Team:Leiden/Human Practices

S.P.L.A.S.H.

Suckerin Polymer Layer to Achieve Sustainable Health

Scroll over people's names to find out more about them!

Egbert Krug is a trauma surgeon at the Leiden University Medical Center (LUMC).

Titus Radstake is the chair of the Dutch Burn Survivor Association (Vereniging van Mensen met Brandwonden, VMB). Being a burn wound victim himself, he could highlight problems from a professional and personal perspective.

Frank Beneker is the head of the skin and bone divisions of ETB-BISLIFE in Beverwijk, the Netherlands.

The Dutch Skin Bank processes and stores human allogeneic donor skin for the treatment of patients with chronic wounds and burns.

The Miserez research group, from Nanyang Technological University (NTU) in Singapore, is led by Dr. A. Miserez. They focus on revealing the molecular, physico-chemical, and structural principles from unique biological materials and on translating these designs into novel biomimetic synthesis strategies.

Prof. dr. M. H. Vermeer is Head of the Dermatology Department at the Leiden University Medical Center (LUMC).

Prof. dr. E. Middelkoop is specialized in Skin Regeneration & Wound Healing at Amsterdam UMC and is the Director of the Association of Dutch Burn Centers.

Hans Peter Mulder is co-founder of the biomedical company Idris Oncology B.V., where they develop medical devices that improve cancer diagnostics.

Prof. dr. M. Sieber is Professor of Biology and Clinical Research at the Hochschule Bonn-Rhein-Sieg.

Dr. R. van Doorn is a dermatologist at the Leiden University Medical Center (LUMC).

Dr. C. P. Tensen is Head of the research laboratory department of Dermatology at the Leiden University Medical Center (LUMC).

iGEM teams from Eindhoven (2018) and Darmstadt (2017)

Dr. P. H. Nibbering is Associate Professor in and researcher of new antimicrobial agents/strategies at the Leiden University Medical Center (LUMC).

Dr. G. P. H. van Heusden is Assistant Professor of gene expression and protein translocation in yeast at Leiden University.

Dr. B. S. de Pater is Assistant Professor of Genome Engineering at Leiden University.

Dr. E. Vijgenboom is an Assistant Professor focused on using Streptomyces lividans as a cell factory for enzyme production at Leiden University.

Linda Dijkshoorn is CEO and founder of EV Biotech.

Prof. dr. P. J. Punt is Professor in Industrial Biotechnology and Chief Scientific Officer at Dutch DNA.

Dr. ir. E. J. van der Zaal is Assistant Professor of Molecular Genetics at Leiden University.

Dr. H. C. van Leeuwen is a senior scientist at the Netherlands organization for applied scientific research (TNO) in Rijswijk, the Netherlands.

Prof. dr. J. T. Pronk is a Professor of Industrial Microbiology at the Technical University of Delft.

EV Biotech is a company with the goal to stir the chemical industry to microbial production using a combination of cutting-edge techniques in Microbiology, Genetic Engineering and Systems Biology.

National Insitute for Public Health and the Environment (RIVM) advises the Dutch government on public health and a healthy living environment.

Korienke Smit is a policy advisor at the RIVM (National Institute of Public Health and the Environment) and coordinator of the Safe-by-Design assignment.

Dr. E. G. M. Kleijn is Associate Professor at the Institute of Environmental Sciences (CML) at Leiden University and focuses on industrial ecology and circular economy.

Nienke de Graeff is a PhD candidate in the Ethics of Biomedical Innovation at the Department of Medical Humanities of the University Medical Center Utrecht (UMC Utrecht).

Eugen Kaprov is a business advisor who provides an early stage venture capital for founders using technology to solve big problems.

Vincent van der Wel is the director of Patient One and Business Development Manager, and project leader at Fair Medicine.

Prof. dr. A. Cohen is a Professor of clinical pharmacology and until 2018 CEO of the Centre for Human Drug Research (CHDR).

Wouter ten Voorde is a research assistant at the Centre for Human Drug Research (CHDR).

