic biology, to put an end to it.

### 7th

**⬤ Second workshop for Inclusivity Special Prize: Aidis, FAISEM, CEPER and APROINLA**

In this second session, we started phase 2 of our Need-based response methodology, which consists of trying to hypothesise and answer the question posed in the first session. To do this, we taught them how to search for valid information using new technologies and encouraged them to make a graphical representation of the results found.

### 8th

**⬤ The video game dialogues were finalised.**

Once all the feedback comments in the dialogues were fully implemented, the content was translated into English, in order to internationalise the video game and extend its reach to a more global audience.

### 10th

**⬤ AflaxOFF Presentation Event**

On Wednesday morning, September 10th, the presentation of the UMA-MALAGA 2024 team took place at the rectorate of the University of Malaga. In this event we had the honour to show our project AflaxOFF before eminent people such as the rector of the University of Malaga, Teodomiro Lopez, the Vice Chancellor of Students, Employability and Entrepreneurship, Juan Carlos Rubio, the Councillor for Youth of the City of Malaga, Maria de las Mercedes Martin, and the Dean of the Faculty of Sciences, Antonio Flores. They presided over the event together with the coordinators of the UMA-MALAGA initiative, Manuel Bernal and Carlos Rodríguez. After an exciting morning, the team went to lunch to talk about the events that took place and plan the next steps to take in this exciting adventure that is iGEM.

### 12th

**⬤ Third workshop for Inclusivity Special Prize: FAISEM**

We continued with the Inclusivity workshops! On this occasion, the sessions consisted of demonstrating in a practical and experimental way the hypotheses that we had established in the previous session. They were the protagonists and the ones who put on the lab coats to learn all the experiments and obtain the results.

### 13th

**⬤ Third workshop for Inclusivity Special Prize: APROINLA, Aidis and CEPER**

We continued with the Inclusivity workshops! On this occasion, the sessions consisted of demonstrating in a practical and experimental way the hypotheses that we had established in the previous session. They were the protagonists and the ones who put on the lab coats to learn all the experiments and obtain the results.

### 17th

**⬤ Final Day of Inclusivity Workshops**

To put an emotional end to this great adventure that has been Inclusivity, on Tuesday 17 we organized a great final workshop in the botanical garden of the University of Malaga which was attended by the centers of AIDIS, APROINLA, FAISEM and CEPER. Throughout the morning a great variety of activities were carried out and the flashcards created during the individual sessions in each center were exchanged. With this day, we tried to foster a support network through science, so they themselves carried out and explained the experiments they had learned to their classmates. We couldn't be happier with how everything went!

### 20th

**⬤ The video game is finished**

![](https://static.igem.wiki/teams/5466/static/notebook/nb-57.png)

September 20th marked the end of the creation process of the video game ‘Los Mohocytes’. It was launched on the AflaxOFF website so that any Internet user could access and enjoy it. In addition, desktop versions were developed on various platforms, allowing those who prefer to play on their own devices to have that option as well. The video game was showed at the Instituto de La cala de Mijas, Researchers' Night and the Pompeu Fabra institute

### 24th

**⬤ Visit to the Victoria Beer Factory**

Last Tuesday 24 September, we had the honour of being invited by the Cervezas Victoria brewery to tour their facilities and learn about their history. The day ended excellently with a tasting of their exquisite beer.

### 25th

**⬤ Talk at IES La Cala de Mijas High School**

On Wednesday, September 23rd, we went to the high school IES La Cala de Mijas where we gave a talk in which we disseminated the project and tested our educational video game. Everyone was delighted.

### 27th

**⬤ European Researchers' Night**

On 27 September, our colleagues Juan Antonio and Lucía Campos held a micro-meeting at the Rectorate of the University of Málaga, as part of the European Researchers' Night, organised by UMA Divulga. During the event, they offered a workshop in which they explained our work using an educational video game.

## 2024 October

### 2nd

**⬤ The wiki of the UMA-MALAGA team is finished.**

The development of the wiki is finished, having integrated all the knowledge in an organised and structured way. In addition, a comfortable, guided and attractive navigation style has been defined, allowing users to explore each element with relative ease.

![](https://static.igem.wiki/teams/5466/static/notebook/fin.png)

{% endblock %}

{% extends "layout.html" %}
{% block title %}ENGINEERING SUCCESS{% endblock %}
{% block lead %}*"Science can amuse and fascinate us all,
but it is engineering that changes the world."*
- Isaac Asimov{% endblock %}
{% block page\_content %}

### **Content**

---

1. [Introduction](#introduction)
2. [First Iteration](#first)
3. [Second Iteration](#second)
4. [Conclusion](#conclusion)

### **Introduction**

---

Throughout its course, the **AflaxOFF** project has experienced a lot of ups and downs. Through ongoing engagement with **stakeholders**, in-depth **literature reviews**, and measurements taken in our own **laboratory**, we have been constantly **redefining** our project, both in its technical and practical dimensions, in order to find the best answer to the problem we are addressing.

