Human Practices

Integrated Human Practices

From the beginning, the Human Practices took a central role in shaping our project. When we started our brainstorm sessions in March, we had no clue which topic we wanted to explore. Yet, we wanted it to be relevant to society. During these first months, we got the opportunity to interact with experts and the public to discuss our ideas. These encounters were of great help in our topic selection. During a visit to the Dutch Institute for Public Health and the Environment (RIVM) and the Rathenau Instituut (Dutch organization for technology assessment), we realized that the lack of a safe platform for working with GMOs outside of controlled environments is a problem on itself that deserves a solution. We were excited to tackle this issue with Gelcatraz, but also wanted to work towards a more concrete application. In the time that followed, we consulted several experts and interacted with the public to get feedback on our proposals. Once we had settled on the wound healing application, we interacted with more stakeholders and experts to shape the design of the wound healing patch. We also performed a stakeholder-value analysis and an ethical analysis of our design, and we considered safety during all stages of the project for safe-by-design. We realize that our project would not have been the same without the Human Practices.

In the following section, we share the insights we gained from interactions with stakeholders and experts during the three main stages of the project: finding a project, finding an application and shaping the design. Here you can explore our journey of integrated human practices.

I. Finding a project

Thanks to the TU/e Contest and various brainstorm sessions, we had settled on the living material project. Although we decided to focus on the adhesin system, we wanted to demonstrate the potential of our platform by applying it to a social problem. To find the ‘perfect’ application, we consulted various experts, interacted with the public and attended a biotech conference. We also reflected on the ethical consequences of our potential applications before settling on wound infections.

February & March

Initially, we considered a wide range of topics, from glucose sensors to solar energy, and from ocean clean-ups to artificial organs. The number of options seemed unlimited, and our search was scattered. As a source of inspiration, we consulted newspaper articles, checked the wiki pages of previous iGEM teams and talked to several professors at our university. When going through the wiki pages of previous teams, we realized that many of them struggled with the use of their creations outside of the laboratory. We also came across an article by Liu et al. (2017), who attempted to design a ‘living material’ by means of several layers used to entrap bacteria. Inspired by this cool work, we decided we wanted to do something similar in order to facilitate the easy use of GMOs. However, instead of using several layers for entrapment, we wanted to equip our bacteria with their own anchor. After several brainstorm meetings, we settled on this challenging subject as the goal of our 2018 iGEM project!

April & May - TU/e contest

Right after we decided to anchor bacteria to a hydrogel, we decided to register for the TU/e Contest . This is an annual ideation contest organized by our university, in which students are encouraged to develop their innovative ideas into start-ups. The contest was intended for students who already had a clear idea that they wanted to bring to the market. Unfortunately, our project was still quite vague (‘adhering bacteria to a hydrogel’). Nevertheless, our participation was still useful. We got to meet companies, participate in workshops and pitch our project to the public. Along the way, we received quite some feedback from the participating companies.

April & May - TU/e contest

II. Finding an application

Thanks to the TU/e Contest and various brainstorm sessions, we had settled on the living material project. Although we decided to focus on the adhesin system, we wanted to demonstrate the potential of our platform by applying it to a social problem. To find the ‘perfect’ application, we consulted various experts, interacted with the public and attended a biotech conference. We also reflected on the ethical consequences of our potential applications before settling on wound infections.

May 14, 2018. Dutch iGEM meetup RIVM and Rathenau Institute.

In May, we were invited to The Hague for a Dutch iGEM meet-up organized by two public institutes: The National Institute for Public Health and the Environment (RIVM ) and the Rathenau Institute, an institute for technology assessment. The theme of the meet-up was Human Practices. As we had only just started our Human Practices trajectory, this day was a great way to get inspired.

May 14, 2018. Dutch iGEM meetup RIVM and Rathenau Institute.

day Our day started with a presentation on the different stages of a project. After the pitches by all six Dutch teams, we got the opportunity to speed date with seven experts on the topics of biosafety, security, bioethics, policy and regulations: Zoë Robaey, Cécile van der Vlugt, Pim Klaassen, Niek Savelkoul, Pieter van Boheemen, Lilian van Hove and Jaco Westra. They provided us with helpful feedback on our plans and we discussed potential applications of Gelcatraz. They encouraged us to focus on previous iGEM projects that could have been improved with our platform. In fact, wound healing was one of the applications they mentioned! It was also great to meet the other Dutch teams for the first time and to learn what they were working on. We would like to thank the Rathenau Institute and RIVM for the inspiring day!

Insights

We should choose a concrete application, because that sells much better and leads to better human practices.
We should try to center our Human Practices around our future application.
The safe use of GMOs on itself is already a relevant problem which we are aiming to tackle with our project.
We should contact as many experts and stakeholders as possible. Do potential users indeed need our product?
As an application, it could be interesting to improve on an old iGEM project.

Follow-up

We checked previous iGEM projects to see which ones could have benefitted from our platform.
We searched for a concrete application of Gelcatraz, eventually leading to our focus on wound healing.

iGEM Team Eindhoven 2018 members Laura, Mick and Yahav (from right 6th to 4th) at Rathenau Institute.

May 16, 2018. Meeting Prof. Dr. Patricia Dankers

As we had decided to develop a living material, we wanted to speak to an expert in the field of biomaterials to ask for some feedback on the design and explore possible applications. We therefore contacted professor Patricia Dankers, a professor in biomedical materials for regenerative medicine. We discussed our preliminary selection of dextran as our hydrogel. Prof. Dankers encouraged us to limit ourselves to this gel instead of also exploring other options, as dextran is known to have affinity for glucose and the production of multiple gels would be time-consuming.

May 16, 2018. Meeting Prof. Dr. Patricia Dankers

May Brainstorm sessions

Inspired by the Dutch iGEM meet-up and by our participation in the TU/e Contest , we were determined to find an application relevant to society. During several brainstorm and literature sessions, we composed a list of possible applications. Inspired by work performed by previous iGEM teams, the experts we talked to during the Dutch meet-up and by our meeting with professor Dankers, we decided to investigate bacterial sensors (malaria, urea, scabies), kidney failure, water filtration, bone infections, vaccines, eczema and wound infections.

May 22, 2018. Netherlands Biotechnology Congress

Our first conference! With the other Dutch iGEM teams, we were invited to present a poster at the Netherlands Biotechnology Congress. During the several talks we learned about new developments in biotechnology and public acceptance of GMOs. The highlight of the day was an intriguing talk by Marc Lynas, a British environmental activist and journalist who explained how he moved from violently opposing GMO crops to supporting them. The conference also offered a platform to discuss our project with biotechnologists and with the overgrad winning team of iGEM 2017, TU Delft. After a few drinks and a good meal, we returned home with a lot of new insights.

May 22, 2018. Netherlands Biotechnology Congress

June 1, 2018. Meeting with an ethicist: Dr. Lily Frank

To examine the ethical implications of our iGEM project, we contacted Dr. Lily Frank. She is an Assistant Professor of Philosophy and Ethics at Eindhoven University of Technology and is specialized in Biomedical Ethics. With Lily we discussed the potential benefits and dangers of synthetic biology, and she pointed us at some of the more controversial issues related to synthetic biology.

June 1, 2018. Meeting with an ethicist: dr. Lily Frank

June 9, 2018. Dutch Technology Week

The Dutch Technology Week (DTW) is all about promoting Dutch high-tech to the public. During this week, many high-tech companies open their doors. It is a good opportunity to raise public awareness about synthetic biology! We arranged a stand during the High-Tech Discovery Route. We made hair gel and slime with kids. While some of us entertained the children, others got the opportunity to discuss our applications with the parents.

June 9, 2018. Dutch Technology Week

The parents seemed to appreciate the ideas of (chronic) wound healing patches, needleless vaccines and the detection (and simultaneous treatment?) of substances in the blood and on the skin. One of the visitors was a dermatologist. She liked the idea of a patch to combat skin infections and encouraged us to develop this idea further. She said that this idea as well as the needleless vaccines would have a huge potential. However, another visitor argued that there is a huge need for treatment of antibiotic resistant skin infections in western countries, whereas a vaccine patch would be desirable for developing countries. Considering the conclusion drawn from our meeting with Lily Frank that ethically, it may be saver to choose an application relevant to Western countries, the skin infections application may be preferred.