Rafaëlla Paulo Teixeira is the founder of Fish4Me, a company that develops an information platform to provides consumers with information about the origin and sustainability of fish.

Integrated Human Practices

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Science and society go hand in hand, and they influence each other in many different ways. For our project, we wanted to improve current burn wound treatments by using the promising biomaterial suckerin. Therefore, it was important to investigate the impact that our project would have on its direct environment and the world. To do this, we included the feedback of a variety of stakeholders, like patients, medical professionals, companies, researchers and many others. Our conversations with them shaped the design of our project, for example by implementing a linker system that can couple antimicrobial peptides, which is lacking in most current treatments. Throughout this page, there are several links to other pages where you can find more information.

On this page you can read about the process of our project and the integration of society into the science:

Click the icons to see our timelines!

Phase I:
Exploring the problem

Phase III:
Development of the product

Phase II:
Suckerin as a solution

Phase IV:
Impact

Abstract: setting up our initial project design

Burn injuries affect millions of people each year. We discussed the current treatment options with several experts and concluded that treatment consists of two phases: initial remediation and aftercare. One of the main initial treatment options is the application of allogeneic donor skin to close the open wounds. However, this treatment has a limited and unpredictable supply resulting in a high demand for alternative solutions. Therefore, we aimed to find an alternative to donor skin. Additionally, we thrived to improve existing methods. We recognized hydrogels as a promising substitute, that still leaves space for optimization, for instance by increasing production efficiency or mechanical properties.

Exploring the immense challenges of burn wounds

In order to contribute to society, several local and worldly problems with high priority were contemplated in the first stage of our project. Since Leiden is close to one of the national burn wound centers in the Netherlands (Dutch Burn Centre Beverwijk), this subject eventually attracted our attention and we decided to explore the problem of burn wounds in more depth. According to the World Health Organization (WHO), more than 300,000 people die of burn injuries, together with the hospitalization of 11 million a year [1]. Several studies over the last decade have shown that approximately 42%-65% of fatalities are caused by bacterial infections [2-5].

• Treating burn wounds is a difficult process that annually causes death and disability around the world. We wanted to investigate the various challenges in this problem to see at which stage we could help improve conditions for the patients.

The underlying complications: life-long effects on the well-being of patients

To understand the challenges of burn wounds further, we spoke to Egbert Krug, and realized that there are actually two main stages of burn wound treatment. Firstly, the initial remediation and care of open wounds bring difficulties due to excessive fluid loss and increased risk of infections. Secondly, the aftercare consists of long-term and recurring treatments, for example, surgical interventions to salvage and replace the damaged skin. We contacted the Dutch Burn Survivor Association for more information on burn wound aftercare, and talked to the chair of the organization: Titus Radstake, who is a burn wounds victim himself. He explained that patients are faced with a long process of restoring and repairing skin and recurring surgeries become a standard of living. In addition, the association brought us in contact with another burn wound victim who suffered from third-degree burn wounds, covering 55% of her body. She explained to us that she had had multiple skin transplantations, resulting in thick scars. Most importantly, she still needs weekly micro-needling therapy to treat the stiff scar tissue, ever since her accident, two years ago.

• Burn wound treatment is extremely complicated, thereby decreasing the quality of a patient’s life. The aftercare could be greatly improved by inventing a strategy to restore the skin barrier during the initial burn wound recovery.

Current primary treatments of burn wounds: donor skin

Once we had insight into the severity of the problem, we started investigating the current solutions and treatment options. Titus Radstake also mentioned that initial treatment involves two parts:

1. covering the wounds with allogeneic donor skin
2. removing skin autografts from unaffected body parts, to replace the damaged or missing skin

To investigate these parts, we approached Frank Beneker from the Dutch Skin Bank. From him, we learned that current treatment is dependent on the highly fluctuating supply of post mortem donor skin. In addition, the available donor skin has to meet various quality criteria before it can be deemed ‘safe’ for treatment purposes. This includes a medical examination, as well as blood- and bacterial tests. The processing results in a low yield of usable donor skin, creating a gap between demand and supply of donor skin.

