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Human Practices

For our Human Practices (HP), we wanted to investigate the complex systems a new development such as this would have to navigate before entering the market.

Human Practices


GutFeeling aims to create a commensal gut bacterium that is able to autonomously secrete medication at a consistent rate, L-DOPA in our case. As it stands, such a treatment is far from actually being used In the following sections, we explain the factors playing into the implementation of live bacteriotherapies such as GutFeeling, following the framework of Multi-Level Perspective and the guidance of various stakeholders..

The first step in tackling HP is identifying your stakeholder network. In our case we build a simplified stakeholder network for the development of new treatment methods for Parkinson's disease (PD) (figure 1). We decided that patients are the starting point and ending point of the network, since their needs for healthcare products produce the impulse for all other stakeholders to act. If the perfect treatment were to exist there would be no further need for additional developments in PD treatment.

Our stakeholder network is as follows. The needs from the patients provides an impulse for regulatory bodies and financing bodies to provide funding for academia to study the disease and potential remedies. The knowledge that is generated by these activities are then translated by biotechnological and pharmaceutical companies into methods, technologies and drugs that can be used by the healthcare system to combat the disease. Before being used in healthcare they have to first be accepted by regulatory bodies, which are a central decider in our network. Public perception of the value of scientific inquiry into PD is a major driving force for regulatory and financing bodies, and is itself strongly impacted by education and marketing activities.

This figure is interactive! Click on any of the elements in the flowchart to learn more.

Multi-level Perspective


After identifying the stakeholder network (see Human Practices, Fig. 1), the next step in HP would be to formulate a strategy to study how our project interacts with the network. We are lucky, as one of our PI's, Terry Vrijenhoek, joined the iGEM project to add his insights and experience in tackling Human Practices (HP) within disruptive fields of life sciences. He observed that many iGEM teams of previous years did not base their HP on a theoretical framework. So he motivated us to attempt this within our project.

The first idea Terry shared with us is the idea of a transition curve, or x-curve (Fig. 2). Basically, innovations aren't used or accepted immediately by everyone. First there is experimentation with the idea which creates improvements or support that accelerates its use. This quicker pace of adaptation results in a point of chaos, as developments come faster than people or governments can keep up with. If the innovation successfully passes the point of chaos it becomes the standard and a long-term part of society. Because the point of chaos is the most crucial point, we decided to focus a large part of our project to efforts that would help live biopharmaceuticals to get past that point. We think the point of chaos is getting approval for the first treatments in human patients.

X-curve for transition

Figure 2: X-curve for the transition of drug based medication to live bacteriotherapies. The four main phases we identified are achieving functional expression and control in the gut, safety trials, initial treatment development, and standardisation of the treatment method

Because not all innovations actually get accepted we wondered how to give our project the highest chance of being translated into society. For this we started looking at different theoretical frameworks from Transition Management. Terry also suggested looking at the broader field of Innovation Science. After selecting some of the most commonly used frameworks, we studied if iGEM teams of previous years had used those or other theories for HP. We think this work highlights that iGEM teams could benefit from using theories from Innovation Science, and is published in Vector 2025. Since we provide an overview of multiple dominant theories from Innovation Science, and provide arguments for incorporation them in iGEM we view it as a contribution. A link to this article can be found on the resources page. Our research made us decide to build our HP based on the framework we think suits our project best: multi-level perspective (MLP).

MLP is a framework that describes how changes in a system can happen over time by looking at three different levels . The central level is what we will call the system. A system is all the stakeholders that together do a certain social or economic task, like energy production or healthcare. If we take the healthcare system as an example, the stakeholders would include hospitals, doctors, patients but also governments that fund those hospitals or regulate health insurance and many others. Changing a system is usually hard, because it needs companies, governments and consumers to change the way they function . This requires new investments, training or infrastructure and is therefore expensive. So MLP states that outside pressure over time forces the system to slowly change. Examples of these outside forces could be societal values or economic trends, for which we will use the term "social and economic background". The social and economic background provides a direction for the system to change, for example to become more sustainable. Then entrepreneurs, start-ups, inventors and scientists try to develop new inventions that enable the system to change, and we will call anyone who does this "innovators".

To give a simplified example of an MLP, based on Geels : for a long time we used coal and fossil fuels to generate energy (system). But due to concerns over climate change and environmental pollution, society and politicians wanted a more sustainable way to produce energy and introduced financial compensation for sustainable energy developments (social and economic background). So people and businesses invented and improved solar panels and wind turbines (innovators). These improved methods for energy generation then got used more and more (system change), and sustainable energy production in Germany went from 3.6% in 1990 to 44.6% in 2022 .

Geels also makes clear that without the financial support from the government this change would likely not have happened, and that many large energy companies did a lot to resist this transition at first. This means that having a good goal alone is not enough to change a system, you need pressure and innovation as well. We think that MLP can help us and other iGEM teams to identify if that pressure exists or should be increased, and what innovations are needed. On this page we will describe our efforts to analyze the social and economic background, the current system and how we tried to adapt this to innovate the way we deliver drugs to patients with chronic illnesses.

Why: We hoped to gain input on our HP approach from a Transition Science expert.

What: Terry provided an explanation of X curve and theoretical frameworks

Takeaway: Terry advised us to involve theoretical frameworks in our topic selection and general HP approach.

Integration: We created our own X curve and used it in the process of topic selection. Besides this, we based our Human Practices on the theoretical framework of MLP as described previously.

Our Human Practices journey started with one of our PIs, Terry Vrijenhoek. He is an expert in ethics, and transition science, among many other things. Transition science is the study of systemic changes, which examines how societal systems shift and adapt over time. Commonly, transition science regards the incorporation of a newly emerging technology.

In our first conversation, Terry Vrijenhoek introduced us to several theoretical frameworks and tools from transition science that we could use to guide our HP process, including the X-curve framework, as well as some approaches to stakeholder identification. He explained that this was an unusual approach to HP within iGEM, and motivated us to try using these theoretical frameworks in our HP.

We chose to incorporate the X-curve framework in our topic selection process. We made the following figure for the societal incorporation of living biopharmaceuticals, which was a central point of many of our topic ideas (Figure 2).

Here the major point of chaos lies between point two and three, that being the moment where living biopharmaceuticals would be approved for use in the human gut.

Having worked this out, we realised that many of our topic ideas regarded developments that fit in the top of the X-curve, while the lower part remains underdeveloped. Because of this, we shifted our focus to developing the base of the curve. Instead of developing a specific therapy, we set out to tackle the roadblocks on the way to implementing living biopharmaceuticals in general.

After this conversation, we delved deeper into several theoretical frameworks, examining which ones align best with not only our goals as a team, but also iGEM's expectations. We settled on Multi-Level Perspective (MLP) as our main theoretical framework to base our HP work on. A more detailed description of this can be found above.

Follow-up

Once we had created this X-curve and adjusted our topic proposal based on this, we scheduled a follow-up conversation with Terry Vrijenhoek. The purpose of this meeting was to get feedback on our project topic and the X-curve.

Regarding the X-curve, he agreed with the steps to implementation as we had identified them. Terry motivated us to consider our influence on the process, both in our research and in our engagement. To do this, we had to analyse the network of stakeholders involved in this process, and determine the barriers and drivers keeping them from or pushing them towards change.

With the help of Terry's feedback, we created an overview of stakeholders in the form of a flowchart (Fig. 2).

Social and Economic Background


Overview

In this section we describe some values and trends that create pressure on stakeholders in our network to change the way medication is produced or delivered. We think that an increasing care for sustainability and affordability of medicine provides pressure on the pharmaceutical industry. The wider field of synthetic biology will benefit from many economic tailwinds. These trends will increase the chances of using bacteria in drug manufacturing and therefore might translate into increased study of live bacteriotherapy.

Pressure from Trends in Social Values

In order to change a system, you first need to know what it should change to. Below we highlight trends in social values that we think might predict what the future will look like. We think that sustainability and equality will become more important in the future, and might also impact the pharmaceutical industry and healthcare.

Increasing Demand for Sustainable Healthcare

In recent times sustainability has become an important social value. In our survey concerns over the environment were the 5th most commonly raised concerns. It is therefore no surprise that the entire pharmaceutical industry has been under increased pressure to transition into a more sustainable industry. Using Scopus we found that now over 3.000 papers are published yearly that contain "sustainable" and "pharmaceutical" in their titles, keywords or abstract (Fig. 3). The unsustainable production of L-DOPA and the mounting pressure on the pharmaceutical industry might create opportunities for change. Pharmaceutical companies are responding to this pressure. For example, Abbvie, a large pharmaceutical company that produces the Duodopa PD treatment, has set goals to reduce emissions, use renewable energy, waste reduction and other aspects in their 2024 Environmental Social Governance (ESG) report.

Sustainability papers trend

Figure 3: Publications that include “sustainable” and “pharmaceutical” in their titles, keywords or abstract over the past 40 years.

Increasing Concerns Over Equal Access to Medication

In addition, concerns about the price and availability of medicine have also increased. Annual treatment in 2018 was around 1.292 to 5.307 dollars for Neupro , which administers PD treatment through a patch that has to be applied to the skin and is effective for a day. Read the section System: Industry, PD medication not taken orally for more details on Neupro. A long-term treatment for PD is Abbvie’s Duodopa, which is a hydrogel that is administered into the intestines through permanent tubing with the help of a pump. Treatment with Duodopa is estimated to be around 60.590 dollars annually. We did an extensive calculation on the potential price of our treatment based on hospital data on fecal transplant cost and other variables, which can be seen in resources. We estimate that our live bacteriotherapy treatment would cost at a minimum 16.000 euros. This would mean that our product would not be able to compete in terms of price-availability compared to daily medication for PD, but does provide a better alternative as a long term solution than Duodopa. Another obvious advantage for bacteriotherapy compared to Duodopa is not needing permanently attached tubing in your body.

