Human Practices

PROLOGUE:

Radboud University 2024 is the first team to ever emerge in our university. So, this implied a lot of struggles and obstacles which we had to overcome as an inaugural team. When looking back on our journey, it has been divided into two main challenges:

Introducing and incorporating iGEM in the Radboud University:

This included convincing Radboud University to join the competition, understanding iGEM, gathering a team and introducing iGEM to radboud university local community, finding sponsors, taking steps to founding iGEM as Radboud University tradition;

Creating a project with an innovative research idea while meeting requirements of iGEM:

This included project organization, finding a project topic, research planning and brainstorming, Laboratory work.

To achieve both of these goals, our team had to contact multiple people, and get known by the university to be able to develop the project and bring it on the international stage in the Grand Jamboree, 2024.

Introducing and incorporating iGEM in the Radboud University

CONVINCING RADBOUD UNIVERSITY TO JOIN THE COMPETITION (MAY, 2023 - SEPTEMBER, 2024)

As the first team of the Radboud University, it has been a difficult journey from the very beginning. Since, we needed to convince the representatives of Radboud that joining iGEM, and kick-starting this project was worth it and a good investment. This required us to find teachers who are inspired to be our PI, Instructor, and Advisor, and to introduce the awareness about the competition among the administration of Radboud.

Dr. Luc-Jan Laarhoven

Education Coordinator - Education Center FNWI - Education Policy and Coordination in Radboud University, PI of the Radboud iGEM Team 2024

The first person who we introduced to the concept of iGEM. Luc-Jan provided constructive feedback on the possibility of conducting the project. His insight provided us with an understanding of which further people from the university we can address to initiate the project, as well as providing us with a strategy and insight on conducting team recruitment. While agreeing to become our PI, he also gave us an insight on how to invite more members to our team - making presentations about iGEM during the lectures -, as well as suggesting to invite Dr. Tom Bloemberg to become our team member and an advisor of our team.

Dr. Tom Bloemberg

Practical Coordinator - Practical Chemistry Prevention Officer (PAM) - Practical Chemistry in Radboud University, Advisor of the Radboud iGEM Team 2024

Tom Bloemberg was enthusiastic about joining our team as the Advisor, also giving us an insight to contact Frank Nelissen - current team member and Instructor of our team.

ing. Frank Nelissen

Lab Assistant - Biophysical Chemistry -Company Emergency Response Officer (BHV) - Biophysical Chemistry - Prevention Officer (PAM) - Biophysical Chemistry

When we contacted Frank Nelissen, he agreed to become a member of our team as an Instructor, as well as giving us insight into the future organization of working in the lab.

Dr. Paul Kower

Director of Education - Molecular Sciences Institute of Education Associate Professor - Synthetic Organic Chemistry in Radboud University

When it was time for our team to apply to join the iGEM competition, we needed to find the right person to address the issue of our team being supported officially and financially by our University. To do this, Dr. Laarhoven suggested that we contact Paul Kower who suggested and helped us to then reach the Science Faculty Board in our university - Karen de Bruijn.

Drs. Karen de Bruijn

Director of Operations FNWI - Faculty Board in Radboud University

As per the advice of Dr. Kower, we have reached out to Karen de Bruijn, a member of the Faculty Board at Radboud University. With her help, the Faculty of Science considered it useful to invest in the Radboud iGEM Team, allowing us to successfully join the iGEM competition, in 2024.

prof. dr. Laura van Niftrik

Vice Dean of Education - Faculty Board Professor - Microbiology at Radboud University

Along the way, some struggles were present in contacting and collaborating with the Science Faculty of Radboud, for which Dr. Laarhoven suggested that we contact Laura van Niftrik, who then acted as a mediator and provided a resolution in this conversation between our team and Radboud Science Faculty.