Insights

We learned about the existence of some useful ethical frameworks.
A fair distribution of the benefits and burdens of synthetic biology both across society and globally is an important task of scientists working in the field of synthetic biology.

Follow-up

We decided against using our living material for malaria-sensing or vaccines-delivery in third world countries.
We explored the ethical frameworks recommended by Lily and used them for an ethical discussion of our work.

At our stand in Dutch Technology Week, team member Mariska is guiding kids to play with our polymer cross linker.

June 18, 2018. Kick-off Safe-by-Design Challenge: Skype with Korienke Smit and Niek Savelkoul (RIVM)

The Dutch National Institute for Public Health and the Environment (RIVM) challenged us to take part in their safe-by-design challenge. This challenge was about integrating safety in different phases of our project.

June 18, 2018. Kick-off Safe-by-Design Challenge: Skype with Korienke Smit and Niek Savelkoul (RIVM)

June 22, 2018. Meeting Dr. Sandra Hofmann

Before deciding on an application, we had a final meeting with Sandra Hofmann. She is an assistant professor in mechanobiology in bone tissue engineering at Eindhoven University of Technology. We had found a paper (Johnson et al., 2018) on bone fracture healing with lysostaphin that we wanted to discuss with her, as we considered the secretion of lysostaphin by our living material.

June 22, 2018. Meeting dr. Sandra Hofmann

We proposed our plan to combat bone infections with bacteria producing lysostaphin, encapsulated in a hydrogel. She would not use it for bone infections, because only a hydrogel on bone is not enough (not load-bearing), and combinations with other materials are very complicated. Also , some interesting alternatives eliminating the need for secondary surgery are already being developed right now. And there are some (non-ideal) alternatives which guarantee a reasonable quality of life, which makes it more difficult to find people willing to take the risk of incorporating living GMOs into their body. However, our hydrogel may still be helpful if we turn it into a composite of hydrogel and granules. But for the scope of this project (limited time), she instead recommended us to focus on skin infections. She believes it is very promising. Furthermore, the need for it will only grow as people live longer. This means they develop more decubiti’s and more metabolic syndromes, which in turn increases the risk of skin infections. These infections even have the potential to spread to the bone, worsening the situation. She also gave us some advice in how to handle the bacterial invasion problem: she suggested to research ion release.

Thank you, Sandra, for your extensive feedback!

Insights

Our living material would not be the best solution against bone infections, as hydrogels are not load-bearing, alternative, GMO-free solutions are already being developed and alternatives exist which offer a reasonable quality of life.
In contrast, Gelcatraz would be very suitable as a plaster against skin infections.
Increasing life-spans and a surge of metabolic syndromes increase the demand for a solution against chronic wounds.

Follow-up

This was the final feedback we ‘needed’ to settle on skin infections as our application.
We researched ion release as a method to prevent infiltration of foreign bacteria into our gel.

June 22, 2018. The final decision

We took the feedback received from Sandra Hofmann, during the Dutch Technology Week and earlier feedback from previous iGEM teams and experts into account to settle on the application we would be working on.

June 22, 2018 The final decision

III. Shaping the design

Once we had decided to focus on wound healing, there were many new questions to be answered by the Human Practices team. How to offer patients suffering from infected wounds the best possible treatment? Is there indeed a need for a new treatment? What requirements does our material need to fulfill? What are the medical details of the wounds? Which bacteria do we need to kill? To collect answers to these questions and many more, we contacted several experts and stakeholders.

July 3, 2018. Plasmacure

In our search for existing and future wound treatments, we came across Plasmacure. Plasmacure is a start-up aiming to cure chronic wounds, especially diabetic wounds, with plasma (fourth phase of matter). They developed a small machine producing plasma which can be connected to a disposable plaster. The plaster is applied to the wound. During a one-minute treatment, the wound is exposed to plasma. This should be enough to kill all bacteria and fungi on the wound. This treatment is repeated ten times. The product is still under development, but the first small clinical trial showed positive results for half of the patients. During a meeting with their CMO Koen Lim, Lim was so kind to sketch the wound market and its problems, to share his solution and to give us feedback on our plans. We also learned about the long and winding trajectory of bringing a medical device to the market. This resulted in our problem sketch.

July 3, 2018. Plasmacure

Furthermore, Lim provided some feedback on our idea to combat skin infections with lysostaphin and shared some requirements our device should fulfill. Most importantly, the patch must be user-friendly such that it can be applied by a nurse at home. It must thus be safe and easy to handle. Koen Lim liked the ability of our patch to be freeze-dried. He also said we should aim to kill the major bacterial strains contributing to wound-infections rather than one strain only. We will thus examine the option to make our bacteria produce more proteins than only lysostaphin.

Insights

Wound care is no medical specialism, which means that care is poorly coordinated. Due to a lack of standards, the quality of treatments differs between hospitals.
Currently, no proper treatment exists for complex wounds. This makes wound healing a “desperate market: one dies with their wounds, but not due to them.”
Wound care is estimated to cost the Dutch health care system 1.5-2 billion euros annually.
Home care nurses play a crucial role in wound care, as most of the treatments take place at home. It must thus be easy for nurses to work with our plaster.
Almost all diabetic wounds are infected. Almost everyone uses antibiotics for that reason. Due to increasing antibiotic resistance, treatments are less effective. A clear need exists for alternatives! An additional reason why an alternative to systemic antibiotics is demanded, is the disturbance of the gut microbiota caused by antibiotics. Diabetic patients generally already have a disturbed gut microbiota, and this is worsened by antibiotics.
The most difficult wounds occur in older people with poor blood circulation. This makes that wounds barely heal, particularly in the legs and feet, regions which suffer most from reduced blood flow. These older patients are usually diabetic patients, as diabetes affects blood vessels. At the same time, the reduced blood flow affects the nerve system, making that patients feel less pain. They may not notice that they have a wound on their feet. In the worst-case scenario, feet or legs must be amputated. In that case, five-year survival rates are even worse than for many cancer patients. Sometimes, heart patients experience similar problems as diabetic patients.
Another group of patients that may benefit from our treatment are burn wound patients. Burn wounds are also often infected.
It may be helpful to stimulate blood flow while applying our plaster, as blood flow has been demonstrated to be crucial in wound healing
We should aim to kill the major bacterial strains contributing to wound-infections rather than one strain only. Lim did not know which strains are most problematic.
We learned about the long and winding trajectory of (clinical) testing required before market launch of medical devices.

Follow-up

We integrated the insights in our applied design.
We contacted the Maasstad Hospital burn wound center to learn more about burn wounds.
We researched the main bacterial strains contributing to wound infections and possible bacteriocins we could use to extinguish them. Maasstad Hospital and Dr. Edgar Peters gave us more insight in which bacterial strains cause the most problems in wounds.
To make it user friendly, we take the wishes and needs of home care employees and patients into account while designing our patch.

July 20-22, 2018. European Meetup Munich

In July, we traveled to Munich for a meet-up organized by iGEM Munich. We had a great time meeting other iGEM teams, exchanging ideas and we received a lot of feedback on our design and potential problems.

July 20-22, 2018. European Meetup Munich

August 01, 2018. Skype call with Lori Goff

Lori develops hydrogel membranes for water filtration. She was so kind to call us to give us feedback on our project. She approved our idea of using our living material in a biomedical context, as she expected the most potential in that area.

August 01, 2018. Skype call with Lori Goff

August 03, 2018. Skype with iGEM Düsseldorf: The potential of quorum sensing as a mean to prevent overcrowding and/or infiltration

During the European iGEM meet-up in Munich, we received some useful feedback from team Düsseldorf. They warned us for the potential problem of overcrowding. For their project, they are developing a quorum sensing mechanism to regulate cell density.

August 03, 2018. Skype with iGEM Düsseldorf: The potential of quorum sensing as a mean to prevent overcrowding and/or infiltration

August 07, 2018. Meeting with professor Menno Prins about biosensing

Menno Prins is a professor at TU/e active in the field of molecular biosensing who develops technologies for protein detection and the study of protein function. We met with him to discuss the potential of our living material in sensing applications. He warned us that bacterial biosensors have two serious disadvantages compared to (bio)chemical or physical sensors: they require very stable environments and detection is slow compared to other sensors: on the time scale of hours versus milliseconds.