Pressure from Economic Trends

As mentioned before, there is a lot of money involved in healthcare and drug delivery. This means that without economic pressure companies might not switch their focus. That means that without significant economic advantages being created in the future, the system won't change. So below we will detail a couple economic developments that might provide a boost to the adaptation of bacteriotherapy.

Increased Knowledge on Genomics, Epigenomics and Gene Editing Tools

More and more knowledge is available on genomics, epigenomics and gene editing tools every year. Together these fields might provide tools to regulate protein production very strongly. Strong regulation of the production of proteins and compounds of interest might reduce concerns over variation in dosage. Additionally, the increased use of gene and epigenetic editing tools is driving down costs of these tools, while they are still getting increased accuracy.

Increased Commercial Use of Synthetic Biology

Another significant trend is that synthetic biology is increasingly being used commercially, partially to the item mentioned directly above. The market for synthetic biology is expected to increase by 17.3% to 20.7% annually in the upcoming years . The pharmaceutical industry is inherently innovative and oriented at getting economic advantages. This could drive exploration of the use of synthetic biology by the industry. In turn this will boost general interest, funding and knowledge for new synthetic biology based technologies. Examples of innovations that would benefit both pharma and bacteriotherapy include improved biocontainment strategies and increased production yield of drugs by microbes. Increased investment into synthetic biology from the pharmaceutical industry could therefore provide horizontal crosstalk that would benefit bacteriotherapy.

Increased Government Funding into Synthetic Biology

Historically, global investment from governments into synthetic biology has been rising, with large parts of continental Europe initially falling behind. But the EU has been providing additional funding to synthetic biology, for example through its Horizon Europe programs . In terms of academic output on synthetic biology Europe is a global leader, ahead of even the USA . These trends are expected to continue with the drive towards a more sustainable biobased economy. Increased funding, innovation and stimulatory regulation will over time benefit the development of new synthetic biology based techniques, potentially including techniques for drug manufacturing and delivery.

The System of Drug Delivery


Overview

In this section we will look at how the current way of drug delivery is done and maintained. We will also look at factors that resist or promote changing drug delivery. MLP analyses this through 5 lenses: the market user, industry, science, cultural and policy & regulation viewpoints .

Market User

Overview

The market users of our product would be PD patients. Initially we were concerned that patients would be hesitant to use GMOs as a treatment, but patients were more open to try it than expected. Based on our survey and interviews we conclude that patients experience a burden of medication, and would feel relieved if our treatment would be successfully implemented. Additionally, the most important factors that should be taken into account when in dialogue with patients would be providing a clear explanation of the treatment and the potential long term effects for the patients themselves.

Identifying Our Users

Our most prominent group of end users and the main demographic of our product is people with chronic illnesses who need to take constant medication to manage their symptoms. Especially those who struggle with medication adherence or who are burdened by their constant need to medicate could benefit from reliable constant medication. After attending the seminar Beyond Movement: The Impact of Dopamine Dysfunction on Neuropsychiatric Functioning in Parkinson’s Disease by Isabelle van Hapert, MSc., and Dr. Marit Ruitenberg, we learned that Parkinson’s patient commonly struggled with medication adherence, especially after the disease progressed to a point where it would cause cognitive decline. Because of this, Parkinson’s was taken as the main example in our project, but similar live bacteriotherapies could potentially also be applied for conditions such as psychological disorders and neurological disorders.

To study how PD patients as well as other members of society think about our project we shared a survey of which the results can be found under resources, and held multiple interviews with various stakeholders.

A Desire for the Best Treatment

We assume that the primary concern of patients is to receive the best treatment possible. At the moment of writing, we can not claim with certainty that bacteriotherapy will prove to be a more or less effective treatment than conventional medication. But, if a bacteriotherapy would exist that is more effective in treating a chronic disease, we think that patients will see that as a reason to adopt that treatment. We found evidence for this with patients voicing concerns over quality of life decreases due to PD:

"(...) the further Parkinson's progresses, the less possibilities there are to keep it liveable. I would seize (almost) any opportunity to increase my quality of life."

Quality of life can be improved through effective medication, but also through improved patient care. This includes patient-doctor communication, patient experience and integration of treatment into the life of patients and relatives . In these aspects we think that bacteriotherapy will have advantages over conventional medication.We assume that patients would have to order and pick up medication less frequently from apothecaries, and would require less assistance by informal caregivers, which came forward as a struggle of some PD patients in our conversation with Kyra Zymnik, who is in part responsible for the care of her grandfather with PD. She noted a constant administration method would relieve her and her family’s burden as caretakers, while simultaneously restoring her grandfather’s independence. Furthermore, once the technique has been established and integrated as a standard practice of healthcare we think that the frequency of hospital visits might also be reduced, due to the increased stability of medication slowing disease progression. This is not the case for the first administered treatments, as those would be strictly monitored for adverse long-term side effects.

Eagerness to Be Relieved of Burden of Chronic Medication

As mentioned before, chronic medication places a burden on patients and makes it easy to forget medication. Around 30% of the participants in our survey also indicated they find it hard to adhere to their medication schedule, with an additional quarter of the participants indicating it is neither easy nor difficult (Fig. 4).

Medication adherence difficulty

Figure 4: Experienced difficulty sticking to a daily medication schedule.

We found that people who indicated to struggle with medication adherence were more likely to feel comfortable with our proposed treatment (Fig. 5). More than half of the respondents indicated that they were at least slightly comfortable with using the treatment method.

Comfort with treatment by adherence

Figure 5: Comfort with genetically engineered bacteriotherapy based on issues with daily medication.

When asked to elaborate on their feelings regarding this treatment, patients clarified:

"[My disease] is a second job, it would be lovely if there were something less mentally burdensome"
"If it works it will give a certain sense of rest. Now, I live every day on the clock."

Overall, we found strong support for the relief of medication burden. This sentiment was shared by Merle, a representative of ParkinsonNL, whose stakeholder interview can be found under Science dimension. She noted that even patients who had no trouble taking their medication on time at all would still benefit from constant administration, as oral L-DOPA administration still has “off periods” where the last dose of medication has worn out, and the new dose isn’t effective yet. Merle explained that even patients with perfect medication adherence suffered from this phenomenon, and would thus benefit from live bacteriotherapy as well.

Reluctance to Change Medication

We initially expected that people would not like to change medication if they are currently taking effective medication. This was because new medication can come with adverse side effects, have lower efficacy than the current treatment etc. However, based on patient and survey responses we expect that patients would consider switching treatments if it was proven safe and effective (quote 1; Fig. 6).

Openness to treatment

Figure 6: Openness to genetically engineered bacteriotherapy based on issues with daily medication.

Why: determine the needs of Parkinson's patients

What: Information about their struggles

Takeaway: medication adherence is a serious issue among Parkinson's patients

Integration: We focussed our project on addressing this issue.

We attended an informative evening organised by IFMSA as a part of their Medicafé series of seminars. In Beyond Movement: The Impact of Dopamine Dysfunction on Neuropsychiatric Functioning in Parkinson's Disease, Parkinson's experts Isabelle van Hapert, MSc., and Dr. Marit Ruitenberg on the non-motor challenges faced by Parkinson's patients.

They explained that PD does not end at tremors and bradykinesia, but also encompasses mental health challenges such as difficulty controlling impulses and increased anxiety. Furthermore, it became clear that one of the non-motor symptoms of Parkinson's is cognitive decline, which can negatively impact a patient's ability to pay attention or think, but can also cause memory loss ranging from occasional forgetfulness to severe dementia.

In some cases, this can lead to a negative spiral where cognitive decline causes subpar medication adherence, and this, in turn, furthers cognitive decline. Isabelle and Marit did note that these extreme cases would not be left to take care of themselves, but would rather be admitted to a care home once the cognitive decline got severe. Still, the same issue would occur for patients experiencing minor forgetfulness, and in fact the most extreme cases would likely start out in this minor way. If we could tackle this problem early, we might prevent severe cases.

Thinking back to our earlier concepts for a living biopharmaceutical, we came to a potential solution for the problem of medication adherence in Parkinson's. We committed to creating a live bacteriotherapy for Parkinson's, where a commensal bacterium could remain in the gut of the patient and secrete therapeutic compounds at a consistent rate.

Why: We wanted to get input from Parkinson's patients. Though we were not able to get an interview with someone who has firsthand experience with the disease, we managed to get a conversation with a grandchild and caretaker.

What: We had a very personal conversation on the lived experience and wants and needs of Kyra's grandfather.

Takeaway: The most important thing for this specific patient is his independence.

Integration: This conversation helped us determine the need for an autonomous drug secretion method that lets patients maintain their independence.

We got in touch with Kyra Zimnik through our personal network. She is an occasional caretaker of her grandfather, Simon Hulshoff, who has Parkinson's. With the permission of them and their family, we had a conversation with Kyra about their experience and struggles with the disease.

Kyra told us how her grandfather had worked as a farmer all his life, and got in contact with various pesticides while doing so. When he was about 79 years old, Kyra noticed her grandfather exhibiting the characteristic tremors and gait of a Parkinson's patient. Having just gotten a lecture on the symptoms in a neurobiology class, Kyra alerted her family to the potential disease. A while later, her grandfather got diagnosed. About this, Kyra states:

"He was a good sport about it. He didn't give much of a reaction when he got diagnosed. At most he got annoyed when he could no longer do certain physical activities. Like a typical Dutch farmer, work was an important part of his life."