UNDERSTANDING IGEM (APRIL, 2024 - JUNE, 2024)

Sriram Kumar, Ph.D, iGEM Mentor

Highly-Pathogenic Respiratory Viruses | Innate-Immune Response | Human-derived Tissue Models | Biosafety, Biosecurity, Policy

As the first team that has ever existed in Radboud University, we have applied for the iGEM mentorship programme to get an insight from a judge and an active member of the iGEM community. Such a member assigned to us was Sriram Kumar who answered our question - what exactly is the goal of iGEM. Prior to talking to Sriram, our team has interpreted it as “the competition of projects”. However, with his input, we discovered that iGEM is not just about making the best scientific work, but more of a combination of different efforts that one can take to building awareness about the problem, taking steps to forming a solution to the problem, describing of the reasoning behind your project all along the way, and trying to challenge one's point of view.

PROJECT ORGANIZATION (OCTOBER, 2023 - MARCH, 2024)

It took us a while to understand how to organize the workflow of our iGEM team and how to implement it in the Radboud University system.

With the great help of Luc-Jan Laarhoven, PI of our team, we managed to establish a workflow. A big help with this was Luc-Jan’s Team Structure session , also used in his Panorama Science 2 course. This session made us reflect on the composition of our team, on the roles in the team which are missing, and which people we should recruit next. It also gave us an insight into project planning and organization.

Rina Vos-Veltman

Controller - FNWI Finance and Control

With the advice of Luc-Van Laarhoven, we thought about the aspect of establishing an account for our team as well as a QR-code for donations to receive financial help from sponsors and supporters of our team. Rina Vos-Veltman was the person whom we contacted to help us with these things, and she gave us insight into the ways of establishing bank account for our team as well as helping us to create our team’s QR-code for donations:

Another thing which Karen de Bruin proposed was establishing iGEM Team as an official establishment. To do this, we contacted Luc-Jan Laarhoven who contacted the Radboud law office who registered our team as officially represented by the Radboud University.

FINDING SPONSORS (FEBRUARY, 2024 - SEPTEMBER, 2024)

On our first steps to trying to find collaborations with sponsors as a new team, we have struggles with finding sponsorships. Our first thought was to contact sponsors through emailing them on their general address. Also, we decided to make a non-confidential deck by the advice of Karen de Bruijn. We polished the video after the feedback from Dr. Luc-Jan Laarhoven, Dr. Tom Bloemberg, and ing. Frank Nelissen, and received the following result.

Ton Korts

Start Up Coach at Mercator Launch/ Radboud University

We have also received an insight to contact Mercator Launch from Paul Kouwer, where we connected with Ton Kortz. He gave us an insight on our sponsorship campaign - approach to companies with a question: “Help the future of the scientific world, help your future colleagues!”. Addressing the issue of the competition to get the skill on the market. This changed the way we composed our emails and addressed our sponsors. Not only that, but Ton Korts connected us with an initiative member of Pivot Park Board - Britta Driessen.

Britta Driessen

Business Development Manager at Pivot Park

Britta Driessen helped us to connect with a company called Ardena, and helped us to persuade Pivot Park to support our project and become our sponsor.

Dr. Frits Mattijssen

Knowledge Transfer Officer | Radboud Innovation Science|Faculty of Science at Radboud University

With help of Karen de Bruijn, our team has reached out to Frits Mattijssen, we also received his feedback on our emailing to companies, and gave us an insight that emailing to the general address of the companies or filling out official forms does not help that much when reaching out to them. What helps is personal connection and conversation face-to-face by being introduced to the company representative. Our next contact confirmed his theory.

With great help of Tom Bloemberg, we were connected with representatives of the newly established company Purifise, which specializes in providing purity for their clients’ products, - Gert Jan Ettema and Robert de Boer. They were conducting certain measurements in our university, and so were asked to help us out with our research. Not only that, but they became supporters and sponsors of our project.

Gert Jan Ettema

Managing Partner at Purivise | optimizing processes and products Business development manager at BioConnection

Robert de Boer

Managing Partner at Purivise; Owner at Rodi Holding BV; CMC-Director at SPL Medical; Interim BD-manager BioConnection

Another very useful advice which our team received was from Sriram Kumar who advised us to apply for iGEM sponsors’ programs. We applied for a grant from Promega and for material supplies from IDT. It was a success; IDT provided us with a basic amount of needed materials and some more for free. And even though we were not selected as winners by Promega, they still helped us out. Faustin Mbuyi from Promega contacted us with an offer of free material supplies which was very useful for our project!