Our **Engineering Success** lies in the search for the **most suitable chassis** for the implementation of our solution. This involved the challenge of **shifting** a molecular machinery initially designed to be functional in one organism, to a whole new one. We believe this pivotage to be a proof of our project's **flexibility** and **resilience**.

### **First DBTL Cycle - A prokayotic-based system**

---

#### Design

Early in the project, once our initial idea was determined, we defined the **requirements** for our engineered organism. We needed to construct an organism which was capable of **detecting AFB1** and
**produce sequestering molecules** upon such recognition.

Firstly, due to the strong biomedical orientation of our project, we thought about ***Lactococcus lactis***, due to its probiotic nature. However, through dialog with advisors and early investigations on the available literature, we noticed a **lack** of **well-characterized tools** for engineering sensing platforms in this gram positive bacterium.

![](https://static.igem.wiki/teams/5466/static/design/cadc-nombres-bueno.png)

General architecture of the EMeRALD platform: scFvs are used as ligand binding domains (LBDs)

Therefore, we decided to perform our **proof of concept** in ***Escherichia coli*** due to the greater amount of knowledge supporting its engineering and its **fast growth** and **genetic manipulability**. In order to render this bacterium responsive to AFB1, we found a promising option in the sensing-response platform **EMeRALD**. This system is based on the modification of the ligand binding domain of *E. coli*’s **CadC** transmembrane transcriptional activator, rendering a receptor **activated** through **induced dimerization** by a ligand of interest.

To ensure maximal capture of aflatoxin B1 by our bacteria upon toxin detection, we came up with the idea of regulating the system through **positive feedback**. We designed a construct where receptor expression is maintained by its own regulated promoter, **pCadBA**. This way, only a few receptors are produced due to **promoter leakage** when AFB1 is absent. Once the toxin is detected, the expression is triggered, significantly increasing the number of receptors on the membrane and **enhancing aflatoxin B1 adsorption**.

#### Build

With our design in mind, we ordered certain parts to start testing them individually in the laboratory. We addressed the building of our genetic constructs with **modularity** in mind and concluded that synthesizing the entire receptors, which differed only in its ligand binding domains, would be a waste of resources and loss of maneuverability, especially for future **design optimizations**. Therefore we decided to **split the receptors into domains**, with CadC and the artificial transmembrane region Leu(16) on one side, and the scFvs as LBDs on the other.

![](https://static.igem.wiki/teams/5466/static/engineering/emerald-standard-recortado.png)

Proposed extended-MoClo for assembly of EMeRALD-based receptors: a novel fusion site is designed based on the sequence of CadC to generate new receptors in a straight-forward way

The whole transcriptional units were intended to be assembled through **Golden Gate**, following MoClo standard and adding a **novel fusion site** for the **scarless** and **in-frame** fusion of both domains of the CDS. This was achieved by taking the four first nucleotides from the constant part, i.e., the signaling C-terminal domain of the receptor, as the fusion site, which were also appended to each of the LBDs.

Prior to this assembly, we **pre-cloned** all of our synthesized parts into **SEVA181** plasmid thanks to flanking **EcoRI** and **PstI** restriction sites. By using an **RFP** expressing device as **dropout**, we were able to select the positive transformants. However, during this stage of our lab work, we faced certain **difficulties** with minipreps, which could affect our downstream assembly efficiency. Therefore, we decided to suspend our wet lab tests until a solution was found.

In parallel to our wet lab activities, we worked computationally to implement and investigate the **model** developed by **Lu & Wang** (2017) on receptor heterodimerization in order to gain insights into the behaviour of our EMeRALD-based system.

#### Test

During our first trial stage, we aimed to evaluate whether the expected functionality of our parts had been accomplished, and gather data on their behaviour for further optimization and increase of complexity in future cycles.

Our tests involved addressing **receptor localization**, **CadC signaling** capacity and **efficiency of AFB1 binding**, by means of coupled-protein **fluorescence** and AFB1 determination through **ELISA**, both in resuspended cell pellets and supernatant.

Despite our pause of laboratory activity, we still managed to extract information on the system’s expected behaviour through our *in silico* model. This was achieved by performing **real-time** and **steady-state simulations** of the system, addressing the relationship between receptor signal and key variables such as ligand and receptor concentrations.

![](https://static.igem.wiki/teams/5466/static/engineering/grafica1.png)

Simulation of a ligand-induced heterodimerization equilibrium (concentration-time); the curves represent the evolution of the different complexes over time: P1, unbound subunit 1; P2, unbound subunit 2; P1L, ligand-subunit 1 monomeric complex; LP2, ligand-subunit 2 monomeric complex; P1LP2, ligand-heterodimeric complex

A first simulation was run with set initial concentrations of both of the receptor subunits and ligand. As expected, the amount of **dimer-ligand associations increases gradually** over time. However, certain subunits remain inactive in the form of **monomer-ligand complexes**.