Kyra describes her grandfather as a family man, always protective of his children, and later of his grandchildren as well. When asked to describe him in one word, she calls him funny. According to her, his personality hasn't changed at all since the disease developed, only the energy behind it can be a bit lower at times.

"In general, the disease doesn't bother him much. The only thing he fears is becoming dependent on others to live. He has made sure that he will never end up in a nursing home. If it gets to that, he would rather undergo euthanasia than give up his independence."

The need to be independent for as long as possible is one commonly seen, especially in Dutch rural farmers' communities. Kyra noted his mindset was typical in this sense. Currently, Simon's next of kin have to ensure he takes his medication on time, which is not something he is content with.

When asked if Simon would appreciate the idea of a live bacteriotherapy, Kyra noted that he might not be too bothered with it at all, as long as it was approved by his doctors. If anything, it would help him maintain his independence.

This conversation helped us determine the need for our project. In later conversations with stakeholders, it was often useful to have an example of a person who would benefit from our work. Still, for proper insight, we needed to reach more PD patients.

Why: We wanted to determine if the patient's needs align with our design, and get the input from as many PD patients as possible. We also wanted to estimate the opinion of the general public, and determine which factors played into their acceptance.

What: We asked questions about acceptance, worries, and desires, as well as questions to determine different groups' acceptance of our project.

Takeaway: People cared about reversibility, autonomy, and long-term safety. Different groups had similar acceptance rates, but it is interesting to note that people who often struggle with medication adherence had higher acceptance rates.

Integration: The need for reversibility and autonomy made us shift our attention to the incorporation of a killswitch. The need for a clearer explanation of our treatment made us put more effort into the education aspect of our project, and inspired us to reach out to a science communication expert.

Although it was incredibly valuable for us to have an insight in the lived experience of a single PD patient, there was no way for us to determine the wants and needs of PD patients in general from this contact alone. Because of this, we created a survey with our most important questions, and distributed it to the general public through our personal networks.

Parkinson Vereniging, a Dutch patient organisation for people with Parkinson's or Parkinsonisms, was so kind to help spread this survey among their members, for which we are immensely grateful.

The complete findings of our survey can be found in the resources tab, but the most important findings are summarised below.

In general, people were reasonably accepting of a treatment such as GutFeeling. Over half of respondents indicating they felt comfortable with the idea of using a live bacteriotherapy (Fig. A) and nearly three quarters indicating they would likely consider using it if it had gone through the proper medical trials to prove safety and efficacy (Fig. B).

Comfort survey results

Figure A: Comfort with genetically engineered bacteriotherapy.

Openness survey results

Figure B: Openness to genetically engineered bacteriotherapy if proven safe and effective.

In general, differences in demographic groups such as age, education level, or career field did not impact the acceptance rates of live bacteriotherapy. However, we did see a difference in acceptance rates between people who did and did not indicate struggling with medication adherence issues. People who struggled with taking their medication on time felt more comfortable with the idea of a live bacteriotherapy, indicating that the main demographic of GutFeeling would also largely be willing to use it.

Comfort by adherence survey

Figure C: Comfort with genetically engineered bacteriotherapy based on issues with daily medication.

To determine potential points of improvements for GutFeeling, we asked what people's main concerns were (Fig. D) and what measures would increase their trust in the treatment (Fig. E). The most important concern was uncertainty about long term effects. People also largely cared about agency, safety, and reversibility. Less people indicated concern about safety for the environment, people around them, or ethical concerns.

The most prominent need was long-term safety studies. After that, people indicate the need for explanations, reversibility, and for the treatment to be backed by trusted authorities.

Main concerns survey

Figure D: Respondents' main concerns regarding genetically engineered living bacteriotherapy.

Industry

Overview

The market of PD treatments is growing rapidly and dominated by large pharmaceutical companies. These would become direct competitors to the bacteriotherapy we are helping to develop. This means that it is quite unlikely to successfully realize bacteriotherapy for PD, unless one of those large companies would actively support its emergence. We do not think these companies are likely to do that, unless they buy rights to the technology, treatment or other intellectual property to guarantee a satisfactory rate of return on their investment. It might be hard to convince them, since they already have large market shares, large prior investments, and the bacteriotherapy has obstacles to overcome in terms of safety and reproducibility of the quality of the treatment.

Global PD Drug Market

Fortune Business Insights estimates that the current global PD drug market is around 7.17 billion USD, and will grow to around 13.46 billion USD by 2032. The main methods of treatment include L-dopa together with decarboxylase inhibitors, dopamine agonists, monoamide oxidase B inhibitors, and catechol-O-methyltransferase inhibitors.

The PD drug market is dominated by large pharmaceutical companies including Roche, Pfizer, Novartis, Teva Pharmaceuticals and Merck. However, there is still a lot of development being done in the PD treatment industry, with wearable diagnostic devices, personalized medicine and disease-modifying therapies being some key innovations. An anonymous ethicist in the biotech industry told us that in many cases development of devices and treatments is done by smaller companies or start-ups, which get bought out by the larger companies if their products are effective. This is usually due to the large cost associated with developing drugs, which can be upwards of billions of dollars . For pharmaceutical companies that already have steady income streams it is therefore usually more worthwhile to do limited research and development (R&D) on their own, but buy the rights to potential promising drugs.

Current Methods of L-DOPA Production are Unsustainable

Because of the identified social value of sustainability we wondered if L-DOPA is produced sustainably. Most of the L-DOPA is produced through chemical synthesis, as yield from natural sources is quite low . Goura et al. describe multiple synthesis routes, of which all but one are chemical in nature. However, all of the employed chemical synthesis routes pose safety risks or environmental risks due to the catalysts or by-products that are used or created . Use of biological agents like Erwinia herbicola has commercial issues, as the storage and maintenance of the bacteria increases production cost, making it less viable. This means that currently employed L-DOPA synthesis is unfortunately still unsustainable and environmentally damaging.

PD Medication That is Not Taken Orally

Most of PD medication is taken orally, but there are also injections and transdermal patches. In particular Neupro is a transdermal patch that releases rotigone, a dopamine agonist. The patch needs to be removed after 24h, and a new patch needs to be applied to another hairfree patch of skin. As reusing the same spot of skin can lead to irritation and skin reactions, a patch of skin can not be used for two weeks for another patch. Overall this medication strategy is still an improvement on many fronts, providing a more stable dose and requiring less effort from patients.

Another method to combat fluctuations in L-DOPA levels is the application of a pump that injects an intestinal hydrogel that contains L-DOPA and carbidopa. This is brought to the market by Abbvie under the name Duodopa. Because of the consistent dosage it can help patients in later stages of PD, when the fluctuations in blood level L-DOPA have a more drastic effect on motor and non-motor symptoms, like fatigue, anxiety etc . A 12 month study showed improved effects of Duodopa treatment compared to standard oral L-DOPA . The treatment comes with a significant drawback however, as it requires permanent wiring to be attached within the body to the intestines.

For bacteriotherapy treatment of PD, these and other modes of medication will be direct competitors. The financial leverage the companies have, and the foothold in the market that these products have, will provide real barriers for market penetration. We think that it is therefore important that preclinical data would be convincing enough to get a major pharmaceutical company involved early on in the development. Especially if the product would get support from one of the major players in PD treatment it would be beneficial, as they can use their current network and brand to boost the use of the bacteriotherapy.

Concerns from Industry About Quality Control

In the interview with Evonik we learned that a prior concern of those companies might be the reproducibility of the quality and purity of the medication. Quality controls are extremely strict within the pharmaceutical industry, due to past catastrophes like the thalidomide scandal. Reaching those high levels of reproducibility might be hard for biological agents and populations, especially if we include variation based on diet, bowel irritation, diarrhea and the use of multiple medications in the case of multimorbidity. These issues in reproducibility might also make it harder to prove its safety for regulatory approval for the treatments, which is already hard to attain since there are no clear guidelines for the approval of live bacteriotherapy at the moment.

Concerns regarding genomic stability

Both Evonik, and an anonymous ethicist voiced concerns over the genomic stability of our bacteria. If we introduce genes to the bacteria that increase the metabolic cost, then it would over time be outcompeted by others. In time this would lead to the loss of our population due to evolutionary pressure. Our modeling has investigated this issue and found that the production of L-DOPA has a limited effect on the growth rate of Pseudomonas putida, when approached with flux based analysis (see Modeling - Flux Based Analysis). It seems that for L-DOPA production there is a need for pyruvate, which in optimal growing conditions is used up for growth. But in our model it does indicate that pyruvate is quickly siphoned away from L-DOPA production, if the bacteria were to prioritize growth.The evolutionary cost of a kill switch is deemed even greater (Stirling et al., 2017). However, in our plate reader experiment and subsequent analysis of the growth rate, we do not see a reduction in growth rate when the promoter of the toxin is not induced (see Results - research line 1).

Our Contribution to the Industry Dimension

After gaining input from science philosopher Nynke Boiten and our survey respondents over the importance of patient autonomy and reversibility of treatment, we tried to investigate a kill switch and a population control system to create build-in safety systems. Additionally, we investigated the potential growth effects our constructs impart on the bacteria to estimate the degree of genomic instability and fitness cost such bacteria would have. For further details on this see (Wet Lab - kill switch). At the moment there are no clear guidelines for regulatory approval of bacteriotherapy with GMO’s, so we researched the guidelines for the use of GMOs and the use of bacteriotherapy and combined them into an updated set of guidelines for this combined usage. This is detailed further below in the “Policy and Regulations” dimension.

Why: We approached Evonik to gain insight into the company's experience with bringing novel products to the market.

What: We talked about the strategic approach to developing unconventional innovations, and got advice on navigating the social and regulational dimensions.

Takeaway: We were advised to look at laws and regulations early on, and gave some specific points of feedback to address in our project.