August 07, 2018. Meeting with professor Menno Prins about biosensing

August 10, 2018. Safe-by-design: Phone call with Cécile van der Vlugt

For the Safe-by-design challenge we had a phone call with Cécile van der Vlugt. She is active in the field of risk assessment and provided us with some interesting feedback on our work done so far. The phone call was very useful to put our work into perspective. For instance, she stressed that although it is good to be cautious, we do not need to see risks everywhere. In the week before the call, we had been researching alternatives to the toxic chemicals we use in the synthesis of our hydrogel gel. She said that in principle there is no need to replace the chemicals, as long as we ensure safe working conditions.

August 10,2018. Safe-by-design: Phone call with Cécile van der Vlugt

Augus 13, 2018. Kill switches: Meeting with Prof. dr. Jan van Hest

Inspired by our conversion with Prof. Menno Prins, we decided to take the safety of our device a step further by integrating a kill switch. This would minimize the risk of bacterial escape even further. By making our bacteria dependent on an artificial amino acid and including this in the medium, we could ensure that bacteria which escape the gel die. Through point mutations, one makes essential proteins dependent on an artificial amino acid. To examine this option, we contacted professor Jan van Hest, who has a lot of experience with artificial amino acids.

Augus 13, 2018. Kill switches: Meeting with Prof. dr. Jan van Hest

Prof. dr. Jan van Hest was so kind to share his knowledge with us. He warned us that some artificial amino acids used for this purpose are still present, in varying quantities, in nature. Kill switches relying on such an amino acid are thus not completely safe. We discussed some potential artificial amino acids to use. Van Hest ultimately advised us to use azidophenylalanine, an artificial amino acid which is not present in nature. Its implementation is also efficient, as the required tRNA and synthetase are easily producible by bacteria. Van Hest recommended us to start with only one point mutation. Multiple mutations would be saver, but could complicate the synthesis of essential proteins, leading to cell dead.

Another topic we discussed with Van Hest, is the option to replace the use of antibiotic resistance genes in R&D by dependence on artificial amino acids. As we ultimately want to use our living material on patients, we do not want to risk antibiotic resistant bacteria escaping into the environment. Van Hest informed us that dependence on artificial amino acids could be a serious alternative, albeit not much used yet. He said that currently researchers simply use antibiotic resistance genes because of their ease of use and wide availability. However, dependence on artificial amino acids as a selection mechanism may be even more reliable than antibiotic resistance, as bacteria tend to get rid of genes that are temporarily unused, e.g. in the absence of antibiotics. As genes related to artificial amino acid dependence generally encode for essential proteins, they do not run this risk.

Insights

Dependence on artificial amino acids is a feasible method to implement a kill switch into our design, and thereby make our living material an even saver platform for the use of GMOs.
For optimal safety we must choose an artificial amino acid not present at all in nature.
One must strike a balance between safety and feasibility while choosing the number of point mutations to make.
Dependence on artificial amino acids may form a saver alternative to antibiotic resistance as a selection mechanism in R&D.

Follow-up

We designed a kill switch to be implemented into our design through the dependence on azidophenylalanine. The results can be found on our Applied Design page.

July – August 2018 iGEM'ers Guide to the Future – Stakeholder value matrix

Inspired by the iGEM’ers Guide to the Future, we performed a stakeholder-value matrix. Over the course of July and August, we reflected on the consequences of our design for several stakeholders. The entire analysis can be found here. From the stakeholder-value analysis, it is concluded that Gelcatraz introduces significant benefits to most stakeholders discussed. Importantly, patients will benefit from the plaster as they will experience less pain, enjoy faster wound healing and thus get a better quality of life. The major safety issues have been addressed under safe-by-design. Medical professionals will also appreciate Gelcatraz as a new tool which will facilitate and ease their treatments.

July – August 2018 iGEM'ers Guide to the Future – Stakeholder value matrix

August 21, 2018. Freshmen’s week @ TU/e

During the introduction week for new students, we got the opportunity to promote iGEM at a fair. We talked to new students from all kinds of disciplines about synthetic biology and the iGEM competition. The students were very curious to hear why we decided to spend our summer in the lab! We also introduced the newbies to our iGEM project and asked them whether they would wear a patch with bacteria on their skin. Interestingly, after sharing some information on the role of the bacteria in our design, they seemed to agree that they would wear the patch if we could ensure the safety of the patch.

August 27, 2018. Burn wound center Maasstad Ziekenhuis

As several experts told us that our patch could have potential as a treatment of burn wounds, we wanted to know more about the options and requirements. We contacted Dr. Jan Dokter, specialized in burn wounds at the Maasstad Ziekenhuis. This hospital in Rotterdam hosts one of the three Dutch centers specialized in the treatment of burn wounds. They treat approximately 300 patients annually, who represent 45% of all Dutch patients with severe burn wounds. Dr. Dokter was so kind to invite us over for a visit.

August 27, 2018. Burn wound center Maasstad Ziekenhuis

After getting changed into a blue apron (burn wounds a very prone to infections indeed), we were welcomed by what seemed to be the entire team of the burn wound department. Dr. Dokter and colleagues told us that the treatment of (antibiotic-resistant) bacterial infections is high on their priority list. This is because burn wounds are never sterile, since they are always colonized by bacteria. The goal of the medical team is to contain the bacteria and to prevent them from developing into an infection, as this could be lethal. Currently, containment is done through systemic use of antibiotics, mechanical cleaning of the wounds and/or antibacterial creams. However, they recently observed an increase in antibiotic-resistant bacteria such as MRSA, meaning that an alternative like ours would be very welcome. They told us they would already order some patches if possible. 😉They also foresaw benefits for the patients, as wound dressings must be changed regularly: two times a week for small burn wounds, and even daily for larger ones. As changing is painful for the patients, it would be great to use a wound dressing like ours which could stay on the wound for a prolonged time while ensuring drugs are administered to the wound.

Although they were very enthusiastic about our product, they also saw some room for improvement. Their advice was to complement lysostaphin with other systems to target different bacteria. Bacteria found by the Maasstad burn wound center in burn wounds include Staphylococcus aureus (including MRSA), Streptococci, Pseudomonas (e.g. P. aeruginosa), Klebsiella, Enterobacter, E. coli, Serratia, Proteus, Citrobacter and Acinetobacter baumanii. However, not all of these are of equal danger. S. aureus is recently getting more problematic because of a surge in dangerous toxin-producing strains found in burn wounds. This is also what drew Dokter’s attention to our project. Other strains considered particularly toxic are Streptococci, Pseudomona, Klebsiella and Enterobacter. General, Dr. Dokter told us that Gram-negative bacteria pose a higher threat to patients than Gram-positive ones. The team also warned us that bacteria such as S. aureus growing in biofilms are particularly challenging to get rid of. They were thus very happy to hear that lysostaphin targets bacteria growing in biofilms.

Another problem recognized by the medical practitioners regarding the current treatment methods is the systemic use of antibiotics which increases the risk of resistance. To find out which bacteria contaminate the wound, they usually culture them. However, this takes four days, forcing the doctors to use systemic antibiotics to prevent the outbreak of an infection. As the systemic antibiotics used are not specific, this treatment contributes to antibiotic resistance while the treatment may not even be effective against the specific bacteria colonizing the wound. Once the results of the culture are known, a more specific treatment is selected. The medical team therefore believed it would be very beneficial if we could expand on our patch by integrating a sensing system, to secrete the appropriate type of bacteriocin only when the relevant bacteria are present. This would decrease the risk of resistance developed against our bacteriocins drastically. They said it would be great if we could include different types of bacteria secreting different bacteriocins in combination with a sensing system, as it would directly improve the treatment efficacy and prevent building antibiotic resistance.

We would like to once again thank Jan Dokter and the entire team of the Maasstad burn wound center for their hospitality and kind feedback. Inspired by some in-depth knowledge of the most problematic bacterial strains and the requirements for wound healing and encouraged by your positive feedback, we will continue our efforts to improve our design even further! 😊

Insights

Burn wounds are always colonized, meaning that it is crucial to control and limit the bacteria growing in the wound.
Burn wounds are colonized and potentially infected by several bacterial strains. Particularly the Gram-negative strains and bacteria growing in biofilms are virulent and difficult to target.
S. aureus is increasingly challenging doctors treating burn wounds because of a surge in toxin-producing strains.
Current burn wound treatments are mostly mechanic in combination with antibacterial creams.
A hydrogel forms a perfect environment for wounds, considering that burn wounds benefit from a gel-like environment. We must check that liquid produced by edema can be absorbed. Burn wounds do not need oxygen to heal.
The use of systemic antibiotics while researching the type of bacteria colonizing a wound contributes to the development of antibiotic resistance, but is not always effective against the bacteria present on the wound. The development of targeted treatments in combination with a detection method for specific bacterial strains would thus be very useful, as it will increase efficacy while lowering the risk of resistance against the treatment.

