Reflection, Responsibility and Responsiveness

Creating the first iGEM team in Aragón

Human Practices is an important part of iGEM projects from the moment it is decided to be part of the competition, and in our case, it was no different. When recruiting the team, as the competition was not known in our university, it aroused interest mainly among biotechnology students. Thus, our team ended up being composed of 10 biotechnology students and 1 physics student. Over time we realized how useful it would have been to have team members specialized in other areas, but that did not make us less multidisciplinary. Although we decided to participate in iGEM because of how much we had enjoyed taking subjects such as Microbiology, Genetic Engineering, and Microbial and Plant Biotechnology, we took care of distributing tasks according to our aptitudes.

Starting such a large initiative from scratch is always complicated, but even more so if it takes place in a region far from the country's biotechnological hubs and in the middle of a pandemic. Our first problem as a team was having to deal with the challenge of getting funding in an environment of companies that were not familiar with the iGEM competition. Therefore, from the very beginning, we invested a lot of time in contacting clusters and companies in Aragón (especially related to biotechnology) to make ourselves known. In particular, the health cluster of Aragón, AraHealth, supported us by spreading our initiative among its members and collaborators. This was of great help to us, as this cluster is made up of companies (biopharmaceutical, biomedical engineering, and healthcare), knowledge institutions (universities and research institutes), and government entities (Figure 1).
Despite this help, we had to undergo many meetings with different companies and institutions until we were able to obtain enough funding. This led to a late registration for the competition, and delays in obtaining reagents and materials for the laboratory work.

Figure 1. Arahealth partners.

Identifying a problem to solve: optimizing synthetic nanobodies

Choosing which project to undertake was not an easy task either. We had several options in mind, but given the context, there was one thing always present in our thoughts: the pandemic. For us, the world would have been very different these past two years if a highly effective antibody against SARS-CoV-2 had been available when the pandemic began. This made us think big and we decided that we wanted to develop a method that would allow us to obtain antibodies quickly, cheaply, and without having to use animals.
We wanted to demonstrate that it was possible to generate a library of antibodies against all possible antigens by recombining the different gene fragments that encode them (V, D, J), as happens in living beings (1). Thus, when an antibody against a given antigen was needed, it would be sufficient to confront the library against that antigen and find the best one. The potential of this idea is obvious, as it would allow any hospital, researcher, or company to have any antibody readily available.
However, after a long period of literature review and multiple exchanges of ideas, we realized that achieving something like this was practically impossible.

First of all, we noted that if we wanted to produce antibodies without using animals and cheaply, it was essential to produce them in bacteria (Figure 2). This meant that we could not produce conventional antibodies, which are too complex to be produced in prokaryotic expression systems (2,3). Therefore, we opted to generate nanobodies, which can be produced in bacteria thanks to their simplicity. This decision was also supported by the multiple advantages (Figure 3) they present over conventional antibodies. In fact, their beneficial economic and biochemical properties (small size, reduced production cost, and high stability, affinity, and specificity) together with the availability of multiple crystal structures have encouraged their engineering (4).


Figure 2. To avoid using animals, we produce the nanobodies by recombinant expression.	Figure 3. Some advantages of nanobodies over conventional antibodies.

Second, by reviewing the literature we learned that synthetic libraries of nanobodies were not as simple to develop as we thought. We found out that these libraries are generated by selecting scaffolds of nanobodies that are known to be stable and well-expressed, and then subjecting them to modifications in the regions responsible for antigen binding (3). Consequently, this information led us to wonder whether our idea of recombining the various V, D, and J fragments could lead to functional nanobodies, so we contacted an expert in the field.

In addition, in databases of nanobody sequences (5), we noticed that the different D segments are highly variable, especially in the closest part to region J. Since there were no conserved sequences, it was not possible to recombine the DJ fragments as we wanted. This was also a problem because one of the three CDRs is located precisely there (Figure 4), and if that section cannot be recombined, an enormous variability is lost.