Integration: We closely followed regulations, for which more information can be found under the "policy dimension" section. We addressed their feedback with dry lab work.

To understand the steps needed to bring our project from the lab to the market, we spoke to Sabrina Metze and Johannes Scheiger from the specialty chemical company Evonik. Sabrina Metze is a biotechnologist with expertise in analytical techniques, and Johannes Scheiger is part of Evonik's strategic innovation research and development department, which focuses on new and unconventional developments. Because of this, we were grateful for his perspective on the quite unconventional development of a live bacteriotherapy.

Johannes recommended looking into laws and regulations early on in the process, and to involve regulatory bodies in the process wherever possible. He explained that we were working with them, not against them, as we both want the same thing: to bring a safe product to the market. Because of this, we set out to follow industry guidelines as much as possible in the design of our product to ensure implementation would actually be possible.

Regarding societal pushback, Sabrina noted that there will always be an anti-movement when an innovative product wants to enter the market. Pharmaceutical companies therefore invest in the necessary marketing to make the majority of the public respond positively. She emphasised the importance of taking the wants and needs of the end user into account when developing a potentially controversial therapy like this one. She argued that convincing that group of our product would ensure having the most important perspective of the general public on our side.

Regarding our project design, they asked us to consider how to prove the safety of our product, as this would be essential in the implementation of it. Not only that, but they asked us to look into previous cases of compromised safety in the biotech industry, and analyse how companies approached such cases.

Science

Overview

Increasing prevalence of chronic illnesses has boosted interest in medicine adherence. Additionally, while current biocontainment strategies and expertise with microbiomes in a healthcare perspective might form technical limitations we think that these areas will be intensely studied in the upcoming future and remaining issues will be resolved.

Increasing Prevalence of Chronic Illnesses

Within the scientific community there has been increasing attention for patients with chronic illnesses. The prevalence of chronic illnesses and multimorbidity has been rising globally, even though there are geographic differences . The rise in chronic illness and multimorbidity seems to be due to the aging of the population and prevention of death through improved healthcare . Both are more common in people above 60 .

Increasing Awareness for Medicine Adherence

A well documented issue that occurs often for chronic illnesses is a lack of medicine adherence by patients. In our survey, we found that less than half of the participants in our survey found it easy to stick to a daily medication schedule (Fig. 4, Market User Dimension). This is in line with literature that indicates that suboptimal adherence to PD medication happens 10-67% of times

One important but sad finding is that Radojević indicated that patients suffering from clinically diagnosed depression are three times higher risk of suboptimal medicine adherence. This highlights that patient-centered care is really important in the case of PD.

There is still much ongoing research into transdermal patches, due to the promising results of Neupro transdermal patches (read System: Industry, PD medication not taken orally for more details). Recently transdermal patches even made national headlines, with a research team in Australia making a transdermal patch that needs replacement only once per week. Through optimizations in the gel composition of the patch they achieved a week-long steady release of levodopa and carbidopa .

We therefore think there will be significant interest within the scientific community for exploration of other options to tackle chronic medication. But this realization made us wonder: is science advanced or interested enough in the relevant fields to help develop this in the upcoming years or decade?

Safety and Biocontainment

Within the field of synthetic biology there has been a lot of attention in recent years for biocontainment strategies and genomic stability of engineered bacteria . This is expected to only increase with what will likely be increased commercial use of synthetic biology. Therefore it is quite likely that this will drive innovation and improvements in those areas, which could be utilized by the party that tries to introduce engineered bacteriotherapy in the future.

Microbiome Research

Another area that is receiving a lot of attention recently is microbiomics, which studies the interactions and effects of different microbial species living in our gut. Insights from this field will also prove valuable to the engineering of a stable population of gut microbiota that might facilitate PD treatment. For example, scientists have shown that specific bacterial strains produce enzymes that break down L-dopa in the gut and have successfully developed inhibitors to this process that increased L-dopa bioavailability . In addition, there has been an increasing call to have standardized and validated microbiota populations that can be used for scientific exploration. In mice models there is a validated population of bacteria to study the microbiome which is called the oligo-mouse-microbiota, or OMM12, and last year scientists have requested a similar validated set should be developed for zebrafish . These developments we predict will positively impact the development of bacteriotherapy.

In stakeholder conversations the role of the microbiota has also been brought up multiple times. Merle, a representative of a ParkinsonNL confirmed that recent research into PD has been shifting towards the microbiome. While excited by our approach of introducing new microbes to the gut, she also warned that disruptions in the microbiota can have dramatic consequences. For that reason current research is more focused on altering the microbiome towards a more therapeutically beneficial one, then introducing a new microbiome. To prevent disruption of the microbiome by overgrowth we therefore decided to develop a population control system for our bacteria.

Fecal Microbiome Transplant

Through stakeholder interactions we found out that it would be beneficial if our bacteria get delivered through a fecal microbiota transplant (FMT; read Policy & Regulation). This means that advanced knowledge on this topic is needed. Luckily, increasing amounts of research on fecal transplants are produced. According to Scopus over 250 articles are published yearly containing the words "fecal" and "transplant" in their title, abstract or keywords (Fig 7). The field is currently advanced so far that the American Gastroenterological Agency recommends FMT in the majority of recurrent C. difficile cases. This provides some assurance that by the time the bacteriotherapy would be developed sufficient knowledge, experience and expertise with FMT is available.

FMT publications over time

Figure 7: Publications containing "fecal" and "transplant" over time

Why: We wanted to gain insight into the current state of PD research, and determine if experts also saw the potential of GutFeeling

What: We talked about current PD research trends and microbiome involvement

Takeaway: Current research is focusing on the gut microbiome; we need to ensure our bacteria doesn't disturb the existing microbiome

Integration: We developed a quorum sensing population control system

We approached ParkinsonNL to get an understanding of current developments in the field of Parkinson's research. We spoke to Merle van Gelder, a research officer who makes decisions about where to spend the funds their organisation has raised.

Merle was pleasantly surprised we were working on a treatment in the gut, as that is actually something current research has started focussing on. Despite affecting the brain, it is theorised that Parkinson's could originate in the gut, potentially due to a disturbance of the gut microbiome. Now, our project doesn't focus on remediating Parkinson's through altering the microbiome, but it is possible that introducing a stable population of beneficial gut bacteria could have more effects than originally anticipated. At the same time, we have to be careful that our bacterium doesn't disturb the existing microbiome further. The exact effects of our bacterium on a human gut microbiome remain to be investigated, as this falls outside of the scope of our project. Still, we were able to conclude the importance of a stable and controlled bacterial population, that would not disturb the larger gut microbiome. Because of this, we started developing a quorum sensing population control system that kept the number of bacteria present in the gut at a stable level.

Regarding our project proposal, Merle was optimistic that it would relieve some burden of the patients. She explained how there are always difficult times in the day of a Parkinson's patient, known as "off periods". These are the moments where there is no active dose of L-DOPA in the system of a patient, such as early in the morning before the first dose starts to work, late at night after the last dose has lost its effect, and at any point between doses. Off periods can also occur when the patient forgets a dose, or the clearance of a dose from the gut is altered by intestinal issues such as constipation or diarrhea. Merle described how advanced cases of Parkinson's can be treated with deep brain stimulation (DBS), but this usually only occurs as a last resort because of the invasive nature of the procedure. Still, many patients who get DBS describe a feeling of relief, as the effects are stable throughout the day and have no off period. DBS also negates the need for a patient to constantly time their medication use, and eliminates patient error in doing so. Merle expressed excitement for our project, because it would give Parkinson's patients relief from these off periods without the invasive nature of DBS.

Before we ended the conversation, she did pose a question for us. She wondered if the constant expression of L-DOPA in the gut would over time decrease the effectiveness of the drug. We did not have an answer for this, but it is a very valid concern. We investigated the issue and found that there are indeed reports of long term L-dopa administration resulting in motor complications known as L-dopa induced dyskinesia (LID). Recent findings show that 53%–59% of patients treated with L-dopa had LID after 10 years of L-DOPA treatment. Because of this, it is important to study the long-term effects of our constant medication approach. In cases where LID is observed after long-term use of our bacterium, it would be beneficial to cease treatment and provide DBS instead.

Why: We wanted to gain input from the Animal Welfare body on our use of zebrafish larvae, and how best to navigate this using the 3R approach.

What: We ended up talking about some choices we made during the design phase of our project that ended up placing a greater distance between our animal-based model and the human application.

Takeaway: We were made aware that our design choices were suboptimal, and a different approach would have been better.

Integration: We were not able to change the bacteria used in our project, but took the input from the Animal Welfare Body into account in our Human Practices.

Utrecht University's own Animal Welfare Body and their internal 3R centre help researchers at Utrecht University and the Utrecht Medical Centre to navigate the use of animals and model systems in their research.

Coming into the meeting with the 3Rs we were expecting to talk about animal usage in the project, since 3Rs office in Utrecht University is responsible for approving animal research focusing on three principles to minimise animal use; Reduction, Refinement, and Replacement. However, the conversation turned to a completely different direction.

The 3R center was content with us using zebrafish larvae (5>dpf), their criticisms were mainly related to the selected strain. They had issues with both e.coli and p.alcaligens, because the use of them adds translational steps to the research. In their mind, for the approval panel it doesn't make sense to use bacteria that is commensal to the fish first and then transition to the human situation. They highlighted that this would increase the number of testing and therefore animals in the future. Instead, we should have started from the application, and picked a bacterial strain that made the most sense there, such as lactobacillus. Only then should we have looked for model systems that fit this approach. As the result of this meeting, we have decided to try and include lactobacillus in our project. However, there was no bacteria readily available on campus thus, we were unable to follow through.