![](https://static.igem.wiki/teams/5466/static/engineering/grafica2.png)

Steady-state concentrations of the different complexes of an heterodimerization equilibrium as a function of total ligand concentration: P1, unbound subunit 1; P2, unbound subunit 2; P1L, ligand-subunit 1 monomeric complex; LP2, ligand-subunit 2 monomeric complex; P1LP2, ligand-heterodimeric complex

When the **steady-state concentrations** of the different species were assessed with **varying concentration of ligand** in the medium, we noticed a peculiar dynamic. In contrast to ligand-monomer equilibriums, where active complex concentration increases with [L] until saturation, the concentration of the dimeric complex was found to experience a **maximum** value at **[L]max**, with a following **decay** due to the accumulation of monomer-ligand associations. In addition, we found this value [L]max to be dependent on total receptor concentration, with a positive relation between both.

Finally, when the analysis performed above was repeated with **two concentrations** of receptor **differing** by a **10-fold**, a substantial difference was observed in both the **concentrations of ligand** at which signal experiences an increase, the **slope** of such increment, and the ratio of **receptor activation at [L]max**, with all of them being greater at higher levels of expression of the receptor.

#### Learn

Based on the results derived from our model, we could reach the following conclusions:

* The concentrations of each of the subunits of the CadC receptor exposed in the cell surface are essential to determine the range of concentrations to which the system is responsive. Therefore, for the following designs, we identified **promoter** and **RBS** **strengths** to be key parameters of our system, due to their effect on protein expression.
* Low activation ratios at low concentrations of the receptors, together with a modified degree of activation with an increasing number of receptors suggest that the system of **positive feedback** might be **difficult to fine-tune.**

In view of the analysis retrieved from our calculations, we decided to search for alternatives to AFB1 adsorption different from the own receptors. While reviewing the literature with such objective, **conflicting information** was found about the **interaction of AFB1 with *Escherichia coli***.

We read some articles indicating that AFB1 was **adsorbed** onto the bacterium’s cell wall, while others supported that it caused **serious damage** to this structure (Chen *et al.*, 2020). These findings raised concerns for our approach: if AFB1 cannot penetrate the cell wall, then it **cannot be sensed** by the receptors; and what is more: if AFB1 alters the cell wall’s structure, then the bacterium’s **viability will be impaired**.

The accumulation of **uncertainties** regarding the use of *Escherichia coli* as our model organism led us to an unavoidable decision: **to ensure our project's viability**, there was a need to **migrate our system to a novel chassis**.

### **Second DBTL Cycle - Migration to a novel chassis**

---

#### Design

After our decision to turn to a new organism different from *Escherichia coli*, we came back to our initial idea of engineering the probiotic *Lactococcus lactis*, which harbors a different surface structure with a thicker cell wall. We attempted to find in this species, **orthologs of CadC** and other one-component systems from gram positive bacteria, such as *Bacillus subtilis*.

Orthologs are genes from different species which share a common ancestor and retain a **similar function**. This concept was originally conceived by Fitch for phylogenetic inference. Nowadays, orthologs are used to study and predict gene function, being fundamental for **gene annotation** and the advancement of **functional genomics**. This approach is of great relevance given that the vast majority of genes in sequenced genomes will never be addressed experimentally.

![](https://static.igem.wiki/teams/5466/static/engineering/ortho1.webp)

Table with orthologs inferred with OrthoFinder for *Escherichia coli* and *Lactococcus lactis* proteomes

To find potential orthologs, we went beyond literature and applied **OrthoFinder**. This software is an innovative method for inferring orthologs of protein-coding genes, outperforming other methods in accuracy and speed, with improvements ranging from 8% to 33%. Unlike other approaches that may omit short genes and incorrectly group long genes, OrthoFinder **normalizes alignment results** based on **sequence length**, allowing it to infer orthologs from incomplete data with **high precision**.

Unfortunately, we were **unable** to **identify** any suitable candidates to engineer a system in *Lactococcus lactis* using OrthoFinder.

In this context of instability, the idea of shifting to the eukaryotic host ***Saccharomyces cerevisiae*** was proposed. Several evidence supported the idea of *S. cerevisiae*, including the ability of AFB1 to reach the cell’s membrane (Su *et al.*, 2023), the deep research around this organism and the existence of a probiotic strain named *Boulardii*.

However, this 180-degree change, **transitioning** from a **prokaryotic** to an **eukaryotic** system which we had not previously worked with, involved migrating our EMeRALD based system to yeast, keeping up with the **same principles** of **functionality** and **modularity**.