Figure 4. V (pink), D (blue), and J (green) fragments recombine to generate new nanobodies. CDRs are represented in grey. As it can be seen, CDR3 is located in the intersection between the junction of fragments D and J, whose sequence variability makes impossible in vitro recombination of V, D and J fragments.

These limitations forced us to adapt our project, but we maintained its essence. Our goal was now to carry out a proof of concept for the generation of nanobodies obtained by recombining CDRs of different antibodies that bind the same antigen. By recombining CDRs directly instead of V, D, and J fragments we ensure that we do not lose variability in regions that cannot be recombined (Figure 5). Furthermore, by starting from antibody sequences that are known to bind to their antigen rather than straight from the distinct V, D, J sequences of the genome, we increase the likelihood of obtaining nanobodies that can be expressed, are stable and functional. Moreover, while the variability obtained in synthetic libraries is limited because only the CDRs are modified by randomization, our library generates much greater variability by mixing whole genetic fragments containing CDR1, CDR2, and CDR3. In this way, the new antibodies that we generate not only present greater variability at a sequence level, but also at a three-dimensional structure level. This is even more remarkable considering that the nanobody fragments we use come from different species.

Figure 5. The goal of our project after taking into account the limitations of our first idea.

Integrated Human Practices

When developing an iGEM project, thinking about the ethical, environmental, and social implications is as important as developing a good experimental design.
We firmly believe that our library could have a very positive impact on the world because it addresses one of the main problems of synthetic libraries: the absence of the antibody affinity maturation process. With our recombination strategy, we try to optimize existing nanobodies to improve their affinity for their target antigen by increasing variability. This strategy not only makes it possible to improve nanobodies in a simple way, but also greatly reduces the costs and resources required, and avoids the use of animals.
However, in order to analyze the risks, strengths, and implications of such a large project, it is important to consult experts and more experienced colleagues, and to take into account their opinions. In the same way, it is valuable to assess how society perceives science and our project because considering their perception allows us to rethink, as well as to design outreach activities according to their level of knowledge.
Below you can see how we have integrated all of this throughout our project:

Brainstorming - time to think big!

Once the team was formed, we spent two months thinking about different project ideas. For each of them, we carried out an analysis of their strengths, weaknesses, opportunities, and threats. Our main options were:

Lactobacillus probiotic to treat histamine intolerance. The idea was to modify a strain of Lactobacillus to degrade histamine, either externally via membrane-anchored enzymes or internally, with the histamine entering via transporters. We discarded this idea for several reasons, including the lack of experience of our advisors with this bacterial genus.
Fluorescent cyanobacteria intended to make neon signs. We wanted to achieve this by expressing the enzyme luciferase. We ended up rejecting this option because of the low light they produce and because there are microorganisms that already do this.
Lindane detection kits. We were attracted to this idea after the problem that occurred in Aragón a few years ago due to contamination with this compound. However, Dr. Emma Sevilla, who is very familiar with the field, warned us about the difficulty of detecting lindane, so we ended up discarding this idea.
Synthetic antibody library generated by recombination of different V, D and J fragments.

Choosing a project

On April 11, after no less than 4 hours of meeting via Zoom discussing the advantages and disadvantages of the two finalist projects (histamine and antibodies), each team member voted for their favorite option. The winning project, as you know, was the antibody library.

Seeking funding

Getting funding has been one of the most complicated parts of iGEM. We contacted more than 60 companies and institutions, so this required a great deal of organization from us. To facilitate the task, we developed a meeting planner (an Excel file) to which we all accessed to update each other on the progress of the situation with each company.