Overall the conversation was focused on making our project as translatable as possible to the human situation, by not only keeping in mind the selected chassis for the treatment but also the future formulations and administrations of the treatment. For example, the regulatory bodies are much more keen on approving treatments involving commensal bacteria as a fecal transplant as opposed to an oral administration when they are presented as therapeutic not supplemental. With this in mind, they also raised the questions like: would the entire population of the commensal bacteria be replaced by the genetically modified ones? How will we determine the stability of the gut microbiome? Who would be eligible for such treatment? among others. We took these points seriously, and tried to address them as much as possible in our Human Practices section.

Culture

Overview
Slowly Increasing Acceptance of GMO Use in Europe

In a review Woźniak et al. claimed that the cultural acceptance of GMO has been slowly increasing in Europe. However, their research does show there are large differences for using different applications. Using GMOs for treatment or prevention of a disease was much more accepted than for example for use in food. This is overall positive for us, as acceptance of GMOs in healthcare was overall very high.

Geographic Location Matters

iGEM values projects that create products that people from different locations and economic status would have equal access to. We noted that the data presented in Woźniak et al. showed geographic differences in GMO acceptance, even between countries that are all within the EU. This might mean that certain cultures or geographies might be less open to bacteriotherapy. This could affect the users, for example by not including it in insurance or by people getting stigmatized. We therefore tried to stimulate acceptance to synthetic biology not just in the Netherlands but also in Portugal, and Romanian communities in The Netherlands, as can be seen in Education.

Survey Showed No Effect of Education on Openness to Bacteriotherapy

Initially we had expected that a higher level of education would lead to more openness to try our proposed treatment. However, based on our survey we have to conclude that that does not seem to be the case (Fig. 8). As can be seen, there is a high level of openness for all levels of education, and no significant variation between them.

Openness by education level

Figure 8: Openness to bacteriotherapy by education level

Increasing Awareness and Education of Synthetic Biology in Society

Culture is maintained and updated through dialogue. Increasingly synthetic biology is part of dialogue in society and incorporated in education. In the Netherlands institutes like The Rathenau Institute play an active role of promoting dialogue between the government, companies, academia and civilians on topics regarding biotechnology, including synthetic biology.

Synthetic biology doesn't reach people through dialogue alone. It is increasingly getting applied in healthcare, agriculture, industry and sustainable alternatives . This means that people from diverse backgrounds are getting in touch with synthetic biology, and this number will only increase. These developments will lead to higher synthetic-biology literacy, which we expect will increase acceptance of GMO based treatment.

Our contribution to the culture dimension

We wanted to add to this literacy by introducing more people to synthetic biology in our education efforts, which were constantly improved with the input from teachers (see Culture dimension stakeholders, Alexandra Vilarinho and Jose Rocha). In fact, Jose Rocha even called our classes a success with a perfect mix of information, activity and fun (see Culture dimension stakeholders; Education page). Next to this, we spoke to Stephan van Duin to learn how to approach people regarding an unfamiliar topic such as synthetic biology and live bacteriotherapies, and took his advice along in our outreach. We were able to establish connections with several artists who explored some core concepts of our project in their artwork, which will hopefully spark even more conversations about synthetic biology in their observers. We were also able to create an interactive educational piece with the input of Lili’s Protolab, which will be displayed at the Dutch Design Week on the 17th of October.

Why: We encountered Nynke at SynBioNL, where we got talking about the philosophical aspect of our project.

What: The conversation touched upon patient needs, and to what extent those were taken along in the project design.

Takeaway: Nynke advised us not to forget about patient autonomy.

Integration: We addressed this by looking into the use of killswitch, as well as seeking out the opinion of patients to ensure their needs were met. This led to the eventual creation of the survey described previously.

We first contacted Nynke Boiten at the SynBioNL event hosted in Utrecht on (Date) by the Centre for Living Technologies. She's a PhD student in philosophy at TU Delft, where she is looking into the conceptualization of life in the context of synthetic cells.

Our conversation was quite enlightening, because we had mostly approached our project from a scientific point of view until then. Nynke asked us to consider the impact a treatment such as this one would have on the patients using it, and what this treatment would mean for their bodily autonomy.

We had been describing the automated nature of our tool as a positive aspect only, however this conversation changed our mindset. Yes, we would be taking away the burden of remembering to take your medication and taking the right dosage, but we also took away the freedom of choice from these patients.

After this conversation, we decided to focus more on the potential reversibility of our treatment, making the kill switch an essential part of our design. Interestingly, this implementation of a kill switch turned out to be an important aspect to several other stakeholders.

Why: The opinion of the general public is an important player in our stakeholder network

What: We discussed our project, and how best to communicate a potentially controversial topic to the public.

Takeaway: This conversation gave us valuable insights in communication strategies.

Integration: We have integrated some of the advice from Stephan in our educational material. Besides tips on communicating with the general public, he also gave us some useful advice on presenting our project, which we have integrated in the wiki.

We contacted an expert in science communication, Stephan van Duin, who has previously contributed to the public perception on controversial topics such as stem cell therapies and vaccinations. We requested a conversation with him to discuss the potentially controversial nature of our project, and how best to communicate it and synthetic biology as a whole to a general audience that might be wary of such developments.

Stephan shared some insights into public perception, including that the distrust of people is rarely directed towards a product. Instead, people are often hesitant to trust the organisation behind a product or development, such as pharmaceutical companies or the government. So although correct and cautiously phrased information on a product can be useful, it does not always address the underlying distrust. Because of this, our motivation should always be clear and clearly communicated: our goal is to help patients, not to make profit.

Stephan recommended we started communications regarding our project with the need, the issue we were trying to solve. From there, we can explain why this is a difficult issue to solve, and how we hope to make it easier. Stephan was happy to hear we had conducted a patient survey and had found willingness of the target group to try our product. He argued that this could be a useful tool in communicating with regulatory bodies for example, as we would have evidence to support the need for our product.

Apart from comments on our project, Stephan van Duin also gave us some advice on presenting our iGEM journey and project on our wiki and at the Grand Jamboree. We are very grateful for these pieces of advice and will be taking them along when writing and presenting.

Why: We hoped to reach a broader audience and make synthetic biology more accessible through design.

What: We discussed possibilities for collaboration and exhibition during Dutch Design Week.

Takeaway: The focus should be on creating something tangible and interactive that represents the main idea of the project.

Integration: We created an interactive model for display at Dutch Design Week with help from Lili's Protolab.

We have come into direct contact with Wing Tai Cheng from BioArt Laboratories on July 1st. We have reached out them with a proposition of a collaboration because their goals and values clearly aligned with what we were trying to achieve as part of education, focusing on accessibility of knowledge to create a more symbiotic relationship between humanity, nature, culture, and technology.

During the first meeting, we were rather lost and discussed with him all the different possibilities of how this collaboration could manifest, settling on exhibiting our project during Dutch Design Week. Wing Tai Cheng, highlighted that this is a 'design week' therefore they are avoiding posters, it shouldn't feel like a conference. The main advice that we received was to make something that is tangible, something that can clearly represent the main idea of the project, what is the design?

This interaction really made us think about our audience and how we can make this an interactive experience. To help us, we have reached out to Lili's Protolab, a workshop that helps students and staff bring their ideas into a functional prototype.

Follow up

While the Dutch Design Week will unfortunately take place only on the 17th of october and we won't see all the products of our labour, we had some check in meeting with Wing Tai Cheng. His feedback was positive, he liked both the design and the interactive aspect of the model, confirming that he believes that the model is of a good enough quality to be displayed during Dutch Design Week.

Why: We needed help creating a physical prototype for Dutch Design Week.

What: We worked with Lili's Protolab to develop an interactive model.

Takeaway: The importance of making our model interactive with clear visual cues to engage the public.

Integration: We created an interactive model that visitors can operate themselves to better understand our project.

As mentioned previously, we went to Lili's protolab to build a prototype/model for the Dutch Design Week. We came into this conversation already having a rough sketch of what we would like the final product to be.

However, during the discussion with Lili's protolab, we had some difficulty explaining the project to people outside of synthetic biology, which really highlighted the need for a model that would make this project as accessible as possible. The main goal that they really highlighted for us is making our model as interactive as possible, with big, clear visual cues to really drive the point home.

Instead of us operating the model, it would be much better for the visitors to interact with it to make the main project ideas more intuitive, and to engage with the public as much as possible.

Why: We reached out to a group of artists as part of our education efforts, but realised that we got some valuable input for Human Practices from them as well.

What: We discussed social stigmas, and cultural views on living bacteriotherapies.

Takeaway: These conversations showed us some interesting perspectives on living bacteriotherapies that we had not considered previously.

Integration: We will be using the artworks from these artists to further encourage conversation about our central topics, as detailed in education.

As part of our education, we reached out to a group of artists to visualise some central concepts of our project. In our contact with these artists, we discussed the cultural context of these concepts, but also of our project as a whole.

During the brainstorming, we have discussed the stigma that exists when taking medications specifically for mental health or involving the brain in any way. There is this belief that these types of medication are unnatural, and that there is a possibility that the chemicals will alter your intelligence or personality. Therefore, it could be argued that we would be exacerbating this fear by introducing another unknown in the eyes of the people. On the other hand, the public might be more accepting, because our bacteria is technically natural. It already exists in the gut and we aren't giving it any novel function, rather enhancing functions that are already present. This way we aren't creating anything that can't already be found in the gut. Still, if the alteration of our personality due to an altered gut microbiome is a major concern of the public, it is something that can be investigated in clinical trials.

Another artist compared our live bacteriotherapy to the interesting example of crows and ravens bringing in rocks to water basins to raise water level, or monkeys eating charcoal to avoid food poisoning. They argued that it is only natural for us to use tools to improve our life. After all, if animals do it, why can't we? This comparison suggests that the general public might be open to bacteriotherapy, as long as we emphasise the function the tool is created for.