![](https://static.igem.wiki/teams/5466/static/design/receptor1-nombres-bueno.png)

General architecture of the GEMS platform: scFvs are used as ligand binding domains (LBDs) and the split ubiqutin system is exploited for signaling: LBDs, ligand binding domains; EpoR, erythropoietin receptor; Cub, C-terminal ubiquitin fragment; NubG, N-terminal ubiquitin fragment with I13G mutation; sTF, synthetic transcription factor

After extensive research, we found a great solution in a system proposed by Su *et al.* (2023) consisting of a synthetic receptor based on the merging of the **GEMS-platform** (Scheller *et al.*, 2018) and the **split-ubiquitin system** (Sinder *et al*., 2010). Such platform harbors an scaffold derived from **EpoR** and ligand binding domains which drive the dimerization and reconstitution of ubiquitin, mediating receptor activation through the release of an **attached transcription factor**.

In order to solve the potential hurdles of our previous design, the functions of **detection** and **adsorption** were **separated** into individual molecules, with the latter being now performed by a **single-domain antibody** (sdAb) expressed in the cell’s surface upon receptor signaling. In addition, to maintain the positive feedback, the transcription factor is also produced in its free form upon receptor activation. Both responses, as well as a fluorescent protein as a **reporter**, were facilitated thanks to the addition of **IGG6** into our set of parts, which would allow us to support **polycistronic expression** of all the proteins without dramatically increasing the complexity of our construction.

#### Build

Using a similar methodology as with *E. coli*, an **assembly strategy** was proposed based on an **extended-MoClo** with two additional fusion sites, determined by the core protein domain of the EpoR scaffold, and permitting the construction of several different receptors by **exchange** of, this time, both the **LBDs** and the intracellular **signaling domains**.

![](https://static.igem.wiki/teams/5466/static/engineering/botes-yeast.webp)

Growth and transformation media for *Saccharomyces cerevisiae: 40% filtered glucosa (top-left); 1M lithium acetate (top-center); YP, Yeast-extract Peptone (top-right); selective SC-ura agar plate (bottom-left)*

Prior to implementing our parts to the new host *S. cerevisiae*, much work had to be done to learn how to **grow** and **engineer** this **yeast**. The first step to this purpose was preparing the **media** required for its growth, including selective media without uracil for selection of colonies harboring a gene complementing **uracil auxotrophy**, as well as the **reagents** necessary for **yeast transformation**. In addition and as importantly, we required a **shuttle vector** that would allow for replication and selection both in *E. coli*, where we would be performing cloning and assemblies, and *S. cerevisiae*, the subject of our assays.

For this purpose, we began transforming plasmids from the **Open Yeast Collection** (OYC) provided in the **iGEM Kit Plates**, with the intention of generating both an **episomal** and **integrative** plasmid through Golden Gate Assembly.

Despite the verification of most of the parts required for the assembly of our plasmid, we had struggles with a few. Time pressure led us to purchase another vector, namely **YEplac195**, to fulfill this gap and be able to continue towards our engineering goal.

Due to the time invested in our project until this point, we decided to order each of our designed **transcriptional units** as a **whole** and not split, in order to accelerate our work.

#### Test

Based on the protocols gathered for **yeast transformation**, we managed to successfully incorporate our YEplac195 plasmid into *S. cerevisiae*. Regarding the testing of our devices, although still ongoing, they are intended to provide us information on the system’s functionality:

![](https://static.igem.wiki/teams/5466/static/engineering/placa.webp)

*Saccharomyces cerevisiae* GM01 transformed with plasmid YEplac195, confering complementation for uracil auxotrophy; SC-ura agar plate

Our receptors harboring **split-mCerulean** as intracellular domains are designed to provide insights on the dimerization capacity of our scFvs, which will require **fluorescence measurements** to monitor the formation of ligand-dimer complexes. The same kind of assay is intended to be performed to test our part **IGG6**, in the context of a **tricistron** expressing the fluorescent proteins **yEmRFP**, **yECFP** and **yeGFP**. For this purpose, the Fluorescence Measurement Calibrant Kit will be used to assess efficiency of each position of the polycistronic gene. To assess the **adsorption** capacity of our yeast, thanks to our **Human Practices** team labour, we count with **ELISA** kits provided by **ProGnosis** to determine the concentration of AFB1 retired from the medium by our modified organism.

#### Learn

After assays, we expect to retrieve essential conclusions from our results in order to **guide** our **following** round of **design**, which will involve bringing together all the interacting parts with the required optimizations, or redesigning a certain part because of its dysfunctionality.

### **Conclusion**

---

In short, the course of our project has been marked by two great stages of development, in which an iterative approach was applied through the DBTL Cycle in order to assess the viability of implementation of our molecular solution to different organisms:

* In the first cycle, an *E. coli*'s based system was built, but the chassis had to be repurposed due to uncertainties on AFB1 toxicity and permeability to the bacterium's cell wall.
* In our second cycle, we pivoted to the novel organism *S. cerevisiae* reengineering our genetic design to keep its functionality and modularity.