Project restructuring

Like we said before, by reading papers about how to build nanobodies libraries we wondered whether our idea of recombining V, D, and J fragments was feasible. We spoke with Dr. Luis Alberto Anel, professor of Biochemistry and Molecular and Cellular Biology at the University of Zaragoza, who recommended us to contact Dr. Laura Sanz, an expert in recombinant nanobodies.
Dr. Sanz is the head of the Molecular Immunology Research Group at the Puerta de Hierro-Majadahonda University Hospital. She told us that synthetic libraries of nanobodies are generated by introducing modifications on scaffolds that are known to be well-expressed thanks to previous studies. This strategy is employed because it greatly increases the chances of the generated nanobodies having a stable structure that allows them to bind to the antigen. Therefore, she told us that the nanobodies obtained by our approach would probably not work and she encouraged us to rethink our strategy.
After this conversation, we spent a few days thinking about how we could redesign our project. We ended up deciding to recombine existing antibody fragments, to ensure that the chances of expression and functionality problems were reduced.

Meeting with SynBio Amazonas

We had a meeting with a team made up of undergraduate and graduate scientists that work with nanobodies too. We thought that it would be interesting to discuss the advantages of working with these types of proteins. They had previous experience in iGEM because they participated in the competition years ago, so they advised us on how to do a good job in the competition.
We started talking about our projects - even though they were not going to participate in this year's iGEM competition, they were working on a really interesting project where they use nanobodies. After that, they gave us advice about some points:

First, they told us to contact experts on the field, and although we had already talked to Dr. Laura Sanz, we took this seriously and we decided to plan more meetings.
We also talked about the modeling part. We explained to them what was our first modeling idea, but after discussing it, they made us rethink this part of the project. They told us to characterize the promoter and see what were the best conditions for it to express the cloned protein in the plasmid. Since that was something that most iGEM teams carry out, even though it was a good modeling idea we decided to develop a different one. We maintained the idea of characterizing the promoter but we wanted to go a bit further by modeling the whole intimin expression system.
As we were already developing our education and communication activities, we discussed the advantages of sharing scientific knowledge with people. In addition, we were recommended to conduct surveys to find out the level of knowledge that people have about synthetic biology and, based on the results, to think about carrying out some activities or others.

Conducting surveys

In order to get feedback about our project from the overall population, we developed a survey and we went out to the streets of Zaragoza and interviewed around 40 people. Moreover, we shared it on the internet thanks to which we made it to more than 400 answers! The conclusions of the survey were:

Synthetic biology is seen as good by two-thirds of those who submitted their answers. However, around 25% find it a bad idea or have a neutral opinion about it. Their arguments are that modifying nature must follow more exhaustive controls, that modified crops can affect the environment in long term and that usually economic aims come before improving human and environmental health.
We are aware that synthetic biology is a controversial topic, as it is clear that if used wrongly it can become dangerous. Nevertheless, while we conducted the survey we tried to show people that great things can be achieved thanks to synthetic biology. For example, it permits obtaining genetically modified bacteria that produce insulin.
More than half of the people interviewed found it difficult to detect which of the news that we showed them were about synthetic biology. Nonetheless, almost everybody agreed that more information about synthetic biology should be found in journals and news in order to increase the general knowledge about it. This shows that people are aware that science is the key to progress. These results gave us the idea of carrying out a series of scientific outreach sessions in different cities of Aragón, during which we conducted some experiments aimed especially at kids and also explained to people the objective of our project.
When we asked them about antibody applications, two-thirds of the people answered that these proteins are helpful to treat infections and in diagnosis, probably due to the COVID-19 pandemic. Besides, around 50% of people knew that they are also used in cancer treatments and contaminants detection.
When asking them what they think about using bacteria instead of animals for the production of antibodies, more than 75% answered that they find it a very interesting idea. These results gave us the motivation to continue with our project.

Figure 6. Some of our team members in the streets of Zaragoza conducting the surveys we prepared.

Figure 7. Results

Expectations vs reality: We did not expect such curiosity about synthetic biology. What shocked us the most was the eldery, they were so interested in learning about science. People were really supportive as they gently encouraged us to carry on with project.