Follow-up

We researched bacteriocins to target Pseudonomas and other Gram-negative bacteria, without affecting our own E. coli bacteria. This resulted in our pyocin S5 BioBrick.
We researched a combination of sensing and secretion. See Applied Design.

At Maasstad Ziekenhuis

Sept 21, 2018. Ethical analysis – update

As we continue to make progress on our project, our understanding of ethical issue also evolves.

Sept 21, 2018. Ethical analysis – update

Sept 21, 2018 Visit to PAMM: Foundation for Infective Diseases and Pathology

PAMM is a foundation for Infective Diseases and Pathology. They perform diagnostics for hospitals in the region and are also involved in prevention. We are indeed very lucky to have PAMM as one of our main partners! PAMM is not only helping us financially, but also offered help with the functional testing of lysostaphin.

Sept 21, 2018 Visit to PAMM: Foundation for Infective Diseases and Pathology

As we had realized that working with S. aureus brings significant safety issues (check our safe-by-design page for more information), we reached out to PAMM for help. To discuss the collaboration, we were invited to visit their laboratories. On Friday September 21st, we met with Kees Verduin and Jeroen Tjhie, two of their microbiologists. We presented them with our progress and discussed some issues we were still struggling with. It was very encouraging to hear that Kees and Jeroen saw a lot of potential in our approach. Afterwards, Kees gave us a tour through their impressive laboratories. It was interesting to notice the differences with our own lab. We were very impressed with the amount of work that had been automated! We also learned a lot about the safety measures they implement in their different labs with varying safety levels. Kees ensured us that our experiments with S. aureus will be nothing special for them in terms of safety. 😉 After this tour, we cannot wait to come back for our own experiments!

Insights

A lot of work in diagnostic laboratories is automated.
S. aureus does not require any extraordinary safety measures when we work in PAMM’s laboratory

Follow-up

We planned several experiments to test the functioning of lysostaphin.

1 / 4

Guided tour around PAMM

2 / 4

Guided tour around PAMM

3 / 4

Guided tour around PAMM

4 / 4

Guided tour around PAMM

❮ ❯

Oct 02, 2018 Skype call with medical doctor Dr. Edgar Peters (VUmc Amsterdam)

To investigate the option of applying our bandage to diabetic wounds, we contacted internist Edgar Peters. Two of his specializations are diabetic feet and infected wounds. Furthermore, he is specialized in infections caused by biomaterials. Perfect for our project!

Oct 02, 2018 Skype call with medical doctor Dr. Edgar Peters (VUmc Amsterdam)

We asked Dr. Peters about his opinion on the feasibility of our project. He believed our project sounded very promising, but he was concerned about regulations which will make its market launch difficult. It would thus be important to demonstrate that E. coli cannot escape the patch. If we achieve this, it would be great to regulate the production and secretion of various bacteriocins through a sensing system. Peters expects that upon the extermination of one bacterial strain, other (malignant or benign) ones may take over. A huge advantage of our GMO-based system would thus be its flexibility to adapt to a changing wound flora. As most current antiseptic bandages are ineffective, this new treatment would be useful.

Insights

S. aureus is the most important infection-causing bacteria on diabetic wounds. Streptococcus comes second, although he has encountered them less frequency lately. Pseudonomas is more important on burn wounds than on diabetic wounds.
Most existing antiseptic bandages are not effective.
It will be important to demonstrate that E. coli cannot escape the bandage in order to conform to regulations.
It is not likely that our plaster will cause inflammation, as it is no part of the body. It may be smart to change the patch on a regularly base, however. Currently, bandages are changed every one or two days. The plaster will probably be required until the wound closes, which may take months.
It is to be expected that once the bandage will have eradicated one type of bacteria growing on the wound, others will take over. Therefore, an integrated sensing system regulating the production and secretion of a range of bacteriocins would be the ultimate solution.
Patients affected by diabetic wounds are usually 60-70 years old, they suffer from peripheral neuropathy, vascular disease and diabetes. From all diabetic patients, 65% will develop neuropathy. 20-25% of those patients will develop wounds on their feet, of which half gets infected.

Follow-up

We will next equip our patch with a bacteriocin against Streptococcus.
We researched options to contain the medium in the patch.

Oct 03, 2018 Visit to ‘Expertisecentrum wondzorg’ (expertise center wound care)

To get more insight into the treatment of wounds, we are not interested in treatments against infections, but also in general wound treatment. We therefore contacted the Expertise Center for Wound care in Oosterhout. We were invited by Dr. Jacques Neyens for a visit.

Oct 03, 2018 Visit to ‘Expertisecentrum wondzorg’ (expertise center wound care)

We were told that there are two types of wounds, and three types of basic ways to distinguish what to do with a wound. The decision about what you need to do with a wound is very simple. It is based on color! If a wound is black, that means that there is necrotic tissue which has to be removed. If the wound is of a yellow color, the wound is not yet clean and there are still some remains of bacteria or tissue left. This means that the wound has to be cleaned, but there is no dead tissue. A red color means that the wound is not contaminated, but still needs to be protected and covered. These are things that we must take into account when designing a patch for wound infections. We were also told that the amount of exudate of a wound is very important. If a wound is too dry, it will not heal. Is there too much fluid? Not optimal either. Treating a dry wound is performed by the addition of a hydrogel to increase the fluid levels of the wound. In that respect, our gel would provide an ideal environment for dry wounds.

After some information about the wound healing process, we asked about wound-related statistics. It turns out that of all wounds, about 80% are acute and will heal on their own without too much care. However, 20% will become chronic and of these 20%, more than half will get infected and thus requires attention. These wounds are estimated to be approximately 100.000 wounds. This means that in the Netherlands, 100.000s wounds annually could benefit from our solution.

Since an antibiotic treatment usually takes around ten days, it would be good to aim at removing the infection in ten days or less with our solution. That way, we can prove that our solution would be better than current treatments.

Insights

Keep in mind that the amount of fluid in a wound is very important.
Also keep in mind that a wound can be in various stages of its healing process.
He saw a lot of potential in solutions that are not directly working towards antibiotic resistance. If we can guarantee that it is safe, it should be a very good solution.
Keep in mind that the condition of the patient also plays a huge role in the healing process.

Oct 04, 2018. Plastic surgeon M.D. Lesley Bouwer

As Koen Lim from Plasmacure had told us that plastic surgeons regularly deal with wound infections, we contacted plastic surgeon Lesley Bouwer. Via e-mail, he answered some questions.

Oct 04, 2018. Plastic surgeon M.D. Lesley Bouwer

Oct 05, 2018. Patient Karel

Via Jacques Neyens (Expertisecentrum Wondzorg) we got in touch with patient Karel. He suffers from diabetic wounds which heal poorly. Karel was so kind to explain his situation and answer some questions.

Oct 05, 2018. Patient Karel

What type of wounds do you have?

Diabetic wounds which heal slowly. One of the wounds on my feet got infected. It took six (!) weeks of systemic antibiotics to get rid of it. This had a great impact on my well-being.

How are your experiences with continuous wound care?

The pain and discomfort caused by the treatments are limited. Except for the consequences of antibiotic treatments.

How much discomfort do your wounds bring you?

A lot, since my wounds are large. Furthermore, they are pretty deep and cleaning them may make cause them to expand.

How long have you been suffering from your wounds?

Since February.

Do your wounds have an impact on your life?

They have a large impact. I can barely walk due to the wounds on my feet. Heel erg veel. Omdat het wonden op de voet zijn kan er nauwelijks op gelopen worden. In February, dead tissue was removed, causing a big hole. After that, my blood circulation reduced, which has been solved by means of a bypass. On August 21st, the wound was cleaned for another time, which has helped the wound to start curing.

How satisfied are you with your current treatment?

Since the wound has started to cure, it has been fine. Every now and then, I am coming for a check-up.

What are your thoughts on the use of living bacteria for the treatment of wound infections?

Considering grubs, living material on wounds is not a bad idea. If it is safe and it works, why not? I think it is better than antibiotics, which cause resistance and a lot of side-effects on the liver and kidneys when used systemically, like in my case.