Faustin Mbuyi

Account Manager in Promega

STEPS TO FOUNDING IGEM AS A RADBOUD UNIVERSITY TRADITION (MAY, 2024 - OCTOBER, 2024)

Our next step to integrate iGEM into the Radboud University was attempting to make it known and potentially inspire a new team of students to start the project anew, and make iGEM a tradition at Radboud! Here are some steps which we made:

1. Educating Radboud on what the competition is via Radboud Newsletter

With an insight from Karen de Bruijn and Luc-Jan Laarhoven, we were suggested to get in contact with Radboud local Science Faculty Newsletter. This way, we could gain publicity and make ourselves and iGEM known in Radboud.

Esther Veerbeek

Contentspecialist - Marketing and Communicatie

With consultation and help of Esther Veerbeck, we managed to formulate and submit two new messages to the science Newsletter, and receive more publicity in Radboud University.

2. Presenting at the students 1st Yearly Bachelor Science Faculty Symposium

When brainstorming the possible ways to reach out to the Radboud iGEM community, Luc-Jan Laarhoven came up with a way to reach out to younger students who could start up another iGEM Team in 2025. This opportunity was the 1st Yearly Bachelor Science Faculty Symposium. By adhering to the advice and insight from Luc-Jan, we have prepared a presentation of our team and of why iGEM is a good opportunity for a student to develop themselves. We received a positive response from the community, and made several people interested in starting a team in the next year.

3. Meeting with Teams in Utrecht

As a great initiative from the Dutch iGEM community, organized by the Center of Living Technologies, our team was invited for the yearly Dutch convention of 2024 iGEM teams. We were invited to join as another Dutch iGEM Team, and used this as an opportunity to present our project and get some feedback from other iGEM teams as well as getting connections. The insight of other iGEM teams made us think about the way of administration of our medicine and its exact mechanism, as well as the method of testing the functionality of the delivery system. Also, our team was advised that developing social media is useful for interactions with other teams and keeping connections. This event was a wonderful look at our project from a different, new perspective.

4. Instagram page and LinkedIn

Also advised by the other teams, we started to actively upkeep our social media pages, which provided us with greater publicity, and provided us with further opportunities to join more exciting events with other teams.

5. Collaborating with other teams

As a result of us trying to expand our publicity, we were contacted by the iGEM team of Linköping who invited us to join an initiative to create iGEM Teams international Lipid Delivery System Handbook Collaboration. As this was a great opportunity to enhance and easen the research of future iGEM teams in this topic, our team dived right into this process, making our contribution to improve the community of science around us.

Together with these wonderful teams we created our Lipid Delivery System Handbook which we are very proud of.

We consider what we did by our social and internal initiatives a great help and support for the future Radboud iGEM Teams, and for the world, since creating a new initiative community of scientists is a way to create a community which creates robust and creative ideas which can improve the world and move the science forward!

Creating a project with an innovative research idea while meeting requirements of iGEM

FINDING A PROJECT TOPIC (SEPTEMBER, 2023 - FEBRUARY, 2024)

Brainstorming our project ideas was not easy, and to answer the question: “Which challenge is iGEM Radboud 2024 going to take on?” During the multiple brainstorm sessions, and getting feedback from Dr. Luc-Jan Laarhoven, Dr. Tom Bloemberg, and ing. Frank Nelissen, we have cast away research topics such as glowing UV mushrooms detector, milk spoiling detector, or mold detector, and stuck with the topic of Haemophilia. When we first started our research, we wanted to target the research of aptamers in Haemophilia patience - an immune response of the people who start to get treated against Haemophilia disease.

Sanna Rijpma

Clinical Chemist at Radboudumc

To answer the question: “Where do we start with the research on aptamer?”, we decided to consult with an expert from Radboud University Clinical Center on Haemophilia A (HemA) disease, Sanna Rijpma, and we have received some useful insight into HemA and the advice on the further project development. As it turned out, a treatment to aptamers is to develop an immune resistance under constant control of the doctors, and since we wanted to create a patient friendly system with which they would not have to leave the comfort of their home, we focused on developing an innovative treatment method of the Haemophilia disease - InfinitiF∞.