This iterating approach led to the construction of **two analogous batches of parts**, of great utility for the construction of sensing-response platforms, both in *Escherichia coli* and *Saccharomyces cerevisiae*.

### **References**

---

Chen, Y., Yang, Y., Wang, Y., Peng, Y., Nie, J., Gao, G., & Zhi, J. (2020). Development of an Escherichia coli-based electrochemical biosensor for mycotoxin toxicity detection. *Bioelectrochemistry* (Amsterdam, Netherlands), 133, 107453. <https://doi.org/10.1016/j.bioelechem.2019.107453>

Scheller, L., Strittmatter, T., Fuchs, D., Bojar, D., & Fussenegger, M. (2018). Generalized extracellular molecule sensor platform for programming cellular behavior. *Nature chemical biology*, 14(7), 723–729. <https://doi.org/10.1038/s41589-018-0046-z>

Snider, J., Kittanakom, S., Curak, J., & Stagljar, I. (2010). Split-ubiquitin based membrane yeast two-hybrid (MYTH) system: a powerful tool for identifying protein-protein interactions. *Journal of visualized experiments : JoVE*, (36), 1698. <https://doi.org/10.3791/1698>

![](https://static.igem.wiki/teams/5466/static/footpages/footpagemetio-1-1.webp)

{% endblock %}

{% extends "layout.html" %}
{% block title %}EXPERIMENTS{% endblock %}
{% block lead %}*"Not all questions are answered,
 but fortunately some answers are questioned."*
- Terry Pratchett{% endblock %}
{% block page\_content %}

### **Content**

---

1. [Introduction](#introduction)
2. [*Saccharomyces cerevisiae*](#saccharomyces)
3. [OYC Shuttle Vector](#OYC)
4. [*Escherichia coli*](#escherichia)
5. [AFB1 Growth Inhibition](#growth)
6. [Protocols](#protocols)

### **Introduction**

---

In order to approach the engineering of our desired biological functions in the laboratory, we made use of well-established and optimized protocols and developed organized experimental workflows to guarantee that our research is effective and reproducible.

The methodology applied involves the assembly and cloning of the desired parts, followed by what our PI Manuel Bernal calls “asking the question”, i.e.: determining whether our organism is performing the function of interest as expected.

Here we describe the experimental designs elaborated for the purpose of our project and gather the protocols applied during the process.

### ***Saccharomyces cerevisiae***

---

To approach the modification of *Saccharomyces cerevisiae* towards responding to aflatoxin B1 in the laboratory, we organized our goals into the following parallel modules:

**![Icono](https://static.igem.wiki/teams/5466/static/experiments/puntoafb1-azul.png) Evaluation of the three testing devices**

This line of work is aimed to assemble and characterize the main parts of our circuit and gain insights on their individual functionality:

**![Icono](https://static.igem.wiki/teams/5466/static/experiments/puntoafb1-azul.png) Proof of concept of the genetic circuit**

This module is meant to build and implement a first version of the whole circuit to provide knowledge on the integrated behaviour of all components.

#### **Evaluation of the three testing devices**

## **AFB1-dependent dimerization of scFv1 and scFv2**

Following synthesis of both transcriptional units, high-fidelity Q5 PCR-amplification is performed to add the corresponding BioBrick prefixes and suffixes to the parts. The required primers are shown in the table below. The two fragments are introduced into YEplac195 by BioBricks ligation and cloned into *Escherichia coli*. Following purification, the resulting plasmid is transformed to *Saccharomyces cerevisiae*. mCerulean fluorescence is assessed with increasing concentrations of AFB1.

Primers utilized for addition of BioBrick prefixes and suffixes to both receptor's sequence

| F1-scFv | Forward | atcggaattcgcggccgcttctagactgtctcagttcgagtttatc | 46 nt |
| --- | --- | --- | --- |
| R1-scFv | Reverse | atcgctgcagcggccgctactagtcatctcgttcagggtaatatattttaaccgc | 55 nt |

![](https://static.igem.wiki/teams/5466/static/experiments/split-cerulean.png)

Experimental workflow to build and test the split-mCerulean-fused AFB1 receptors

**Assay procedure**

* 5 mL of YPD are inoculated with *Saccharomyces cerevisiae* cells transformed with YEplac195+split-mCer plasmid and grown O/N at 30ºC with agitation.
* The culture is diluted in YPD to a final volume of 50 mL and OD600 of ~0.1, and is grown for about 3-5 hours to an OD600 of ~0.6.
* A 96-well microplate is prepared by pipetting 200 μL of the grown culture to each of the wells. 22,22 μL of a stock AFB1 solution of 2 μg/mL is pipetted in the first column and decimal serial dilutions are made in the following columns by mixing 9 parts of culture with 1 part of the previous dilution. The last column is left untreated as a control. 7 replicates are made for each final concentration of AFB1, with the last row including culture mixed with the acetonitrile solvent without toxin.
* The fluorescent intensity is assessed by using BioTek Synergy H1 Multimode Reader. Excitation light is set to 433 nm and fluorescence intensity is measured at 475 nm; a measure is taken every 2 minutes for 30 minutes at 30ºC with 150 rpm orbital agitation.