Outreach talk

Beatriz Latre has a degree in Chemistry from the University of Zaragoza. Currently, she is the head of the Scientific Culture Unit of the Institute of Chemical Synthesis and Homogeneous Catalysis (ISQCH) and Institute of Nanoscience and Materials of Aragon (INMA), developing innovative projects that lead the chemistry and science of materials to the society. Some projects in which she has participated are Crystallization Contest at School, De Copas Con Ciencia, Science Demonstrators, and Play Tools among others. Now she is involved in her latest project, a videogame @HiScoreScience.

Education and project communication has been one of the most important aspects of our project. We wanted to approach science to everybody, with a special interest in our region, Aragón. This is why we organized experiments in the streets of different cities, we participated in the European Researchers' Night and we spread information about our project on social media.
In order to better develop all these activities, we had a long conversation by Zoom with Beatriz Latre. This meeting took place while we were already doing some of these activities and helped us to better organize educational activities, and realize some mistakes we had made while doing them. Here are some of the conclusions we drew, which helped us a lot and we hope they can also help future iGEM teams:

Setting objectives for the educational and divulgation activities is necessary before starting them. Sometimes these objectives are trying to reach more people so your project gets known and understood, or in order to get financial support. Other times, it is just a way to spread knowledge and to help people to take a close look at science. This led us to consider using our next outreach sessions to promote our crowdfunding, which we did.
It is important to know who is the target public since designing an activity aimed at children and their parents is not the same as designing an activity aimed at teenagers. Once the public is selected, the information provided should be clear and with a low level of difficulty so everybody can understand it. If while performing the activity someone has a high level of knowledge about the topic and wants further information, we should provide it even part of the public won't be able to understand it. As a result, we decided to bring our laptops to the next outreach sessions with graphical sketches and videos about our project, in order to provide more advanced explanations to anyone who wanted to learn more about our project.
Trying to involve the public while explaining a difficult concept is a good option to help them understand it. We put this into practice in the European Researchers' Night , for which we created an activity to explain to the general public how we develop new nanobodies by mixing fragments of different nanobodies. In this activity, people learned how the huge variability of antibodies is obtained, by mixing cards with different words to form new sentences.
A good manner to involve the highest number of people is to perform different activities in different cities. This is something we wanted to do from the very beginning. We made science experiments in the streets of five cities in Aragon: the three capitals of the autonomous community, and two other cities where some of our teammates are from.
It is important to make good planning of the different activities to be done, which allows organizing activities that are complementary at the same time. This helped us recognize a mistake we had made, which was that the first days we carried out outreach sessions, we had not yet launched the crowdfunding. This made us realize that we should have prioritized launching the crowdfunding before, so we could tell people about it while doing the experiments. Once it was ready, we brought QR codes to our sessions that led directly to the website.

Having this conversation with Beatriz Latre was really helpful, and the next activities we prepared were better organized and performed. Moreover, she facilitated our participation in the European Researchers' Night and she shared our social media with even more people, which was of great help.

Expert feedback: Interviews

You can read the whole interviews here:

Dr. Esteban Martínez:

Dr. Esteban Martínez is a researcher in the field of microbiology and synthetic biology and works at the National Biotechnology Center (CNB), in the Systems Biology Department. Besides, he is from Zaragoza - what a coincidence! His research projects focus on providing Pseudomonas putida with new characteristics and, in some of them, nanobodies are involved. He provided us with the plasmids we are using in the wet lab (pSEVA228-I, pSEVA238-I, pSEVA228-AT), so we took advantage of the meeting and asked him for some tips that could help us with the project design.

Having the opportunity to talk to Esteban helped us a lot in the development of the project. Here we show some of the things we learned and the pieces of advice he gave us:

When developing new synthetic biology technology it is important to take into consideration bacterial fitness. Usually, having one copy of the construct is less stressful for the bacteria than several, so he advised us to integrate our constructs into the bacterial chromosome in the future.
In the evaluation of nanobodies' binding affinity, it is crucial to have a high throughput selection method so even if many of the newly generated nanobodies are not able to bind to their target, those that can bind will be detected rapidly.
As we were having issues when trying to purify low copy plasmids (pSEVA228-I and pSEVA228-AT), we asked him for help. He told us how he usually purifies these plasmids and we decided to apply his protocol, which gave us quite better results!
Finally, he congratulated us on our project idea because he thinks it will have a very positive impact as nanobodies have lots of applications, and being able to improve the characteristics of a given nanobody by recombination is a great step forward.