RESEARCH PLANNING AND BRAINSTORMING (APRIL, 2024 - JUNE, 2024)

When it came to a step of brainstorming the research procedure, our team received a lot of feedback from Tom Bloemberg (Provided planning for the LNP part, and insights into analytical techniques regarding LNPs) who provided an insight of the importance of elaborate research planning. It took us several attempts to improve our research planning to get Tom’s and Frank Nelissen (Provided planning for the mRNA part, and insights into analytical techniques regarding mRNA) approvals and access into the student labs. The planning involved planning of the research timeline, procedures, and scientific model selection, since the budget of our team was limited. But in the end, we came up with the idea of the following research plan: Radboud iGEM Team Research Planning. Radboud iGEM Team Research Planning

Luuk van Summeren

Employee of the Molecular Sciences Practical Training at Radboud University

In the midway of our research progress, we encountered a problem of our expected lipid materials for LNPs to be too expensive for our budget, for which we consulted Luuk van Summeren who gave us insight into our planning, as well as into our ionizable lipid synthesis results: he suggested to analyze NMR more precisely to make sure that the compound that we look for is correct, and if the results are not very precise to conduct MS analysis as well as elemental analysis. On the matter of lipid particles, he agreed with Tom Bloemberg that a model system of LNPs is necessary, and suggested that we contact Moussa Boujemaa on the following matter to get a better insight on the matter of LNP structure. After all of the acquired feedback, our team came up with the following alternative model compared to the LNPs from the original reference article [1]: LNP material choice. lipid materials for LNPs

Moussa Boujemaa

PhD candidate - Systems Chemistry Department

And so, the next person who improved our insight into LNP formulation was Moussa Boujemaa. With his vast knowledge in this field of research, he provided us with useful insight into the LNP preparation and practical tips when we still were setting up the research of lipid carriers.

Dr. Kevin Neumann

Assistant Professor at Systems Chemistry Department, Radboud University

Kevin Neumann, also from the Systems Chemistry Department of our University, shared his insight into the LNP preparation, practical tips when we still were setting up the research of lipid carriers.

Remi Peters

Intern at Radboud University

Remi Peters provided us with insight into the types of LNPS analysis and characterization, when we still were setting up the research of lipid carriers.

Kevin Venrooij

PhD student at Synthetic Organic Chemistry, Radboud University

Kevin Venrooij, with his specialization being alternative treatment of autoimmune diseases, reactive b cells, t cells, chemically made peptides, and peptide targeting for certain receptors, provided us with practical tips when setting up research, insight into the cost calculation and time estimation for the LNP formulation procedure, when we still were setting up the research of lipid carriers. He especially made us consider potential immune responses of the patient on the external LNPs.

Gert Jan Ettema and Robert de Boer

Gert Jan Ettema and Robert de Boer provided us with an insight on our plan, and research tracking - they suggested a table tracking method which we applied for our project. On the matter of research, they challenged our opinion, and made us decide on using cheap cells, like HeLa or HEK cells (which we used in the end), and not LSEC cells which are very rare and expensive. They gave us an insight on the ethanol injection procedure which we used in the end, and suggested on the future plan of action if we were to do the nasal spray excipients: to work on its formulation, dosage, and apply a single use spray pack to avoid side-effects of the overdose. They also suggested that we analyze LNPs with TEM, DLS machine (size and Z-potential), and encapsulation efficiency, all of which we applied in the end. We got an insight from them which we applied that the mRNA degradation can be prevented by reducing pH, and storing them in solution with alcohol and aqueous phase.

Zainab Javed

PhD candidate - Physical Organic Chemistry

Dr. Wojciech Lipiński

Postdoctoral researcher at Radboud University

Zainab Javed and Wojciech Lipiński provide us with an answer to the following topics (questions were brainstormed together with Tom Bloemberg): Cheapest and most accessible lipid stocks (we found DSPE PEG2000 to be the most suitable candidate), any common mistakes in the LNP synthesis procedure (sonication of lipids before use helps to break down the aggregates that they form), a possibility of not including mRNA in LNPs (it is possible not to add mRNA to the aqueous phase), common analysis of LNPs (DLS, potentially TEM), the length of procedure for LNP formulation (1 day), storage of LNPs (Lipid stock: -20; Lipids: RT; LNPs: -80). Not only that, but they provided us with the necessary lipids from their lab for our experiments, which we are very grateful for.