Insights

The wound care process itself is not too bad. However, the wounds are usually large, resulting in a relatively large infection. This means that it takes a lot of antibiotics and a lot of time to resolve this infection. A large, systematic antibiotic treatment has a large impact on the body. A more local, surface solution would thus be better.
Karel is fine with the idea of using living bacteria as a treatment.

Follow-up

We will do everything we can to prevent systemic side effects of our treatment.
Karel’s positive feedback motivated us to work even harder. 😊 We are glad he is not taken back by the concept of adding living bacteria to the wound.

Conclusions

The discussions with experts and stakeholders were a valuable part of our iGEM trajectory. Thanks to their continuous input, we learned for which applications our living material would be most relevant. This made it much easier to settle on a wound healing patch as an application for Gelcatraz. A further set of interactions with medical doctors, experts on infectious diseases and others helped us to get a clear overview of the problem at hand. This helped us tremendously in the product design process . It was extremely motivating to experience the enthusiasm of medical doctors when we told them about our project. This inspired us to work even harder! These experts also had interesting advice for improvements on our design. As a result of the stakeholder and expert interactions described above, we took several decisions and made adaptations. Summarized:

The choice for dextran as our hydrogel of choice was confirmed by a discussion with Prof. Patricia Dankers.
Input from RIVM experts, Dutch biotechnologists, dr. Sandra Hofmann, and an anonymous dermatologist made us decide on wound healing as our field of application. This decision was further informed by a discussion with ethicist Dr. Lily Frank, during which we realized that a product for our own country is preferred over one aimed at developing countries considering a fair distribution of risks.
We researched the chemical safety of our hydrogel and its synthesis after advice from Lori Goff and risk assessment expert Cécile van der Vlugt. We demonstrated the chemical safety of the final product and proposed some adaptations to improve the future safety of the synthesis.
Thanks to a visit to Plasmacure, we decided to research the requirements for a patch against burn wounds and against diabetic wounds. This resulted in visits to the Maasstad Hospital (burn wound center), Expertise Center Wound Care, and in calls with a patient suffering from chronic wounds and an expert on diabetic wounds and infectious diseases. We learned that besides S. aureus, Streptococci and P. aeruginosa would be our main targets for diabetic wounds and burn wounds, respectively. We designed an additional BioBrick (pyocin S5) to target P. aeruginosa and we selected an existing BioBrick to inhibit Streptococci.
Several stakeholders stressed the importance of safety. Therefore, we designed a kill switch as an additional safety level, based on advice from Prof. Jan van Hest.
The doctors in the Maasstad Hospital and from the VUmc made us realize that a sensing mechanism to regulate our secretion system would be a great asset. We subsequently designed one.
Wound healing professionals emphasized user-friendliness as a design requirement for the plaster. We integrated this wish in the future scenario of our plaster, with a freeze-dryable, sterilely packed plaster ready for use. We will make sure the plaster can stay on the wound for a few days to limit patient suffering, but not longer to minimize the risk of inflammation.
We used PAMM’s expertise and facilities to work safely with S. aureus.

References

Johnson, C. T., Wroe, J. A., Agarwal, R., Martin, K. E., Guldberg, R. E., Donlan, R. M., … García, A. J. (2018). Hydrogel delivery of lysostaphin eliminates orthopedic implant infection by Staphylococcus aureus and supports fracture healing. Proceedings of the National Academy of Sciences of the United States of America, (12), 201801013. https://doi.org/10.1073/pnas.1801013115
Liu, X., Tang, T.-C., Tham, E., Yuk, H., Lin, S., Lu, T. K., & Zhao, X. (2017). Stretchable living materials and devices with hydrogel–elastomer hybrids hosting programmed cells. Proceedings of the National Academy of Sciences, 114(9), 2200–2205. https://doi.org/10.1073/pnas.1618307114

Ethics Discussion

The best that most of us can hope to achieve in physics is simply to misunderstand at a deeper level.

▲

Getting started: Meeting with an ethicist

To examine the ethical implications of our iGEM project, we contacted Dr. Lily Frank. She is an assistant professor of Philosophy and Ethics at Eindhoven University of Technology and is specialized in Biomedical Ethics. With Lily we discussed the potential benefits and dangers of synthetic biology, and she pointed us at some of the most controversial issues related to synthetic biology. For instance, Lily strongly recommended us not to choose an application which must be tested in developing countries. Instead, we should test our product close to home where the required infrastructure for safe testing is available. Furthermore, we learned that the relative novelty of synthetic biology as a field combined with slow politics complicates risk assessment, as many of the potential benefits and risks are still uncertain or have been ignored.

Ethical frameworks

Lily was so kind to suggest some ethical frameworks to get started with our ethical discussion. She particularly recommended frameworks resulting from the European project SYNBIOSAFE (2009) and from work performed by the US Presidential Commission on Bioethics in 2010 (1). As Europeans, we were quite interested in the European view on the SynBio case. However, we found the European framework a collection of potential issues and concepts rather than a framework useful for an ethical analysis. As Lily Frank shared our preference for the American framework, we decided to use this one for our analysis. In the discussion, we also touch upon some points extracted from SYNBIOSAFE.

We realize that some of the positions taken by the US Presidential Commission are themselves debatable. However, we consider it beyond the scope of this ethical analysis to debate the framework. In the next section, we will briefly explain the used framework before applying it to our project Gelcatraz.

Analysis

The American report New Directions: The Ethics of Synthetic Biology and Emerging Technology (2010) was a result of six months of work commissioned by president Obama, in response to the development of the first self-replicating artificial bacterial cell in 2009 (1). For six months, they consulted approximately thirty experts and received written comments which discussed the risks and benefits of synthetic biology. The commission concluded that synthetic biology should be approached with “prudent vigilance”. This means it is not necessary to implement new regulations or even discontinue research in the field of synthetic biology, but that the developments of the field should be continuously monitored. They invite scientists and public policy makers to assess the risks, benefits and moral objections of new projects and their implications, and they strongly advice to engage stakeholders, scientists and the public in this debate. In fact, such evaluation is well captured by iGEM’s Human Practices process.

The commission found five guiding principles for the evaluation of synthetic biology processes and products: public beneficence, responsible stewardship, intellectual freedom and responsibility, democratic deliberation and justice & fairness. Below, we discuss our project in the context of each of these five values.

1. Public beneficence

The value public beneficence is defined as the maximization of public benefits and the minimization of public harm. This principle should be applied to the individual, institutional, community and public levels. The commission advises to be vigilant about harms.

Our entire human practice trajectory was performed with the aim of optimizing public beneficence and minimizing the harmful side effects of our design. For instance, we ensured optimal entrapment of bacteria in the gel, designed a kill switch to minimize the risks associated with bacterial escape, added an additional bacteriocin to our secretion system to eradicate additional malignant bacteria and researched the options for replacement of toxic chemicals during the synthesis of the hydrogel. This process is documented on the safe-by-design page. The final bandage resulted from input from stakeholders and expert on our designs. This process was further supported by a stakeholder-value analysis we performed based on the iGEM'ers Guide to the Future.

We believe that our product benefits individuals as well as society, by improving patients’ quality of life, improving public health, lowering the prevalence of antibiotics resistance and eventually reducing health care costs. These benefits are extensively explained on the applied design page. For instance, we enabled the secretion of active lysostaphin by E. coli. This eliminates the need for expensive laboratory equipment other than the standard equipment available in modest laboratories for the production and purification of lysostaphin. This will allow scientists with all budgets to produce lysostaphin of high purity. Currently, drugs sold in developing countries are often of lower purity than drugs used in developed countries (2). Hopefully, patients in developing as well as developed countries will benefit from a treatment with highly pure lysostaphin.

We identified the main risk introduced by our design as the escape of GMOs into the environment. We however believe that we minimize this risk using a proper adhesin. We consider the addition of a kill switch to our system in the future. This will add an additional layer of safety to Gelcatraz. However, it will make the system more expensive. This may not be desirable as it may prevent scientists with a limited budget from using our platform. In contrast, without the kill switch, the low costs of our system and its user-friendliness make Gelcatraz accessible to any scientist with basic equipment.