Carmen Lafoz:

Safety

Dr.Rosa Monge:

We talked to Dr. Rosa Monge, an industrial engineer, entrepreneur in the field of bioengineering, and founder of the company BEOnChip. Her doctoral thesis was based on the development and application of microtechnologies to design and manufacture microfluidic devices for cell culture in a biomimetic environment. Based on her thesis, she decided to found the spin-off BEOnChip, which depends on the University of Zaragoza. In 2017, she was recognized by the MIT Technology Review magazine as “one of the ten best talents under 35 years of age in Spain.
The collaboration between engineers and biologists is the key of this company to design organ-on-chip devices that are true to the physiological environment of the body. They create the next generation of in vitro testing platforms, which allow the performance of experiments in vitro that were impossible or only possible in vivo before. Therefore, their products reduce the cost and time span required to develop new drugs, cosmetics, or chemical products. They firmly believe that more and better in vitro research is needed in order to reduce the ethical and economic burden of animal research.
The main relationship of this company with our project is that they defend in vitro tests instead of the use of animals: their devices mimic the environment of the human body, just as we try to simulate the process of generating antibodies that occurs naturally. Besides, the platforms they create reduce costs and time. Here are some of the conclusions that we drew from the meeting with Rosa:

When developing a new product it is important to follow some steps to ensure success: carry out a market analysis, understand what need(s) your product or service is going to solve, communicate with your prospective clients and have alternative plans.
She thinks that our approach is very promising as reducing impacts is fundamental for the future; either reducing animal experimentation, doing things faster, or more sustainably.
The Organ On-Chip devices designed by BEOnChip can make a great synergy with our project as we could use them for nanobody selection, after immobilizing antigens and testing our newly formed nanobodies against them.

Susana Gamen:

We interviewed Dr. Susana Gamen, Director of I+D+i of Operon, a company dedicated to the production of antibody-based lateral flow immunochromatography products. She did her undergraduate studies in Chemical Sciences at the University of Zaragoza, and then she specialized in Biochemistry. In Operon, they produce monoclonal antibodies for their products using mouse hybridomas.
We interviewed her face-to-face because it was a great opportunity to see up close what the process of creating antibodies in a company is like, in addition to applying the advice she gave us when it comes to carrying out our project.
We learned a lot from the interview. These are some points that we extracted:

Even though we knew that it is important to purify our generated proteins, Susana gave us some tips about how to optimize this process and to avoid trying some protocols that would not have paid off.
A very important thing to take into account that she told us was about testing the final product. She said that when you transfer something to the market it is very important to verify that the product works with real-life samples. In our case, even if our antibodies worked with the purified antigen, it would be very important to check whether they still recognize the antigen in natural samples.
She told us we were working in the right direction and that every step that we were taking was well developed, and could work when brought to a large scale in a bioreactor.

References

Chi X, Li Y, Qiu X. V(D)J recombination, somatic hypermutation and class switch recombination of immunoglobulins: mechanism and regulation. Immunology. 2020 Jul 1;160(3):233–47.
Sandomenico A, Sivaccumar JP, Ruvo M. Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments. Int J Mol Sci. 2020 Sep 1;21(17):1–39.
Muyldermans S. A guide to: generation and design of nanobodies. FEBS J. 2021;288(7):2084–102.
Muyldermans S. Nanobodies: Natural single-domain antibodies. Annu Rev Biochem. 2013;82:775–97.
Single Domain Antibody Database [Internet]. [cited 2021 Oct 17]. Available from: http://www.sdab-db.ca/

Team:UNIZAR/Human Practices

Human practices