• The main treatment of burn wounds, allogeneic donor skin, has limited and unpredictable availability. Therefore, there is a demand for alternative methods that could substitute donor skin as a therapy.

Current primary treatments of burn wounds: hydrogels

In addition to donor skin, trauma surgeon Egbert Krug informed us that burn centers are constantly looking for novel dressings. In particular, he mentioned hydrogels as a promising substitute. We learned about the two prominent hydrogels: Aquacel, which allows vertical transportation of fluid, and alginates, which are used to cover wounds. These are already on their way to replace donor skin as a treatment option. Although hydrogels can mimic biological tissues, they often lack the ability to self-heal. This limits their use in biomedical applications and researchers have been attempting to improve these mechanical properties [6].

• Hydrogels are already used in treatment to keep wounds moist and cover skin wounds. However, they lack mechanical properties that can be improved on, to make a hydrogel that actively stimulates wound healing.

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Abstract: project specifications and design

For the design of our project, we focused on suckerin as biomaterial for hydrogel formation. We evaluated the advantages of the modular molecular makeup of suckerins, as well as the disadvantages that this protein could pose in medical use. We further fine-tuned the project design to include the concerns and advice of stakeholders regarding the problems of immunogenicity, scarring, and bacterial infection. As a solution, our final product will contain a cleavable linker system that could be coupled to compounds such as wound healing agents, painkillers, or antimicrobials peptides.

Suckerin as a replacement for skin

We continued our search for an alternative and improved burn wound treatment through literature. In the last decade, (bio)materials are widely studied regarding tissue engineering. Silk is a promising candidate, due to its biocompatibility, controllable biodegradability and elastic strength [7]. In addition, its mechanical structure is suitable for different structures ranging from hard to soft tissue. Further investigation, led us to the work of the iGEM teams SDU-Denmark (2016) and UCLA (2015), on hydrogels from spider silk. However, spider silk is unfavorable for large-scale production and molecular engineering, due to the size and genetic composition of the protein. Knowing this, we discovered with similar advantages as spider silk, but without the restraints in production [8]. This protein, called suckerin, originates from the Humboldt squid Dosidicus gigas. Like silk, suckerin can resemble different tissues by being both strong and flexible [9,10]. In contrast to spider silk, suckerins have a smaller molecular weight and simpler genetic composition. To get more insights into the qualities of suckerin, we contacted the Miserez research group from Nanyang Technological University in Singapore. This group has been studying suckerin for several years regarding production in recombinant microorganisms [11]. Suckerins offer promising opportunities as a biomaterial for hydrogel formation, thereby serving as an alternative and improved treatment for burn wounds.

• Previous iGEM teams have investigated hydrogels composed of spider silk. This biomaterial is promising due to its mechanical properties, but can cause problems during molecular engineering. We found a similar protein called suckerin, that has similar properties and is more advantageous for production in recombinant microorganisms.

The advantages of suckerin

In the Humboldt squid, suckerin is found in its sucker ring teeth (SRT). These teeth are mainly used in predation, and thus need a range of mechanical properties. Suckerins provide these by consisting of an alternating amount of two main amino acid sequences. The first one determines the strength and rigidity of the protein, whereas the other specifies the flexibility. The ratio of these two modules determines the mechanical features of suckerin proteins. During our conversations with Egbert Krug and Maarten Vermeer, we learned that the modularity of suckerin would be a great advantage to our hydrogel. Using the suckerin protein allows us to tailor the final product and make a range of hydrogels with different flexibility and rigidity. These different properties make suckerin-based hydrogels more versatile in use, for example in different body parts. To integrate this knowledge into our project design, we aimed to produce different suckerins. We directed our focus to the production of suckerin-12 and -19, since these are best studied regarding genetic recombination and hydrogel formation [10,12]. In addition, we incorporated suckerin-8 and -9, which are very rigid or flexible, respectively [10,13].