Merlijn van Haren

PhD candidate at Radboud University

In out time of struggle with finding the right equipment for the LNP purification, we received invaluable help from Merlijn van Haren, who provided us with insight into dialysis membranes and LNP purification techniques, as well as invaluable help with provision of necessary lab equipment - dialysis tubes, all of which we applied in our further research procedure.

LABORATORY WORK (MAY - SEPTEMBER, 2024)

During our lab work, we have met multiple complications and were puzzled by many questions such as: where do I store this material, how is it better to deal with this chemical, how should I do this analysis, “what do I see on the gel?!”. With these and many more things we have received a lot of help and support from Frank Nelissen, our iGEM instructor, who was there to support us by answering these questions,supervised wet lab work in the mRNA production part of our research, and provided critical feedback along the way. We not only had the struggles with analysis of the results, but also with constantly getting access to university facilities and laboratories, for which we were lucky to address our advisor, Tom Bloemberg, who was there to not only give us access, but to also provide his critical thinking to support us on our planning and decision making regarding the acquisition of certain materials. Tom also helped us a lot by supervised wet lab work in the LNP production part of our research.

We have received a lot of support not only from Frank and Tom, but also from the other staff members of the Radboud University Student Lab facility. They provided us with access to university facilities and helped us with multiple other things. Luuk van Summeren was there to help us with the brainstorming of our laboratory work planning; technical training and insight in laboratory work, help with getting needed supplies for the lab. Luc provided us with an insight of how to use Fluorescence Plate Reader, which we used to do the analysis for mRNA encapsulation efficiency of our LNPs.

Pepijn Geutjes

Amanuensis Molecular Sciences

Pepijn Geutjes provided us with his Insight in sonication procedure, as well as explaining to us the COSY-NMR analysis. He operated the NMR machine from which we received data required to analyze the ionizable lipids in our research.

Michel Giesbers

Education assistant and warehouse employee

Michel provided us with the needed glassware for our research and gave us insight into the better option of glassware for a certain experiment.

Ishani Bhattacharya

Supervisor, Synthetic Chemistry Labs

Ishani provided us with the needed support and encouragement during the lab work, as well as answering our immediate questions in the lab which helped us to keep up the pace when performing the experiments in the lab.

Rob Mesman

Staff Scientist - Microbiology

Rob Mesman conducted the TEM analysis for our team, and provided his insight into what results we see after the analysis of the LNPs and transfection. With his input we understood that it is important to add lipids into the aqueous phase upon mixing during the ethanol injection, and that it is important to have higher concentration of lipids, since otherwise they mostly form lipid clusters.

Dustin van Doeselaar

PhD candidate at the Systems Chemistry department of Radboud University

With invaluable help of Dustin van Doeselaar, we have received an insight into the DLS analysis method, since Dustin provided us with a special DLS machine training and helped us to get an access to the DLS machine booking system. Dustin gave us his opinion on our analysis results, such as behavior of correlogram and the correct analysis mode, which helped us to reach a more certain conclusion about our research results.

Martin Emmaneel

PhD candidate - Biophysical Chemistry

Finally, with the invaluable help of Martin Emmaneel, we managed to perform transfection of our LNPs and mRNA, and with his reference GFP we received useful insight that the targeting and LNP procedure should be improved in the future.

We are convinced that by performing multiple collaborations within, and from the people outside of Radboud University, we made the iGEM competition more renown not only among the students and administrations of Radboud, but also among multiple Radboud research groups. This, in the future of other Radboud iGEM Teams will make their path to success easier, since they will receive more help and support from all the members of Radboud community, which will also unite our university in a creative project on a way to develop innovative solutions for improving lives of many across the world!

References:

1. M. Kim et al.,Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver.Sci. Adv.7,eabf4398(2021).DOI:10.1126/sciadv.abf4398