2. Responsible stewardship

The committee believes that scientists involved with SynBio must think and act for the advancement of all, thus also for those who cannot talk. Examples of the latter are nonhuman species, the environment and future generations. Challenges recognized by the commission are firstly our incomplete and uncertain understanding of the potential risks and benefits of synthetic biology, and secondly the risk of intentional misuse of SynBio technologies. Once again, the commission stresses the importance of prudent vigilance here, rather than being action-oriented or extremely cautious.

Insofar as we can judge with our current knowledge, the plaster as we designed it seems to globally advance people. Patients suffering from chronic wounds or burn wounds will benefit from the plaster, and so will other people as our design diminishes the burden of antibiotic resistance. It may be argued that animals also benefit from the latter, as they also suffer from bacterial infections. Moreover, the plaster could replace current antibiotics administered to livestock. Things get more complicated when we consider Gelcatraz as a platform for GMO use outside of the laboratory. By making working with GMOs safer and more convenient, we may accelerate the use of GMOs in regulated environments as well as in less controlled environments. The desirability of the widespread use of GMOs could be the topic of a separate ethical discussion and is beyond the scope of this analysis. Nevertheless, we should only launch our system for use outside of laboratories if the safety of our device can be demonstrated and regulations should ensure that only GMOs classified as ‘low-risk’ are allowed. When people use Gelcatraz within a laboratory environment, they do not need special equipment to work safely with the platform.

Another reason why prudent vigilance would be important comes from the dealing with novel technologies which have not yet been thoroughly tested. Interestingly, SYNBIOSAFE highlighted the need for new risk assessment methods in three cases: a) when larger DNA-based circuits are built based on individual ‘parts’, b) when new minimal organisms are designed and c) when alternative biochemical structures are involved (3). The first case is generally applicable to iGEM with its emphasis on BioBricking. Although we do build new circuits, their complexity remains limited. This means that the functioning of the circuits should remain predictable, limiting the additional risks involved. We also tested our constructs thoroughly. The latter two cases are not directly related to our project, but the third one may be if we implement an artificial amino acid-based kill switch. Although we would implement the kill switch to enhance the safety of our design, we should be aware of the level of unpredictability added by such a novel biochemistry. The authors do not elaborate on the type of risk assessment methods they envision. However, the need for new risk assessment methods means that we must take a vigilant stance during the design process.

3. Intellectual freedom and responsibility

Here, the risk of malevolent ‘dual-use’ should be recognized. That is, the abuse of SynBio technologies for dubious cases. The commission does not believe that this risk is high enough to justify the limitation of intellectual freedom. Instead, it calls for “regulatory parsimony”: no more surveillance than strictly required to ensure justice, fairness, security, and safety, as this may hamper progress of science and technology and may even prevent the development of safeguards. SYNBIOSAFE is more careful with respect to dual-use. They believe that the risk of intentional misuse has been neglected in previous discussions.

In this context, it is problematic that we cannot control the type of applications that we will facilitate with Gelcatraz. This means that we may potentially facilitate the use of GMO for harmful ends. However, the question is whether people using GMOs for dark purposes will benefit from our design, as we designed Gelcatraz with an emphasis on safety. People aiming to abuse GMOs probably do not care much about safety and may therefore not turn to our platform.

SYNBIOSAFE also addresses the possible diffusion of synthetic biology to amateur biologists. Considering that Gelcatraz has been designed not only for safety but also for convenience, it may offer an interesting platform to amateurs. This may be worrisome if amateurs are not aware or do not care about appropriate safety measures. We therefore agree with SYNBIOSAFE’s call for regulation and training in biosafety techniques. However, our platform has been designed as a platform for the use of already manipulated and tested bacteria. This means that only ‘amateurs’ capable of the initial steps needed to, for instance, equip their bacteria with the required adhesin. We therefore do not believe that our platform will attract fully inexperienced users.

4. Democratic deliberation

The presidential commission stresses the importance of an ongoing public debate and the search for common grounds on a topic so controversial as synthetic biology. The commission argues that decisions need not to be binding, as new information may challenge those decisions. Instead, the deliberative process should be ongoing. However, the commission seems to assume that the general public knows enough about synthetic biology to formulate an opinion. We think that often, the required knowledge is missing. Therefore, it may be good to inform people about the field of synthetic biology before asking them for their opinion. It is important that the provided education is objective. We believe that the democratic deliberation process is very much encouraged by the iGEM competition in the form of education and outreach, and feel that it is reflected by our outreach efforts.

5. Justice and fairness

The commission calls for a fair distribution of benefits, burdens and risks across society and globally. We opted for an application which is relevant for people all across the globe, as Staphylococcus aureus is everywhere (4). To fairly distribute the risks, Gelcatraz as a platform and the resulting wound plaster must be extensively tested in the Netherlands before making it available to other countries. That will ensure that (clinical) tests are performed in a setting with all the required safety precautions. Once Gelcatraz is considered safe to use, it can be adopted by anyone with access to a laboratory with standard equipment. As no special equipment is required for the safe use of Gelcatraz, scientists with all budgets may benefit from its use. As discussed above, patients will globally benefit from lysostaphin secretion and its integration in a wound healing plaster.

Although the main benefits of our design will be enjoyed by a certain group of patients, the entire (Dutch) society and even world will benefit from reduced antibiotics resistance. Escape of bacteria, although unlikely, would mainly affect patients and their surroundings. The effects would however be minimal thanks to the several layers of security (weakened, benign bacterial strain unable of survival outside of the hydrogel). From the safety analysis performed for the safe-by-design challenge, it can be concluded that people exposed to our platform for their jobs (e.g. medical doctors, nurses, people working in waste processing or manufacturing) will not suffer from this.

Conclusions

In this ethical analysis, we reflected on our Gelcatraz design with respect to five values: public beneficence, responsible stewardship, intellectual freedom and responsibility, democratic deliberation and justice & fairness. We conclude that our product benefits individuals as well as society, and that its benefits and burdens are well-distributed. We identified some issues that need to be addressed, such as the use of Gelcatraz by amateurs and the yet unidentified risks introduced by synthetic biochemistries. However, we believe that these risks are mostly covered by the several safety levels we integrated in the design.

References

Gutmann A. Synthetic Biology : Emerging Technologies. Hastings Cent Rep. 2011;41(4):17–22.
Caudron JM, Ford N, Henkens M, Macé C, Kiddle-Monroe R, Pinel J. Substandard medicines in resource-poor settings: A problem that can no longer be ignored. Trop Med Int Heal. 2008;13(8):1062–72.
Schmidt M, Ganguli-Mitra A, Torgersen H, Kelle A, Deplazes A, Biller-Andorno N. A priority paper for the societal and ethical aspects of synthetic biology. Syst Synth Biol. 2009;3(1):3–7.
Rossolini GM, Arena F, Pecile P, Pollini S. Update on the antibiotic resistance crisis. Curr Opin Pharmacol [Internet]. 2014;18:56–60. Available from: http://dx.doi.org/10.1016/j.coph.2014.09.006

Safe-by-Design

The Dutch National Institute for Public Health and the Environment (RIVM) challenged us to take part in their safe-by-design challenge. This challenge was about integrating safety into different phases of our project. To get started, we wrote a proposal which we discussed with RIVM experts over Skype. We got to realize that safety goes beyond technical issues and that it is important to consider safety from the initial ideation stage to the final implementation.

For this project, we broke safety down into eight different issues, which we had recognized with the help of some stakeholders and experts (see our integrated practices). Once we had identified the main issues, we consulted more experts and stakeholders to think of possible solutions. An overview of our Safe-by-Design trajectory can be found in the infographic below. The details are provided underneath.

The best that most of us can hope to achieve in physics is simply to misunderstand at a deeper level.

▲

Design trajectory

Start of the trajectory

Experts: Korienke Smit and Niek Savelkoul (RIVM)

We wrote a proposal in which we outlined our safety plans. In a subsequent Skype call, we received feedback on our proposal from two RIVM experts. Their main feedback was that we should only discuss safety in the context of our own project, rather than focusing on general safety-related topics.

Bacterial escape

Problem: We need a safe method for working with E. coli which prevents its escape into the environment.

Experts: Prof. Jan van Hest, Cécile van der Vlugt (RIVM)

Solution: Our hydrogel-adhesin system would prevent leakage. Incorporation of dependence on an artificial amino acid ensures that E. coli would not be able to survive outside the gel.