• Suckerin consists of two alternating modules, which translate into various degrees of rigidity and flexibility. The advances of synthetic biology provide the possibility to switch around and experiment with these modules. We can further improve our product and make it more versatile by testing the production of different suckerins.

The main problem with suckerin: immune response

Esther Middelkoop, Hans Peter Mulder and Martin Sieber addressed the problem of a possible immune response against a non-human protein, so we started searching for a solution. After our conversations with these experts, we concluded that the immune response to suckerin would limit its use for deeper wounds. Together, they recommended us to conduct several in vitro and in vivo biological tests, to investigate the response of human cells to suckerin. One of the suggested in vitro experiments was the addition of a suckerin layer onto human skin cells. Interestingly, a previous study performed this assay regarding suckerin-19 and showed negligible cytotoxicity [14]. After further communication with the Miserez research group, we discovered that they had conducted biocompatibility assays showing that suckerin did not induce an immune response. Since biocompatibility assays are unavoidable during clinical trials, these data are promising for suckerin-based medical devices. Therefore, we incorporated this into the future prospects of our results.

• Several experts addressed to risk of an immune response against suckerin and advised us to incorporate several biocompatibility assays in our project design.
• Biocompatibility experiments performed by the Miserez group indicated the absence of an immune response against suckerin-19, which is promising for suckerin-based medical devices.

Addressing the challenges of scarring tissue

In our conversation with Titus Radstake and other burn wounds victims, we discussed the scarring of transplanted and damaged skin, which causes additional pain and discomfort in patients. They mentioned that this scar tissue is very stiff and therefore accompanied by many physical challenges and daily discomfort due to pain, itching and restricted movement. As a result, some patients experience insecurities that can cause difficulties in work and social related situations. Therefore, we decided to look into possibilities to incorporate this challenge into our project design. As suckerin is a protein, it would, in theory, be possible to create a fusion protein by adding the sequence of a compound of interest to the genetic sequence of suckerin [15]. With the help and suggestions of Egbert Krug, we designed a system where we could couple suckerin to compounds like growth factors. We hypothesized that such a system could stimulate the healing of natural skin by the inclusion of compounds involved in wound healing, such as growth factors.

• One disadvantage of current treatments is the development of scar tissue as an effect of skin replacement and damage.
• In order to combat the above-mentioned disadvantage, we want to incorporate compounds that stimulate the growth of healthy skin tissue instead of scarring tissue. This would increase the quality of life by decreasing the amount of aftercare.

Linker system with antimicrobial peptides

During the development of our linker system, to couple compounds to our hydrogel, another challenge was brought to our attention by Maarten Vermeer, Remco van Doorn and Kees Tensen, namely, avoiding an infection during treatment of the open wound. They suggested to look into the possibility of adding antibiotics to the hydrogel, to prevent infections. In addition, they suggested the inclusion of painkillers to alleviate pain and discomfort. Previous iGEM teams have investigated the possibility of adding antibiotics to hydrogel treatment of open (burn) wounds, so we looked into their achievements. We learned that there are many safety issues and concerns with coupling antibiotic-producing bacteria to a gel. Due to the constant exposure to the antibiotics, resistance can occur. Even though antibiotic-producing bacteria will not be incorporated into our final product, resistance could still occur when antibiotics are embedded in the gel. This concern was also mentioned by Peter Nibbering and he recommended adding antimicrobial peptides (AMPs) instead. In addition, we decided to include a controlled release mechanism, to minimize the chance of resistance.

• Infection of open burn wounds is a challenge during treatment.
• To improve the patient’s welfare, we focus on making a hydrogel that can be easily removed together with a linker system to which antimicrobial peptides can be coupled. Since current treatments are not effective in preventing infections, our product will be an improvement compared to these treatments.
• Considering resistance to these AMPs, we added a controlled release mechanism.

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Abstract: designing and optimizing methods of production

For the design of the experimental part of our project, we closely cooperated with experts in a variety of relevant fields, including biologists and bioinformatics. Incorporating their advice and feedback, we established efficient production and purification conditions and methods, as well as an optimized upscaling approach based on a statistical model.