## **Detoxification potential of VHH synthetic receptor**

A single transcriptional unit expressing the Nb28 VHH is pre-cloned into pJet1.2 using the CloneJet™ PCR Kit from Thermo Fisher. The insert is subsequently transferred to the shuttle plasmid YEplac195 by EcoRI-PstI digestion and ligation. The resulting plasmid is transformed to *E. coli* and *S. cerevisiae* after purification.

![](https://static.igem.wiki/teams/5466/static/experiments/surface-vhh-exp.png)

Overview of the workflow proposed for the building and evaluation of a yeast strain constitutively expressing high-efficiency surface VHHs

**Assay procedure**

* 5 mL of YPD are inoculated with *Saccharomyces cerevisiae* cells transformed with YEplac195+Nb28-Aga2P plasmid and grown O/N at 30ºC with agitation.
* Overnight culture is diluted to OD600 ~0.1 to a final volume of 2 mL. Six flasks are prepared.
* The flasks are incubated at 30ºC with agitation until exponential phase is reached, OD600 ~0.4. At this time, the cultures are inoculated with 1 μL of AFB1 serial dilutions, ranging from 2.0 μg/mL to 2.0·10-5 μg/mL.
* Cultures are incubated for an additional 1 hour.
* The cultures are transferred to a sterile microcentrifuge tube and cells are pelleted at 3000 g for 5 minutes.
* Supernatant is transferred to another tube.
* AFB1 concentration in the supernatants is determined by using Bio-Shield B1 ELISA Test from ProGnosis.

## **Polycistronic expression of three FPs mediated by IGG6**

The tricistronic expression cassette is split in half for synthesis and the two fragments are assembled onto pSEVA281 through Gibson Assembly reaction. Next, cloning to YEplac195 is performed, with final yeast transformation and fluorescence visualization through confocal microscopy.

![](https://static.igem.wiki/teams/5466/static/experiments/igg6-bueno.png)

Workflow proposed for the assembly and qualitative testing of a IGG6-mediated FPs tricistron

#### **Proof of concept of the genetic circuit**

The whole circuit is split into 5 fragments of similar length. Fragments 1 and 5, containing E and P restriction sites, respectively, are digested and ligated with an EP-digested YEplac195. The resulting linear fragment is used for a Gibson Assembly reaction together with F3, F4 and F5. Thanks to the homology regions, a vector is assembled containing the whole circuit (PoC). The plasmid is transformed to *E. coli* and subsequently to *S. cerevisiae*, with the following assays for both the measurement of reporter fluorescence and AFB1 capture, as described above. In this case, emission and detection wavelengths are set to 584 and 607 nm, respectively, and the assay is performed overnight with measures every 10 minutes.

![](https://static.igem.wiki/teams/5466/static/experiments/tocho-god.png)

Workflow designed to assemble and test a first version of the whole genetic circuit

### **OYC Shuttle Vector**

---

To assemble a yeast episomal (YEp) and integrative (YIp) shuttle plasmid for the team, the following parts were chosen from the Open Yeast Collection (OYC) provided in iGEM Distribution Kit Plate 2:

Parts from the iGEM Distribution Kit Plate 2 chosen for assembly of YEp

| Sc2micron | ScURA3-marker | AConL-start | AConR-end | OYC-bridge-AGAC-GCAA | OYC-CamR |
| --- | --- | --- | --- | --- | --- |
| D1 | O16 | I12 | A22 | C18 | E18 |

Parts from the iGEM Distribution Kit Plate 2 chosen for assembly of YIp

| ScHR5'HO | ScURA3-marker | AConL-start | AConR-end | ScHR3'HO | oriT | OYC-CamR |
| --- | --- | --- | --- | --- | --- | --- |
| K14 | O16 | I12 | A22 | M14 | O24 | E18 |

The parts comprising the transcriptional unit were taken from the ones available in our laboratory, including a constitutive promoter, strong RBS, double terminator and GFP coding sequence. The origin of replication was chosen to be pUC due to the thermo-sensitive nature of the one available in the OYC. It was PCR-amplified from pSEVA181.

All the parts from the OYC were transformed directly from the Kit Plate 2 to NEB® 10β and the plasmids were purified using PureYieldTM Plasmid Miniprep System from Promega. They were verified through BsaI digestion and agarose gel electrophoresis.