More information

As we described on the Product Design page, the risk of environmental escape limits the use of genetically modified organisms. Environmental escape not only is a risk that should be avoided out of public and environmental safety reasons, but also contributes to public fear of GMOs (1). We consulted several RIVM experts who confirmed that escape risk is a limiting factor in the advancement of GMO technologies. Environmental safety threats related to GMO escape includes the damaging of ecosystems, the risk of horizontal gene transfer and effects on biodiversity (2).

Gelcatraz has been designed with the intention of minimizing escape risk. We aim to provide a device which makes working with GMOs inside and outside of the laboratory more convenient and safer. Our adhesion system prevents bacterial leakers by securely anchoring E. coli to our device. Experimental results indicate that our adhesin decreases leakage by several orders of magnitude. Although leakage is low thanks to the adhesin, it is not yet zero. To minimize the consequences of a leakage event, we designed a kill switch for our E. coli.

Antibiotics reduction

Problem: The use of antibiotics in R&D does not pose a major threat, but antibiotic resistance in general is an urgent problem requiring alternative solutions.

Experts: Prof. Van Hest, Plasmacure, PAMM, Maasstad Hospital, Cécile van der Vlugt (RIVM)

Solution: Secretion of bacteriocins as an alternative to antibiotics

More information

To research the problem of antibiotic resistance and the need for antibiotics reduction, we approached several experts. All of them highlighted the urgency of the problem. Plasmacure as well as the doctors in the Maasstad Hospital explained that S. aureus and P. aeruginosa are increasingly becoming resistant against commonly used antibiotics. This makes treatment more difficult and even puts patients’ lives at risk. We also learned that the use of systemic, unspecific antibiotics contributes to the development of antibiotic resistance, as described under Product Design .

To contribute to a solution for the treatment of antibiotic resistant infections, we designed our living material to secrete bacteriocins as an alternative to antibiotics. Details on their functioning can be found here. Immunity to lysostaphin has not been commonly observed, but it possible (11). We aim to reduce this risk by integrating a quorum sensing system in the design. This way, the relevant bacteriocins will only be secreted in the presence of S. aureus and/or P. aeruginosa.

Cécile van der Vlugt, risk assessment expert at the RIVM, told us that the use of antibiotics in research and development is not an urgent problem, as the quantities used are low, and the types of antibiotics use are generally basic ones. For those antibiotics, many alternatives still exist in the unlikely case of resistance developed due to research work. Nevertheless, we asked professor Van Hest about the possibilities of replacing the commonly used antibiotic resistances with a dependence on artificial amino acids. He said that, albeit not yet used, dependence on artificial amino acids as a selection mechanism may be even more reliable than antibiotic resistance. The reason is that bacteria tend to get rid of genes that are temporarily unused, e.g. in the absence of antibiotics. As genes related to artificial amino acid dependence generally encode for essential proteins, they do not run this risk.

In conclusion, our project offers a solution to the treatment of infections caused by antibiotic resistant bacteria. We already reflected on ways to prevent resistance against our own bacteriocins. Finally, we explored how we as scientists may limit the use of antibiotics in R&D to prevent the development of additional resistance.

Working with Staphylococcus aureus

Problem: S. aureus is a pathogenic BSL2 species. How do we prevent unsafe situations for team members while testing our bacteriocin?

Experts: PAMM

Solution: Collaborate with PAMM to use their well-equipped laboratories and expertise on working with these dangerous bacteria.

More information

Staphylococcus aureus is a pathogenic species. It is one of the most important infections-causing bacteria in humans and it is easily transmitted through human contact and via the air (3,4). The pathogenicity of S. aureus requires us to take preventive measures before handling it. As we did not have access to the ML2 lab facilities required for use of S. aureus, we contacted our partner PAMM for help. They performed the experiments with S. aureus for us. As a recognized diagnostic laboratory, they work with S. aureus on a regular base and know which precautions to take.

Good Manufacturing Practices

Problem: Our hydrogel production requires toxic catalysts and yields a hazardous intermediate product. How to ensure a save synthesis and polymerization process and prevent the presence of toxic intermediates in the final product? Furthermore, chemical waste should be minimized.

Experts: Cécile van der Vlugt (RIVM), Lori Goff

Solution: Extensive dialysis of methacrylated dextran removes all toxic components. The use of a novel, radiation-based crosslinking method and alternative catalysts could reduce the toxicity of the synthesis and polymerization. The gel is biodegradable.

More information

Our hydrogel production requires toxic catalysts and yields a hazardous intermediate product. How do we ensure a safe synthesis and polymerization process and prevent the presence of toxic intermediates in the final product? Good Manufacturing Practices (GMP) are regulations which ensure the safety and quality of food and medicine. As part of GMP, it is thus important to protect the chemical safety of Gelcatraz and the safety of its production process. Furthermore, chemical waste should be minimized, and the costs should preferably be low. Below, we provide the results from our research into these topics. In short, our research revealed that all toxic components are removed by extensive dialysis of the methacrylated dextran. Furthermore, a leakage test showed that no leakage occurs from the gels over a period of two weeks. The use of a novel, radiation-based crosslinking method and alternative catalysts could reduce the toxicity of the synthesis and polymerization reactions. The gel is biodegradable.

Toxic chemicals and intermediates

Dextran itself is biocompatible and safe, but its synthesis and polymerization require several toxic chemicals. Furthermore, some toxic intermediates are produced. While handling these chemicals, we take all required precautions such as working in the fume hood and wearing protective gloves. However, it would be even better to replace them by less toxic equivalents. We researched replacement options for two steps: the synthesis of methacrylated dextran and polymerization of methacrylated dextran into gels.

Synthesis of methacrylated dextran

For the synthesis of the gel, we use some toxic chemicals, the most important being glycidyl methacrylate (GMA) and 4-dimethylaminopyridine (DMAP). GMA is carcinogenic but required to functionalize the hydroxyl groups of dextran. We handle it with syringes to avoid exposure. Furthermore, we remove all traces of GMA after the synthesis and confirmed this with H-NMR. The final product is thus completely safe. DMAP is a base that acts as a catalyst for the coupling of GMA to the dextran molecules. It is toxic if swallowed and fatal in contact with skin. In the absence of a catalyst, no GMA incorporation could be detected (5). However, we researched alternatives to catalyse the reaction. Triethylamine and pyridine are no reasonable alternatives due to the associated high conversion times (5). Another alternative for DMAP could be tri-ethylenediamine, also known as DABCO. DABCO is like DMAP an organic base and therefore it can theoretically catalyse the methacrylation reaction. However, unlike DMAP, DABCO possesses less toxic properties and DABCO is much safer to handle than DMAP. DABCO is from an economical point of view also a better choice as its commercially available for a lower price. A downside to using DABCO as a catalyst for this methacrylation reaction is that it is less basic than DMAP (6). As the deprotonation of hydroxyl groups by a base is the driving force for the reaction, the reaction time will most likely increase when DABCO is employed as a catalyst instead of DMAP. A comparison of the performance of DABCO in our reaction is required to make an informed choice between DMAP and DABCO.

Methacrylation of the non-hazardous dextran results in molecules with very toxic and carcinogenic properties due to the reactive methacryl groups (7). No material safety data sheet exist for methacrylated dextran. To safely handle this toxic viscous oil, it is kept in the fume hood at all times and only handled using piston pipettes or syringes. To purify the methacrylated dextran from the organic mixture, we dialyse it against demineralized water. To verify the complete removal of the DMSO, DMAP and GMA, the 1H nuclear magnetic resonance spectrum of the dialyzed reaction product was compared with the spectra of pure DMAP and GMA. After 72 hours of dialysis, trace amounts of unidentified by-products could be observed in the NMR spectrum. After one week of dialysis, no more trace amounts of any reactants or byproducts can be observed. The obtained spectrum matches with the one reported in the literature (8). The spectrum of purified dextran can be seen in the hydrogel results.

Polymerization of methacrylated dextran

To form the hydrogel, the methacrylated dextran polymers are crosslinked by radical polymerisation in water. The polymerisation is initiated by tetramethylethylenediamine (TEMED) and ammonium persulfate (APS). TEMED catalyses the homolytic scission of APS to generate radicals which crosslink the methacrylate groups, resulting in the formation of a hydrogel. APS and TEMED are both flammable and irritable. Because TEMED is a catalyst, we only work with small amounts of the substance (10 microliter at a time) using pipets, reducing the risks associated with this compound. TEMED is vital for the liberation of radicals from APS. In the absence of TEMED, polymerisation at room temperature by solely APS is slow, so at -20°C the polymerisation would be even slower (5).