Producing suckerin in microbes

Since suckerin proteins originate from the sucker ring teeth (SRT) of the Humboldt squid (Dosidicus gigas), one could argue to harvest the protein from these animals. However, this is accompanied by various disadvantages, since the Humboldt squid is a species that has developed a high level of intelligence and plays an important role in marine biodiversity [16-18]. Therefore, overfishing the Humboldt squid from its natural environment will result in a negative impact on the ecosystem, animal welfare, and marine biodiversity. Most importantly, SRT consist of multiple suckerins, so a fine-tuned harvest of one specific type of suckerin would be difficult. To avoid these negative impacts, we decided to shift from animal-based production to microbe-based production, a method that is of increasing interest [19]. This leaves the Humboldt squid and its habitat unaffected, Most importantly, it still allows for large-scale, efficient production of specific suckerin proteins.

• Using the natural suckerin protein negatively impacts animal welfare, marine biodiversity, and its medical applications. To minimize this impact, we decided to produce the protein in microorganisms.

Selecting a production organism

Next, we investigated which microorganism would be best suited for production. After conversations with a range of experts, including Paul van Heusden, Sylvia de Pater, Erik Vijgenboom, Linda Dijkshoorn and Peter Punt, we had a selection of recommended host organisms that would be suitable for suckerin synthesis. These included Saccharomyces cerevisiae, Escherichia coli, Streptomyces lividans, Bacillus subtilis, and Aspergillus niger. Moreover, we contacted the Miserez research group, who successfully managed to clone suckerin-19 into E. coli [11]. Regarding the input from the various experts, we decided to use the E. coli strain Rosetta as the main production organism. In our conversation with Sylvia de Pater, she confirmed that this was indeed a good strain for production of eukaryotic-derived proteins. Since E. coli is a gram-negative organism, we decided to include a gram-positive organism in parallel. After discussion with Erik Vijgenboom, S. lividans was chosen, since this strain can transport suckerin to the extracellular compartment, as the cell wall prevents the protein from getting stuck in the periplasm. Furthermore, we learned from our discussion with Peter Punt that post transcriptional modification of eukaryotic proteins could pose an issue during production in prokaryotic host organisms. Therefore we included S. cerevisiae as eukaryotic production organism.

• From a variety of recommendations, we chose E. coli, S. lividans and S. cerevisiae as our respective gram-negative, gram-positive and eukaryotic production organisms.

Suckerin production and purification

After finding a suitable production organism, we investigated purification methods. Both Bert van der Zaal and Hans van Leeuwen warned us about inclusion bodies, which are aggregations of produced protein within the cell. This separation complicates subsequent purification and protein extraction. In addition, proteins inside inclusion bodies can not interact with the machinery to fold them properly, resulting in non-functional proteins. However, we found that this would not be a problem for suckerin, as the protein self-assembles and would therefore remain functional [9]. We used an inclusion body purification protocol, adapted from Ding et al. [11]. Furthermore, Dr. van der Zaal and Dr. van Leeuwen recommended several steps to improve purification:

1. Dr. van der Zaal proposed to include a His-tag as this allows for more targeted purification. We decided to include a His-tag in the suckerin sequences used for upscaled production (suckerin-19 and suckerin-12).
2. Dr. Van Leeuwen suggested trying to build suckerin out of multiple small peptides. We implemented this by creating general sequences of the two modules based on the protein sequences of suckerin-12 and suckerin-19. 

Furthermore, we learned from Sylvia de Pater that it would be best to include an inducible promoter in our plasmid design. This provides control over the production and ensures that the bacteria can grow to exponential phase without the burden of protein (over)production, which would be especially important for the upscaling of suckerin production.