### ***Escherichia coli***

---

Despite being left behind along the project, an experimental design was also applied for the modular assembly of EMeRALD based receptors and transcriptional units during the early stages of our project.

The different protein domains conforming the receptors and other genetic elements are synthesized with flanking EcoRI, PstI and BsaI restriction sites. The first ones are leveraged for ple-cloning in pSEVA181, while the second allows for the assembly through Golden Gate of the desired receptors, which can then be stored for downstream uses.

![](https://static.igem.wiki/teams/5466/static/experiments/cadc-exp.png)

Workflow designed to assemble and test a first version of the whole genetic circuit

### **AFB1 growth inhibition**

---

We designed an assay to evaluate the inhibition exerted by AFB1 on the growth of the different species utilized in our team, which we were able to perform once AFB1 was delivered to our laboratory.

* 5 mL of the corresponding medium are inoculated and grown O/N at the adequate conditions. YPD at 30ºC with agitation is used for *S. cerevisiae* and LB at 37ºC with agitation for *E. coli*.
* The culture is diluted with more medium to a final volume of 25-50 mL and grown for about 3-5 hours.
* A 96-well microplate is prepared by pipetting 200 μL of the grown cultures to each of the wells. 22,22 μL of a stock AFB1 solution of 2 μg/mL is pipetted in the second column and decimal serial dilutions are made in the following columns by mixing 9 parts of culture with 1 part of the previous dilution. The last column is left untreated as a control and the first one harbours sterile LB. 5 replicates are made for each final concentration of AFB1, with the last rows including culture mixed with the acetonitrile solvent without toxin, as well as a control of LB+acetonitrile.
* Optical density at 600 nm is monitored by BioTek Synergy H1 Multimode Reader every 10 minutes, at the corresponding temperature and agitation, for 24 hours.
* Data of samples is normalized to the controls and curves are adjusted to a logistic model.

### **Protocols**

---

Here we list the protocols applied in our laboratory during our project. The procedures for basic molecular biology techniques and *Escherichia coli* manipulation were taken from the work previously developed in our laboratory as part of the 2022 StarchSTEM project. We also add protocols for *Saccharomyces cerevisiae* cultivation and transformation.

![](https://static.igem.wiki/teams/5466/static/footpages/footpageagarro.png)

{% endblock %}

{% extends "layout.html" %}
{% block title %}RESULTS{% endblock %}
{% block lead %}*"The beginning of knowledge is the discovery of something we do not understand."*
- Frank Herbert{% endblock %}
{% block page\_content %}

### **Content**

---

1. [Introduction](#introduction)
2. [AFB1 dimerization](#dimerization)
3. [AFB1 detoxification](#detox)
4. [IGG6 tricistron](#igg6)
5. [Circuit PoC](#poc)
6. [AFB1 Growth Inhibition](#growth)
7. [Conclusion](#conclusion)

### **Introduction**

---

Since the beginning of our journey, one of our main motivations has been to go beyond theory and validate the functionality of our genetic designs in the laboratory. Through a process of constant effort and learning, and despite the difficulties encountered, we managed to make certain progress into our investigation.

## **AFB1-dependent dimerization of scFv1 and scFv2**

![](https://static.igem.wiki/teams/5466/static/results/gel-scfvs.png)

Agarose gel electrophoresis of PCR-amplified scFv1-EpoR-N-mCer (1st lane, ~2700 bp) and scFv2-EpoR-C-mCer (2nd lane, ~2400 bp)

**Achievements**

Both of the transcriptional units synthesized were successfully PCR amplified using a high-fidelity Q5 polymerase. Agarose gel electrophoresis revealed the presence of amplification and amplicons of the expected size: ~2700 (scFv1-EpoR-N-mCer) and ~2500 (scFv2-EpoR-C-mCer).

**Future considerations:**

Downstream steps of the workflow were not addressed due to time limitations. Once obtained the TUs with the corresponding Bb prefixes and suffixes, the workflow can be continued as designed.

## **Detoxification potential of VHH synthetic receptor**

**Difficulties**

The initially proposed pre-cloning into pSEVA281 was not achieved. When an RFP dropout was used, a low effiency was observed in the ligation. The few potentially positive white colonies were picked and inoculated into liquid media. However, in two different sets of pre-cultures adding up to 9 tubes, no growth was observed after 24 hours on either of them. We hypothesized that there might be a problem of plasmid stability, which would compromise antibiotic resistance. We decided to utilize the CloneJET PCR Kit for efficient pre-cloning.

![](https://static.igem.wiki/teams/5466/static/results/placa.jpg)

Replica plate elaborated for colony PCR screening of pJet1.2 transformants

![](https://static.igem.wiki/teams/5466/static/results/mutacion-sec.jpg)

Sequencing results from 3 clones of pJet1.2+VHH

**Achievements**

The transcriptional unit from synthesis was successfully pre-cloned into pJET1.2. The screening of transformant colonies through colony PCR resulted in a great number of samples with amplification of the expected size ~1600. 4 of the colonies were pre-cultured for plasmid purification and 3 of the resulting plasmids were sequenced. The results of all the analyses permitted verification of clones 1 and 3, while the second presents an insertion mutation (T) at position 622, which falls at the nucleotide -60.