Why: Our education team reached out to these teachers for guidance on our activities.

What: We were seeking collaboration and advice on how to facilitate our education events for the kids.

Takeaway: We learned that structure is incredibly important and we need actual frameworks. Furthermore, active participation is more effective than passive learning.

Integration: We integrated this advice in our education design of "Build your own bacteria game", "The Plasmid Game", STREAM festival event.

We met with Alexandra Vilarinho and Jose Rocha before visiting a school in Portugal. The goal of the meeting was mostly to establish a timeline, feasibility and make sure that the content meets the learning goals that they have set for that month. What started as a meeting discussing logistics, turned into an unprompted brainstorming session that has triggered events that transformed our education, taking it to a new level.

While Alexandra Vilarinho and Jose Rocha are both biology high school teachers, their classes are vastly different. Alexandra Vilarinho teaches grades 10-12th, these students have previous biology experience, 11th and 12th graders have selected this subject as a part of their exams, indicating that they already have at least some interest and curiosity for biology. Rocha, however, teaches 9th grade. The students are just transitioning from middle school to high school, they haven't had the "true biology experience yet", it is also a mandatory course, meaning not everyone there enjoys the subject. Plasmids, promoters are still abstract to them. This was quite challenging for us to understand because in our eyes, how can this be so difficult? As we started to discuss, we talked about what is and what is not appropriate per grade level. How can we make the same activity adaptable? What principles should we use to guide our activities? We came to a conclusion that structure is very important. Some ideas we wanted to introduce seemed abstract, so we needed to give a simplified but solid base that we could build up. For example, we really needed the students to have the basics down for the plasmid game and clearly understand how we can be critical about certain bacteria design choices. So, we added examples, check-in questions, and gave context, since the plasmid game allowed them to connect their knowledge to the real world.

As we started making these connections, we got a glimpse into the frameworks that educators use to make sure that students retain as much information as possible, repeatedly highlighting the need for active creation and participation, as opposed to passive learning. This conversation has left us inspired to look deeper about different pillars of education; what are the current practices? What are the theoretical frameworks? This among other questions really pushed us to develop much more meaningful and structured educational materials.

Follow up: The lessons in the schools were an overwhelming success! We have achieved the goals that we outlined in our education section. We managed to make "The plasmid game" adaptable to all high school grade levels, and after the class majority of students were able to answer formative questions presented by their teachers.

While working on education, we dove a lot deeper into the pedagogy and education theories and pillars than we initially thought we would. We ended up building all of our activities involving kids and teens to match said pillars, making sure to analyse which ones we actually achieved.

Feedback: Jose Rocha told our education team that the class was a major success. A good mix between entertainment, content and activity. A real success by our education team, that highlights the importance of cooperation! Read the education page for more information.

Policy & Regulation

Overview

Overall, the Dutch government is trying to modernize healthcare, by increasing digitization, use of AI and reduction of on-site treatment and involvement of healthcare professionals. The last two of these factors could provide opportunities for governmental support for live bacteriotherapy. At the moment, however, we could not find evidence of clearly formulated strategy or governmental pressure to integrate bacteriotherapy, long-term alternative methods of drug administration or other innovations for chronic medication.

Cost and Modernization Trends in Dutch healthcare

Healthcare is one of the most important sectors in society and a recurrent topic of debate in politics. As we are located in the Netherlands, we will base a large part of our analysis on healthcare in the Netherlands. In the Netherlands the government spent 113.5 billion euros on healthcare in 2024 . In addition, the Dutch government is actively involved with changing and improving the way healthcare is done. This also includes larger transitions, like the use of generative artificial intelligence for the use of administrative tasks in healthcare (Regeerprogramma 6a Zorg, Thema 2). However, chronic medication is not currently one of the main themes for the government program. Still, theme 2 “Prevention of an uncontrollable labor market shortage” does state the intent to reduce the involvement of healthcare professionals in patient care. It includes digitization of healthcare and technological innovations that enable patient care with less involvement of healthcare professionals. We expect that live bacteria-based medication will in the long term be more effective and less time consuming to healthcare professionals than traditional medication. This means that the government could be open to supporting live bacteria-based medication.

Improvement in the Quality of Dutch healthcare Through Multi-year Agenda

The Dutch government also has a multi-year agenda to improve the quality of performed healthcare, called the "Meerjarenagenda" . The Dutch government does not currently include in the multi-year agenda alternative modes for chronic medication or improvement of PD treatments . The main themes that were on the multi-year agenda in previous years are aimed at mental health, dental health, cancers and COPD . The current items are also aimed at mental health, COPD and dental health and some others .

Efforts by Dutch Government to Reduce Price of New Medicine

One aspect of medication that the Dutch government has been getting more involved in is the pricing. In 2016 minister Schippers formulated a letter to the parliament with a vision on medication prices. In this she explains that the Dutch government had little control over drug prices because pharmaceutical companies get exclusivity rights for their product, so there are no alternatives. In addition, the Netherlands holds only around 2% of the global market share for such drugs, providing little leverage in price negotiations with international pharmaceutical giants. So a list of recommended actions were presented to the parliament to increase control.

In 2023 minister Kuijpers provided an analysis of the effectiveness of the vision, and concluded that the government's grip on drug prices had increased a little, but was short of being called "under control". Based on these reports the government is still trying to make medicine more affordable. These developments can be both a driver or a barrier to the adaptation of bacteriotherapy. The first bacteriotherapies would be high-risk investments for the pharmaceutical companies to develop, and they would therefore likely put an expensive price on the treatment. At the same time, there is also the case that a large one-time expense is better than a medium but repeated expense. That makes it hard to estimate how likely governmental support is for an innovative approach to drug administration.

Why: We hoped to get some insight in the IP landscape, and certain regulations that might apply to our work.

What: We talked about the potential opportunities for IP protection in our project.

Takeaway: The main question we were asked is "Is the problem big enough to justify this level of engineering complexity?"

Integration: Based on this question, we performed a QALY analysis to justify the need for our product.

We came in contact with Utrecht Holdings following a Dutch iGEM event organised by the Center of Living Technologies in Utrecht, where the need for intellectual property was highlighted. Utrecht Holdings is a Knowledge Transfer Office in Utrecht University that focuses on helping researchers bring their inventions to the market by managing intellectual property, providing consulting, among others.

We met with Bas Nagelkerken (manager IP at Utrecht Holdings) and Daniella Counotte (Business Developer at Utrecht Holdings) to better understand the IP landscape and regulatory implications of our work. Specifically, we discussed the novelty of our approach, the potential for patent protection, and the broader value of such a project.

Both Nagelkerken and Counotte were positive about the interview and have provided a lot of insights. First of all, it was important for us to realise that a project with similar level of engineering and complexity has multiple technical problems, which require technical and innovative solutions, that can also be patentable. Therefore, our project offers multiple opportunities for protection; the plasmid design, engineered strain, and possibly the delivery method itself, for example.

We also explored the implication of bringing such a system to clinical trials or market use. Nagelkerken and Counotte, discussed with us key regulatory bodies (EMA and FDA) and discussed how the classification of our system (therapeutic vs. food additive vs. living biopharmaceutical) would shape the regulatory process. As the result of this discussion we came to a similar conclusion as during our 3Rs discussion; we should use commensal or generally regarded as safe strains, and use fecal transplant delivery to make a regulatory journey simpler.

Counotte offered important insights into how to frame the problem for both the public and for regulatory or IP audiences;

"Is the problem big enough to justify this level of engineering complexity?"

We have decided to therefore conduct a potential Quality Adjusted Life Years (QALY) assessment for Parkinson's patients in the EU to determine at what point QALY gains are no longer worth the price as the result of the production complexity. However, since we are trying to use genetically modified bacteria as a drug delivery system we can also consider that it can be assessed in other diseases where patient compliance and steady drug delivery are unmet needs.

Why: We contacted COGEM to gain some insight into regulatory systems that might apply to a live bacteriotherapy.

What: Due to the specialisation of COGEM, we only talked about environmental safety, not patient safety. Still, there were some important things to keep in mind.

Takeaway: The two barrier approach identified previously was confirmed to likely be enough for FDA and EMA approval.

Integration: We were unable to meet the two barriers for safety in our project, but we see it as a decent standard to strive for when attempting to bring live bacteriotherapies to the market.

COGEM, or the Netherlands Commission on Genetic Modification is an independent scientific advisory body to the Dutch Government, offering input on the risk of genetically modified organisms on the environment, as well as considering social and ethical implications. We approached them for a conversation to discuss current regulations affecting our project, as well as their ethical perspective on our project.

We spoke with Jose Dekker, and Clara Posthuma, a scientific staff member and scientific secretary within the organisation. They explained how COGEM specialises in environmental safety, and could thus provide no input on the safety to the patient. Regarding safety to the environment, they asked if we had considered the effects of bacteria escaping the gut. For our project to get approved for use in current regulations, they explained, we would either have to prove or reason that our bacterium would not be able to leave the gut, or that it would not have any effect on the environment if it did get out.

We were able to use our previous research into this topic to argue that currently, the approval process for living biopharmaceuticals in both the Food and Drug Administration (US) and the European Medicines Agency (EMA) is on a case-by-case basis, therefore there are no concrete properties that such a product should have. In the context of the US regulatory bodies, the requirements for safety are a bit more clear than in the EU. The current stance is that the GMO needs to have two levels of independent barriers that can be in the form of two kill switches, or bacteria being anaerobic and an additional kill switch, that will prevent survival, spread and/or function anywhere outside the designated area.