• After feedback from different experts, we developed our project design to have:
• E. coli strain Rosetta as a suitable organism for production of eukaryotic-derived proteins
• Addition of a His-tag to the protein sequence to enhance purification
• An inducible promoter to maximize and control suckerin production






Figure 1: A visual summary of our design for suckerin production. Using the feedback and recommendations of our stakeholders, we will produce suckerin in E. coli Rosetta, with a plasmid containing the inducible P_Lac promoter and His₆-tag.

Evaluating a method of upscaling

To scale up our production method, we first investigated whether it would be possible to let E. coli secrete the protein. After talking to Paul van Heusden, we decided not to further pursue this secretion possibility, as this would be a difficult system to realize in the limited time we have. Secondly, the use of bioreactors was considered for increased bacterial growth and subsequent higher quantity of produced suckerin. For this, we contacted Jack Pronk and he made clear that upscaling of the process would have to be done in steps, starting with shakeflasks before further upscaling to bioreactors. In order to optimize the production process and find the optimal conditions for large scale production, we cooperated with EV Biotech to model the metabolism of suckerin-producing E. coli in different media.

• Suckerin production will be initiated in shake flasks, after which bioreactors are used to upscale protein. Growth conditions in the bioreactors were optimized using our model.

Team members during their visit at the headquarters of EV Biotech, to talk about the possibilities of modelling our suckerin production.

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Abstract: analysis of the impact of our project

To comprehend the impact of our suckerin-based hydrogel on society, we talked with a range of stakeholders. Each gave us feedback and new ideas to integrate into our project design. Since our final hydrogel will be considered as a medical product, safety was a crucial aspect of the project. Furthermore, we investigated the sustainability, as it may give an advantage of biodegradability of the suckerin proteins. Lastly, we summarized the stakeholders that were involved in previous phases.

Safety of suckerin

Together with scientific prospects mentioned in phase 3, we addressed our scientific responsibility in safety of the project. First, we participated in the Safety-by-Design assignment from the RIVM. We discussed with Korienke Smit and her colleagues how to identify and address risks and uncertainties in our project. From this we learned, for example, that the environmental safety of our product is not solely dependent on the final product, but is also present in the production process. We implemented their feedback in our safety page, as well as an infographic displaying a visual summary of the safety of our project design. Furthermore, to increase the assurance for clinical implication, Esther Middelkoop suggested to keep the hydrogel as simple as possible. Inclusion of more active ingredients leads to a longer and more complicated track to validate their safety, non-toxicity and effectiveness. Therefore, to keep our final product as safe as possible, we would at first only test the addition of FDA approved drugs. By ensuring the safety of the product during all stages, the chances of getting the final product on the market increases.

• By participating in the Safe-By-Design assignment of the RIVM, we anticipated the safety risks of our project design. Furthermore, in our initial tests we will involve FDA approved compounds to test the linker system.

Sustainability and considerations

In addition to the responsibility of making a safe product for the consumer, we also recognize that our suckerin-based hydrogel should be good for the environment. Therefore, we sought to integrate the sustainability of our product, by looking into the biodegradability of suckerin. During our talk with René Kleijn, we discussed that this could be an important feature and has potential, as it is a protein. For instance, Dr. Kleijn told us that medical plastics are not recycled due to safety reasons, therefore everything is burned instead. In addition, biodegradability would also be an important wound dressing feature for the patient, as Egbert Krug and Esther Middelkoop informed us that removing membranous dressings like hydrogels is often painful. If suckerin indeed biodegrades in a suitable time-frame, our product could degrade naturally and disappear over the course of the treatment. This discards the need to be removed and burned. This concept would be similar to the degrading of stitching fiber after wound closure. Therefore, more research on suckerin biodegradability is needed. We decided to include recommendations for experiments to explore the biodegradability of suckerin proteins.

• The sustainability of suckerin has potential and should be further explored, as it could provide additional advantages to our suckerin-based hydrogel.