Despite the mutation not affecting the CDS, which would have resulted in a change in the reading frame, the mutation might cause a change in the promoter strength. Thereby, clones 1 and 3 are chosen for downstream experiments.

![](https://static.igem.wiki/teams/5466/static/results/pcr-vhh.png)

Colony PCR of pJet1.2+VHH (lanes 17-36)

## **Polycistronic expression of three FPs mediated by IGG6**

**Difficulties**

The assembly of both fragments by Gibson Assembly was not achieved. In a first attempt, a very few transformants were visible, yet no colonies were observed in the positive control. Therefore, the result was discarded. A possible reason for such result could be the insufficient competence of the cells used. Therefore, we decided to repeat the experiment with commercial NEB® Stable competent cells. This time, more colonies were observed, both in the sample and positive control.

Screening of the transformants by colony PCR, however, led to no positive result, given by the absence of amplicons of the expected length (~ 3000 bp) in the samples, as well as no amplification in the positive control (pSEVA281+RFP).

![](https://static.igem.wiki/teams/5466/static/results/colony-igg6.jpg)

Colony PCR of 36 colonies transformed with the product of IGG6 Gibson Assembly Reaction

**Future considerations**

Despite the inconclusive results from our last attempt of assembly, we believe that part of our troubles come from a reduced efficiency of the Gibson reaction. Due to the usage of the fragments from synthesis directly, which have a concentration of 10 ng/μL, with the volumes established in the kit’s protocol we are only able to achieve the suggested concentrations by minimum. Prior amplification of the fragments through PCR could help concentrate the DNA and ensure a high fragment-vector ratio, which is recommended for higher efficiency. This could also be achieved by increasing the total reaction volume. In addition, to reduce the noise originated from undigested
plasmids, gel purification followed by clean up could help minimize the amount of undesired plasmids. Altogether, we expect these ideas to lead us to a positive result
in order to continue the workflow.

## **Proof of concept of the genetic circuit**

**Future considerations**

The assembly of this cassette was decided not to be approached until a simpler Gibson Assembly reaction had been achieved. However, due to the requirement of greater amounts of pmol per fragment in a reaction involving 4-6 fragments, a previous PCR amplification appears compulsory to meet the required DNA concentrations.

## **AFB1 Growth Inhibition**

![](https://static.igem.wiki/teams/5466/static/results/graph1.png)

*Saccharomyces cereivisae* growth in YPD with concentrations of AFB1 added from 2 µg/mL to 2·10-9 µg/mL. X-axis: time (h); Y-axis: OD600 nm

![](https://static.igem.wiki/teams/5466/static/results/graph2.png)

RFP-expressing *Escherichia coli* growth in LB monitored through optical density, with concentrations of AFB1 added from 2 µg/mL to 2·10-9 µg/mL. X-axis: time (h); Y-axis: OD600 nm

![](https://static.igem.wiki/teams/5466/static/results/graph3.png)

RFP-expressing *Escherichia coli* growth in LB monitored through fluorescence intensity, with concentrations of AFB1 added from 2 µg/mL to 2·10-9 µg/mL. X-axis: time (h); Y-axis: fluorescence intensity

Based on the results from the growth inhibition assays, aimed to provide more evidence regarding AFB1 toxicity on *E. coli* and *S. cerevisiae*, we found that the effect was more apparent in the yeast species.

A slightly declining trend is observed in the growth rate of *S. cerevisiae* with increasing concentration of the toxin. However, no serious impairment is observed, supporting the use of this organism in the context of our project.

Meanwhile, *E. coli* growth does not seem to be signficantly affected under the experimental conditions tested, as revealed by both types of measurements. However, the assay performed does not provide information on the cell wall's permeability to AFB1. Thus, more experiments would be required to determine the viability of an *E. coli*-based receptor.

### **Conclusion**

---

During the course of our project, various have been the changes of ideas and difficulties which have led us to a delayed start in the laboratory activities regarding our final design. Until that point, we managed to generate useful parts for future endeavours, including parts from the EMeRALD platform and for the construction of a novel shuttle vector.

Regarding our testing designs, we managed to complete the first steps of the proposed workflows. However, due to time limitations we could not see our project grow to its whole grandness during this iGEM season.

We understand that regardless of the passion and effort put into a scientific goal, research does not always turn out as expected. Nevertheless, we have the determination to keep working in this line and hope to find ways to keep up with this project.

![](https://static.igem.wiki/teams/5466/static/footpages/footpageagarro.png)

{% endblock %}