Jose and Clara explained how market authorisation is provided by the EMA, according to their standards, even if COGEM would advise against it. Once a product is authorised for the market by the EMA, that authorisation is valid in the entirety of Europe. They advised that EMA approval is usually easier to achieve by getting approval from the FDA first. Because of this, we are confident that the 2 barrier approach as advised by the FDA is enough for market approval by EMA as well.

This conversation confirmed the need for a better and safer bacterium. Although we were not able to work with Lactobacillus, we highly recommend use of it for patient safety.

Why: We were interested in a conversation with a spokesperson from a certain organisation.

What: As the conversation went on, we got personal input from this person as well, which did not necessarily align with the views of this company. In the end, what we ended up using for our integrated Human Practices had less to do with the company, and more with the advice from this person. Because of this, the company will not be named, and the person will remain anonymous to avoid association with this company.

Takeaway: We got some useful insights on the policy dimension that were mentioned previously, as well as confirmation that our project needed to be justified from the needs of the patient.

Integration: We wish we could have followed the advice to organise a focus group, but were unfortunately not able to do so given the time of the project. We did integrate the input from this stakeholder in our description of the policy dimension.

We got into contact with an ethicist and evolutionary biologist in the biotech industry, who would prefer to remain anonymous. We had a conversation about changes in society and policies, and what would need to happen for our product to be incorporated. They touched on the difference in pace of developments and change in policy. In general, developments happen faster than policy changes. However, they assured us that this does not mean there can be no development on the academic side. On the contrary, developments in academia can accommodate for policy change, as research generates knowledge on safety needed to properly set up laws and regulations.

Our contact did mention that disruptive developments such as our own could only really stand a chance if we could justify the need for it. We discussed our survey and the input we got from people with Parkinson's and other long-term or chronic illnesses. To this, the person confirmed we had a solid reason for our project. They did also suggest the use of focus groups, but we were unfortunately not able to set one up within the remaining time of our project. Still, we appreciated the advice, and will be passing it on to Utrecht's next iGEM team as something to pursue earlier on in their project.

Innovators


Overview

Improvements are still being made in long-lasting PD treatment and on L-DOPA manufacturing. A lot of innovation is happening within the field of synthetic biology, with improved biocontainment strategies and epigenetic regulation being of high importance to the potential implementation of bacteriotherapy.

Our project hopes to contribute by providing a proof of principle that drugs can be produced by bacteria in the gut of living beings. We also provided an extensive model that can study the metabolic effects of L-DOPA production and other metabolites in Pseudomonas species, and even extended it with genetics-metabolics integration through gene product reactions (GPRs). Additionally, we thought about different biocontainment strategies and tested them. We studied the social acceptance of GMO bacteriotherapy, and increased the general knowledge on it and synthetic biology as a broader field. In addition, we studied the forces that could drive or prevent live bacteriotherapy implementation in healthcare from an Innovation Science perspective.

Innovation Outside of Our Project: Long Term PD Treatment Delivery

As mentioned before, a lot of attention is still being put in solving medical adherence and blood level fluctuations for treatments of PD (read System: Industry, PD medication not taken orally). Currently the two designs that are getting primary attention are trans-dermal patches and slow-release hydrogels. Recent studies have mainly tried to further improve the transdermal patches.

Innovation Outside of Our Project: L-DOPA Manufacturing

Due to the toxic by-products and negative environmental impact of the chemical synthesis routes of L-DOPA , research is still being done to improve its synthesis (read System: Industry, Current methods of L-DOPA production are unsustainable). Most of the research however, continues to be focused on chemical synthesis, instead of using bioactive agents.

How Our Project Helps to Innovate

In our project we identified a major hurdle: getting approval for the first bacteriotherapy in patients. We studied values, trends and barriers that impact its chance of success with our HP based on a theoretical framework from Innovation Science. As we think HP is crucial to translating iGEM projects, we published an article in Vector journal about it and have added an explanation of our approach as a contribution.

From our survey and stakeholder interactions we found out that the biggest contributor to acceptance by patients would be a clear explanation of how our treatment works and its long term effects. So our education team set out to learn from others what they need to understand our synthetic biology approach, while teaching them the basics in return. In addition, we identified a large gap in regulatory guidelines for assessing the safety of a bacteriotherapy, so we provided one of our own by combining existing guidelines on GMOs and fecal transplants (see contributions).

Many stakeholders provided input on safety and biocontainment strategies. We tried to develop both a kill switch that would reverse treatment, as well as a population control system that would prevent overgrowth and infection. We failed testing the latter in time due to an incorrectly synthesized DNA sequence, but we tested the kill switch. Our results showed that the kill switch is not as universal as we originally thought, and requires a narrow metabolite composition. With this we showed that an arabinose kill switch would not be practical for the implementation in live treatment.

Within iGEM we are relatively unique in that we use zebrafish as a model organism. This meant that we had to reinvent the wheel somewhat in how to use it, both technically and within the iGEM competition. The things we learned are compiled in a zebrafish guideline for iGEM teams and can be found in the contributions.

Another way we are unorthodox is by approaching L-DOPA production from a synthetic biology perspective. Usually small drug molecules are synthesized chemically, but we engineered a commensal bacteria to produce it and proved it could do it while being in a zebrafish gut. In addition, we supported this by building an extensive metabolic model for Pseudomonas species. Our model now also includes gene products and can therefore be used to study the effect of genetic modifications like knock-ins or knock-outs.

We also took concerns from the synthetic biology field about genetic instability due to metabolic cost of exogenous protein production into account. Modeling showed the metabolic cost and approximated its effect on the growth rate. In support we utilized and compared three common growth models to identify the one that best suits both E. coli and Pseudomonas growth data, that we obtained ourselves. After identifying Gompertz as the best of the models we also included the most up-to-date parameter estimation method we could find, which was released only last year. Therefore we have added our growth model as a contribution as well, which we think can be used by a wide audience that would like to validate the growth of their bacteria.

Why: We hoped to get the perspective of a grant reviewer and translational medicines expert on our project setup.

What: Edward Nieuwenhuis discussed our project proposal and design, giving valuable feedback on both.

Takeaway: Edward advised us that we need a back up bacteria strain, evaluate strains behaviour in a human situation, establish clear L-DOPA quantification protocols, and that dosing control needs to be established.

Integration: We have chosen e.coli as our backup strain, developed additional L-DOPA quantification methods based on HPLC.

Edward Niewenhuis is a renowned researcher, pediatric gastroenterologist, the Chief Medical Officer at Princess Maxima UMC, and many more.

We decided to reach out to Edward very early on in our project for multiple reasons. We knew that Edward is really passionate about new projects, ideas, and translational research and has a lot of experience with zebrafish, specifically zebra fish gut.

We wanted to get his opinion as a reviewer, wanting to hear his opinions about our proposal, the structure of the project, experimental set up, etc. Additionally, Nieuwenhuis has a lot of experience securing finances for research, since it was our very first year doing iGEM we wanted to get as much help as we could get.

We left our discussion with a lot of feedback, and questions from him that really made us see the project from a point of view of a reviewer and experienced research. The feedback and the questions were detailed and numerous, however, the most important ones were:

Will the P. alcaligens stay in the zebrafish gut? If they don't, how will we resolve it?

We have conducted test trials with non-modified P.alcaligens, based on the imaging we determined that the bacteria were indeed staying in the gut of the zebrafish larvae. In our proposal we have added a section where we state that if the P.alcaligens doesn't stay in the gut, we will use e.coli since we saw in literature that it doesn't readily get washed out.

How will the modified bacteria interact in the human gut? Will it be pathogenic?

While we weren't planning on putting P.alcaligens/e.coli in the human gut, since they are pathogenic bacteria, instead we would use a different commensal human bacteria, we still decided to look into it. We came into contact with another researcher on campus, Silvia Mihaila, to discuss this.

How will we measure the amount of L-DOPA produced?

Our initial idea about L-DOPA quantification was to look at the colour change caused by L-DOPA degradation that results in melanin. Since the fish larvae are clear, we could observe them turning more brown/less clear over time. While the method is visual, we were encouraged to have other quantification methods as well. Thus, we have to develop additional quantification methods for HPLC and LC-MS/MS.

Regarding securing funds, Nieuwenhuis recommended to be short, direct, and always ask for more than we need. Unfortunately since we are not an established iGEM team and we are a student group, approaching companies that academia tends to approach in this field would be difficult. We were encouraged to reach out to companies that were on campus, apply for student research grants (with instructions on how to write them), and use crowdfunding.

It was also worth noting that this was an incredibly positive interaction, we received a lot of positive feedback about our project design and approach, which was incredibly affirming that we were doing things correctly.

Why: We sought out the perspective of one of the leading in vitro modeling specialists, to discuss our approach.

What: Silvia Mihaila helped us establish the best model for us, as well as how and where we can run our experiment.

Takeaway: We concluded that the advanced transwell caco-2 cell model is the best suited for our goals. In vitro models can answer a lot of questions and can be an excellent translational step, but they are complex and require standardization.

Integration: We continued developing transwell models with the help of Francesco Suriano and Karin Strijbis, two contacts we received from this conversation.

Silvia Mihaila is an Assistant Professor of advanced in vitro models of disease in the department of pharmacology. We contacted her following the conversation with Edward Niewenhuis, with the hopes of getting some input about using an in vitro model.

After listening to our project proposal, Mihaila was rather optimistic and intrigued, even helping us in our education outreach. A lot of her questions were similar to those posed by Niewenhuis previously, however she questioned our initial approach of having the in vitro model being an optional addition and not a clear translational step.