Involved and affected stakeholders

Ethical considerations: We wanted to explore the ethical debate surrounding genetically modified organisms (GMOs), especially since the suckerin protein is produced by a GMO and used as a medical product. We first created a survey in collaboration with the iGEM teams from Oxford and Copenhagen to see the general viewpoint on the use of GMOs in a medical setting. The survey showed that people were more open to genetically engineered medicine when externally applied, rather than internal products. More interestingly, the participants had an overall positive attitude towards genetically engineered medicine. Regarding these results, we also explored the public opinion on synthetic biology in our open discussion event. Furthermore, we wanted to see where our project would fit into this debate, by talking to Nienke de Graeff. However, during this discussion, it became clear that it would be more important to focus our attention on the involvement of the stakeholders surrounding our project. Nienke suggested that we focus on carefully considering all stakeholders. This would make sure we can really cooperate to implement their input and consider the social responsibility of our project and product. An overview of these stakeholders is summarized below:

Stakeholders	Summary
Patients	We decided to start with the most affected group which would directly experience our implemented product, namely people with burn wounds. Our aim was to understand their struggles with the current methods of wound dressings, as well as their wishes and preferences with regard to new methods. To achieve this we came in contact with a burn wound victim who suffered from third-degree burn wounds, covering 55% of her body. Her story highlighted the continuous discomfort due to pain, itching and restricted movement from scar tissue. Moreover, she mentioned her weekly therapy sessions to treat her scars, indicating the intensive aftercare of burn wounds. We talked to Titus Radstake, who told us that wounds are currently covered with allogeneic donor skin to close off wounds, thus avoiding infections and excessive fluid loss as much as possible. We thrive to improve this by including a linker system to which beneficial compounds, such as growth factors, can be coupled. This set-up would stimulate wound healing and decrease scarring.
Providers of current treatment methods	Before we could implement our own project into the real world, we had to investigate the current treatments, struggles, and opportunities for new research. To further investigate this, as well as integrate the point Mr. Radstake had made about the current use of allogeneic donor skin, we talked to the head of the Dutch Skinbank, Frank Beneker. This conversation provided us with information on the current burn wound treatment with allogeneic donor skin and, more specifically, where opportunities for improvement lie.
Medical professionals	Trauma surgeon, Egbert Krug told us that one of the important features of wound dressings would be easy removal. This was also mentioned by Esther Middelkoop, who informed us that removing membranous dressings is often painful for the patient. However, hydrogels are known to be non-adhesive to skin cells, but instead stick to the skin due to the fluids in the wound. In this way, for use on large (burn) wounds the pain caused by removing the hydrogel will be minimized. Nonetheless, biodegradability is an interesting feature for other applications, therefore we included further research on this in our future prospects of our results.
Other providers of biomedical products	From Hans Peter Mulder (Idris Oncology) and Vincent van der Wel (Patient One) we learned the possibilities within biomedical companies, and they gave us advice on how to get the product to the market. We developed their feedback further in our entrepreneurship program.

Developing the product for the market

Lastly, after all efforts to create a product, we developed a marketing strategy to put our product into the real world. The Leiden Bioscience Park provided us with a platform to reach entrepreneurs and business people. With the help of Eugen Kaprov, we developed a unique entrepreneurship programme for our suckerin-based hydrogel. This included our marketing plan for our unique selling points, as well as calculating the costs and investments for large-scale production. During our conversation with Vincent van der Wel, we further developed and defined our marketing strategy. For instance, mr. Van der Wel mentioned that we should patent our product in various countries, instead of focusing solely on the Netherlands and other countries in Europe. As a medical product, our suckerin-based hydrogel has to go through clinical trials to prove its safety and effectiveness. Therefore, we sought advice from Adam Cohen and Wouter ten Voorde from the Centre for Human Drug Research (CHDR). Most importantly, with our experiences and acquired knowledge, we developed a comprehensive roadmap consisting of three phases to guide future iGEM teams in setting up their own business plan.

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Future Prospects

Alternative applications for suckerin

Even though our main aim was to create a suckerin-based hydrogel, the protein itself can be used for a wide variety of applications. During our conversations with experts in different fields we discovered several options. 

We knew from feedback of René Kleijn that the suckerin protein is not suitable for bulk plastic production, but rather for more specialised applications. One possibility that we explored is the use of suckerin as a sustainable plastic in the fishing industry. We reached out to Rafaëlla Paulo Teixeira , who told us that suckerin, due to its strong and elastic properties has potential as a material for fishing nets. However, future experiments should first examine the degradation of suckerin in fresh- and saltwater, as well as its performance under high-pressure conditions.

Additionally, we got the advice from Peter Nibbering to look into the possibilities of application on other forms of open wounds. For example, chronic or diabetic wounds are similar in their treatment to burn wounds. A suckerin-based hydrogel could also be a solution in solving these open wounds. However, further research is required, focusing on the specific differences between the wounds and their treatment process. For example, the compounds linked to the hydrogel can be adjusted to target the specific components involved in these wounds. In addition, the use of different suckerin proteins could be optimized for different wounds.

Guidelines for future iGEM teams

Our project, like other projects within iGEM, underwent various changes as we were confronted with new challenges and knowledge. In this section, we want to explain our process, to give some concrete examples of what you can do with Human Practices. In this section, we hope to aid future iGEM teams in tackling the integration of feedback and stakeholder views into their projects.

For our human practices we used a combination of resources. First, we consulted literature to get familiar with our topic, exploring opportunities and which researchers or research groups are working on this. Secondly, the results from previous iGEM teams have been of great importance. By investigating their project aims and results, we could identify what worked (and equally important, what didn’t work). For example, we learned much about the ethical implications of making a bandage from bacteria from the Eindhoven 2018 team and about the challenges of spider silk production from the SDU Denmark 2016 team. Lastly, we interviewed a large number of experts with a wide variety of backgrounds. To get a coherent story, it was of major importance for us to go through the process step by step, making sure we could back-trace where decisions came from. Something we didn’t manage to complete (but wished we had) is to involve a larger area of stakeholders, such as people from other countries, ethnicities, religions or education. This would give us a more diverse insight into the perception of our hydrogel, and other perspectives of our project design. Click below to see our tips for future teams!

How to build your human practices network: Tips for future teams

• Make use of different sources to corroborate your choices. In our project we had the disadvantage that there are no suckerin experts in the Netherlands. Instead, we explored the literature to find an expert on this topic and contacted them. Besides this, we contacted local experts who work with natural biomaterials and therefore could hypothesize on the potential of suckerin.
• There has been a great deal of iGEM projects: use their knowledge! At the start of the project, you can look at wikis of teams working on similar ideas to familiarize yourself with the stakeholders involved. For example, we looked at the work from teams, like Eindhoven 2018, with a similar project on biomaterials or medical applications, and got inspired from their stakeholder analysis.
• Use the network of your stakeholders. For example, after talking to the Dutch Burn Survivor Association, we asked if they could reach out to anyone who would be willing to share their story with us. In the end, we got a reaction from several people, with whom we further collaborated.
• Try to broaden the horizon of your project by involving stakeholders outside of your own environment/city/country. Imagine the implementation of your project on a global and local scale and from there work your way through every stakeholder affected by it. You can even make a list, prioritizing the different stakeholders you want to involve.
• Be creative with how you involve human practices into your project. It’s a part of more aspects than you think! A good way to approach this is to organize a discussion with the whole team, and brainstorm together. This way, each part of the project is represented and each responsible person/group can think of how to integrate human practices into their part of the project. Analyzing your project halfway through gives you an overview of what is still missing. One useful resource for this is the iGEMmer’s Guide to the Future.
• Human practices can affect your project from beginning to the end. Contacting researchers before you start your lab work, can improve your planning and they can help to prioritize experiments. Besides this, since time is limited, researchers that utilize similar techniques in their work, can provide you with insights in more efficient methods or even offer you equipment.
• Combine public engagement with integrated human practices. Even though integrated human practices and public engagement are different categories, we found that the combination of both can yield a surprising synergy. By interacting with ‘the general public’ and asking for their opinion during events, you can get an insight into their perception of your project. In turn, this can help you to find new perspectives.

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