By the end of the conversation, we have decided to go with a transwell gut model. It is a relatively new model that uses caco-2 cells that are able to produce mucus, mimicking the human intestinal lining. This model allows us to study how the bacteria resides in the mucus, how do our bacteria interact with the cells (are they being pathogenic and are destroying the mucus and cell layers? Or are they able to interact with the human cells without being destructive?). Caco-2 cells were particularly important because L-DOPA is very polar and requires active transport, which caco-2 cells are capable of.

We have discussed introducing P.alcaligens to a human in vitro model, weighing out reasons for and against it to determine if doing so is a logical step in our research. While P.alcaligense is an opportunistic pathogen, testing the engineered strain is still an appropriate strain in the development pipeline. We can obtain information valuable for research and dry lab like; evaluate bacterial behavior, compound transport, L-DOPA production, and assess bioavailability. Even if this strain isn't intended for human use, these experiments offer a baseline for comparing in the future, suitable chassis candidates and can support chassis design and biosafety assessments.

Additionally, we talked about what role advanced in vitro models play in research, what are some difficulties that we can encounter, and how we should select a model.

Complex in vitro models can give us a lot of information in a rather short amount of time and ultimately make a difference in how we can translate our research into human situations. However, we have also found out how difficult using in vitro models could be. Currently, there is no concrete standardization of in vitro models. This can pose a challenge because it can result in varying findings across the labs and/or companies.

Recognizing these challenges, we've been eager to learn from others in the field. Mihaila, kindly gave us a contact who could offer help running our experiment in an advanced transwell model.

Why: We wanted to confirm the ability of our produced L-DOPA to pass the gut barrier with the use of a transwell model, and needed external guidance for these experiments.

What: Both Francesco and Karin were valuable contacts in designing, executing, and troubleshooting transwell experiments.

Takeaway: Francesco and Karin suggested a toxicity experiment, and helped adjust the experiments to cause less toxicity to the cells.

Integration: With their help, we were able to conduct transwell experiments described in the wet lab section.

Karin Strijbis is an associate professor and Francesco Suriano a postdoc researcher, both work in the Department of Infectious Diseases and Immunology. We have come in contact with them through Silvia Mihaila, after the conversation described previously.

In the first meeting, we found out more about the transwell model, how it works, what is the general process of establishing such a model, and what kind of work they run in the lab. We discussed the time line of the experiments, and we decided to run a series of tests that match the timeline of Francesco Suriano, as he would be supervising us.

First of all, we decided to first determine whether or not the working mix that we are using to produce L-DOPA is toxic for the cells, especially since we are planning to add ascorbic acid to prevent L-DOPA oxidation, looking at LDH levels produced by the cells and use TEER to test barrier integrity. For this experiment we are going to be using simpler transwell wells that don't have mucus in them.

We want to test both our genetically modified E.coli and P.alcaligense, with Lactobacillus as a positive control and their own E.coli as negative control. One of the key difficulties that we have identified is that E.coli is anaerobic and P.alcaligense is aerobic, while we can create an anaerobic environment in Suriano's lab, it is damaging to the cells. Therefore we will have a limited time to conduct our experiments. We will be evaluating the barrier integrity (TEER), cell viability (LDH), and examine the cell-bacteria interaction under the microscope.

Follow up 1 - Toxicity Experiment

After the first experiment we have discovered that our working solution is toxic, in hindsight that is not a surprise because it was hyperosmatic and our incubation time was 4 hours. During the post-experiment discussion, we have made the decision to conduct a toxicity trial using different concentrations of our mix, introducing caco-2 cells. We will be reducing incubation time to 1 hour, since as Francesco Suriano informed us it is a more optimal incubation time for when we are going to infect the transwell model. Additionally, we will try to introduce our genetically modified bacteria to the medium normally used in that lab to determine whether or not it can produce L-DOPA without the mix.

Importantly, we came to the conclusion that if we don't end up finding a way to produce quantifiable amounts of L-DOPA in the transwell model, we will still go ahead with the experiment. We can still evaluate the interactions that our bacteria has with the caco-2 cells, both E.coli and P.alcaligens. Since it is interesting for understanding how different chassis strains interact with the intestinal lining in terms adhesion, barrier integrity, and overall suitability for gut based therapeutic delivery.

Why: We sought expert input on molecular cloning because Wilco's experience in synthetic biology provided a valuable perspective on troubleshooting and improving our wet lab workflow.

What: He supported us during cloning challenges, from transformation troubleshooting to plasmid design, Golden Gate optimization, and screening strategies.

Takeaway: Through Wilco's mentorship, we learned how to approach cloning failures systematically, optimize assembly conditions, and adopt efficient screening methods that saved significant time and resources.

Integration: His guidance directly shaped our wet lab strategy, from moving away from unreliable kit plasmids to ordering custom gene blocks, optimizing Golden Gate Assemblies, and efficiently validating clones.

At the start of our project, we planned to use the iGEM distribution kit for plasmid construction, but our first attempts at transformation in E. coli DH5α yielded no colonies. To troubleshoot, we turned to Wilco Nijenhuis, a senior researcher specialized in molecular cloning and synthetic biology. With his expertise, he helped us systematically test our protocols, including positive controls to assess the quality of competent cells. Together, we concluded that the iGEM kit likely contained too little DNA for reliable transformation.

This led us to shift towards designing our own plasmids and ordering custom gene blocks from IDT/Twist. Additionally, Wilco advised us during Golden Gate Assemblies, recommending longer incubation times and overnight assemblies for higher efficiency. He also guided us in designing proper primers and in the PCR setup for generating new overhangs. When faced with screening large numbers of colonies, he suggested colony PCR as a rapid alternative to sequencing everything. To further reduce cost and effort, he recommended using restriction digestion as a pre-screen before sequencing, ensuring that only the most promising clones were sent for sequencing and validated.

Additionally, Wilco trained us in the use of Benchling to analyze sequencing results, align reads with our constructs, and interpret how specific errors might affect functionality. His input allowed us to validate our assemblies more efficiently, saving both time and resources.

Why: We worked with Jelle because of his expertise in zebrafish research and hands-on knowledge of working with live vertebrate models.

What: He trained us in zebrafish handling, experimental design, bacterial inoculation, and imaging, while ensuring adherence to biosafety and ethical standards.

Takeaway: Through his mentorship, we gained the practical and conceptual knowledge needed to reliably test our constructs in zebrafish and generate in vivo data.

Integration: His support enabled us to establish a robust and reproducible zebrafish workflow that directly validated our bacterial designs in a living host. We gained valuable technical skills and improved our ability to connect wet lab results with in vivo outcomes.

Our work with zebrafish required expertise far beyond our team's initial experience, and for this we worked closely with Jelle, a postdoctoral researcher working at the zebrafish facility. He was essential in translating our bacterial constructs into meaningful in vivo experiments. From the beginning, Jelle trained us in handling zebrafish larvae and maintaining the embryos under the requirements of animal care protocols. He supervised our zebrafish trials, teaching us the five-day workflow: egg collection, antibiotic treatment, bleaching, dechorionation, bacterial inoculation, and preparation for microscopy. After training, we performed most steps independently, while seeking his advice for troubleshooting microscopy, software, and imaging issues.

Jelle's input extended beyond zebrafish handling. With his background in molecular cloning, he also advised us on plasmid design, primer selection, and experimental strategies. Together, we developed plate reader assays to validate our functional gene blocks, measuring OD and fluorescence to assess secretion, uptake, and cleavage. He further helped us analyze imaging data using Fiji/ImageJ and guided the visualization of fluorescence and colonization results.

Why: We sought input from Frederik, PI of the extracellular vesicle biology group working with zebrafish, to benefit from his expertise and his perspective as a cell biologist.

What: He advised us on experimental design, provided feedback on zebrafish-related work, and helped with practical and organizational aspects of iGEM, including forms, deadlines, funding, and reporting.

Takeaway: Frederik's involvement ensured our zebrafish experiments were scientifically rigorous while also helping us navigate the organizational and administrative challenges of iGEM.

Integration: His guidance influenced both the research direction and the effective management of our project.

As PI, Frederik combined scientific expertise with practical oversight. He played an important role in the early stages of our project by helping us brainstorm and refine research directions. Throughout the project, we could turn to him for discussions about experimental strategies and approaches. He provided feedback on our experimental results, helping us interpret data and plan next steps. Beyond the science, Frederik supported us in navigating the organizational side of iGEM: ensuring ethical and biosafety requirements were met, forms were completed properly, and deadlines were respected. He also advised us on funding opportunities and overall project management.

A Warm Shoutout to Our Stakeholders

How Our Stakeholders Helped Us Innovate Within Our Project

As any iGEM team, we faced many challenges during our project. With help of Wilco Nijenhuis we solved many problems regarding cloning, ranging from struggles with bacterial transformation to incorrectly synthesized DNA sequences. None of us had experience with zebrafish, so the lab supervision of Jelle van den Bor and help with safety forms from him and Frederik Verweij were extremely appreciated. In addition, we received a lot of help from Silvia Mihaila, Francesco Suriano and Karin Strijbis for the use of transwell systems. Terry Vrijenhoek helped us with understanding the value of HP and a literature based approach. Ewart Kuijk always provided insights on how to improve our experimental approach and story cohesion. Josephina Groenenboom provided valuable insights as a previous iGEM member and judge on how to successfully complete an iGEM project. As the first iGEM Utrecht team in 8 years we initially struggled with acquiring sponsors, but Edward Nieuwenhuis gave us many tips that turned our fortunes. Ben Bouchet also helped us with acquiring critical sponsors like Qiagen and ThermoFisher. Lili’s Protolab and the Dutch Design Week offered us a wealth of support for the development of Scorch and other educational tools. A wide variety of stakeholders provided us with input on their concerns, ranging from investability, feasibility to personal worries.

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