Recently, an alternative approach to crosslinking methacrylated dextran polymers into a hydrogel is has been developed, using radiation instead of the APS TEMED system. This approach circumvents the use of toxic crosslinkers. If the methacrylated dextran monomers are pure, the crosslinked hydrogel is devoid of unreacted small molecules and no further purification is necessary. Additionally, if a high enough radiation dose is applied, typically 25 kGy, the gel is sterilized simultaneously allowing immediate seeding. Crosslinking by radiation is therefore a very promising option for dextran hydrogel synthesis on an industrial scale (7).

Hydrogel leakage test

1H nuclear magnetic resonance spectroscopy has been used to verify that during storage in demineralized water, no dextran or other starting products leak from the hydrogel. Trace amounts of organic residue were detected but could not be assigned. Besides the solvent peak, there is no overlap between the spectra of DMAP, GMA and that of the storage water as can be seen below.

Figure 1 The H-NMR spectra of DMAP (top), GMA (middle) and the storage water (bottom) do not show any overlap. This shows that the final dextran gel does not contain any toxic DMAP or GMA.

Waste production

Hydrogel synthesis

Only aqueous waste is produced during the synthesis of methacrylated dextran and the polymerisation into a hydrogel. Aqueous waste is water with small amounts of organic molecules dissolved in it. In our case these are organic molecules are the solvent DMSO, DMAP, unreacted GMA and methacrylated dextran, NaOH, HCl, synthesis byproducts and byproducts formed during the radical polymerisation (including unreacted methacrylated dextran). None of the waste products can be recycled due to dilution or contamination with other compounds.

Especially during dialysis, a large amount of aqueous waste is generated. Approximately 50 liters of aqueous waste is produced for 80 mL of hydrogel. The amount of waste can be drastically reduced by changing using a smaller dialysis volume. We used a dialysis volume of 4 litres to accelerate the dialysis. If a smaller volume would be used and equilibrium between the dialysis bag and the medium is established each time, we expect that we can halve the amount of waste, at the cost of a longer purification duration.

After use

The dextran-GMA hydrogel is biodegradable and is therefore safe to use in ecological environments. The bacteria are equipped with a kill switch which will also kill the bacteria after the patch has served its function and is thrown away. In this fashion waste production is kept at a minimum.

Costs

We are using a dextran hydrogel for our system. Medical-grade dextran is expensive. However, the production of one hydrogel only requires small quantities (100 μg). Therefore, the estimated dextran costs for making one 400 μL hydrogel in patch form is around €0,65. Adding other reagents will bring the total costs to approximately €1,00 for one hydrogel in small scale conditions. These costs can be optimized by upscaling. Modifying the bacteria is a one-time expenditure. Afterwards they can be cultured and re-used for a long period. Culturing the bacteria can be performed on a large scale. Costs of medium are very low. Overall, our living material can be produced very cost-efficiently and upscaling would further reduce cost. Before seeding our hydrogel with bacteria, they can be modified for expression of different proteins. By also culturing these bacteria that express infection-reducing enzymes, patches that fight infections can be created by a very modest increase in costs.

Conclusion

Although we use toxic chemicals for the synthesis of the hydrogel, they are removed by extensive dialysis of the methacrylated dextran resulting in a biocompatible end-result. Furthermore, a leakage test showed that no leakage occurs from the gels over a period of two weeks. This means that the chemicals do not yield any risk for users of the bandage. The use of a novel, radiation-based crosslinking method and alternative catalysts could reduce the toxicity of the synthesis and polymerization reactions, which would be beneficial for manufacturers. The gel is biodegradable, and its production is relatively cheap.

Patient safety

Problem: We should not only cure patients, but also avoid their infection with E. coli and inflammation of the patch.

Experts: Plasmacure, Maasstad Hospital , Dr. Edgar Peters, Dr. Jacques Neyens, M.D. Lesley Bouwer

Solution: Dextran is biocompatible, the used bacteriocins seem safe to humans (clinical trials still required). Our E. coli strain is harmless.

More information

To address this issue, we consulted several experts from the field of wound healing, diabetic wounds and burn wounds. The enzymes that will be secreted by our patch need to be safe in human systems. Lysostaphin is one of these enzymes. Lysostaphin efficacy has been extensively tested in murine and rabbit models, confirming its effectiveness against S. aureus causing several types of infections (e.g. nasal, orthopaedic implants, abscess lesions, aortic valve endocarditis) (9–11). In those cases, lysostaphin treatment was reported to be non-toxic (11). Clinical studies must be performed to confirm these statements. Therefore, clinical trials are necessary to show that lysostaphin causes no harm when it enters the body through the wound. Although most bacteriocins have no effect on human cells in low concentrations, less is known about higher concentrations. Besides lysostaphin, our E. coli will produce pyocin, a bacteriocin against Pseudomonas aeruginosa. Pyocin has been shown to be effective against P. aeruginosa lung infections in mice, with its required dose 100-fold lower than the commonly used antibiotic tobramycin (12). It still needs to be determined whether it is safe in humans, but there is no indication it will be.

The hydrogel that we use is biocompatible and has been used in humans before. However, GMA that is necessary for the synthesis is highly toxic and carcinogenic. Dialyzing the methacrylated dextran product for one week ensures complete removal of GMA. 1H nuclear magnetic resonance spectroscopy has been used to verify that during storage in demineralized water, no dextran or other starting products leak from the hydrogel . Therefore we know it is safe to use for an application on the skin. The trace amounts of organic material that do leak out could not be assigned to any known reagents or (by)products but it was collected from the 50 mL of storage water. This means that the presence of organic materials in the storage water can be neglected as their concentration is extremely low.

The strains of E. coli (BL21(DE3) and BLR) that we use do not present a direct threat to human health, plants or microorganisms, since it is an biological agent of category 1 (13–15). This means that it is very unlikely that they are harmful to humans. Besides the bacteriocins that are secreted, E. coli has been modified to express a number of different proteins. As mentioned above, these proteins are harmless.

In conclusion, to make our gel user friendly and safe, we used a hydrogel that is not hazardous and is safe when in contact with living organisms. Our E. coli strain is harmless. However, it must still be confirmed that the used bacteriocins are safe to humans. To this date there is no indication that it the bacteriocins would be any more hazardous than conventional antibiotics.

Occupational safety and health

Problem: We must make sure that medical professionals work safely with the plaster.

Experts: Maasstad Hospital , Dr. Edgar Peters

Solution: The plaster will be convenient to apply. The gel is biocompatible, free of toxins and E. coli is non-malignant.

More information

Good working conditions are conditions which ensure that people can work healthily and safely (6). Therefore, it is important to make an inventory of the possible existing hazards. Thinking about our “hydrogel patch”, possible hazards are the dextran material, the diffusion of lysostaphin and pyocin and the bacteria that are inside the gel. As we discussed above under patient safety, both of theseboth molecules seem safe to humans. The same applies to the E. coli strain used. Obviously, it remains important to wash hands before and after handling the patch or touching the wound.

Moreover, to work healthily and safely, is it also important that the gel can be applied easily. Therefore, it should not require much physical work and should not be harmful for the nurses. It would be beneficial if the plaster looks normal, something that feels familiar to the patient and nurse. It should not give them the idea that there are living modified bacteria inside the plaster itself to prevent scaring patients or medical professionals.

From our conversations with medical professionals, we learned that the plaster will need to be replaced regularly. To prevent pain or inconvenience to the patient or nurse, the plaster must thus be easy to apply and to remove. As the plaster will be delivered in a sterile package ready for application, the application should be straightforward and easy. This requirement is thus met. Overall, it can be concluded that our plaster will not make the work of nurses or doctors any more difficult or dangerous than it currently is.

Infiltration

Problem: We want to prevent foreign bacteria from invading the gel to avoid them from competing with E. coli for resources.

Experts: Dr. Sandra Hoffman, iGEM Düsseldorf 2018, PAMM, Dr. Edgar Peters

Solution: The risk of infiltration is relatively low thanks to the secreted bacteriocins. We may lower the risk by means of a quorum sensing system and the regular replacement of the plaster.

More information

Several experts and stakeholders asked us whether the risk exists that foreign bacteria infiltrate Gelcatraz. Infiltration would form a serious safety hazard. What if malignant bacteria colonize the material and use it to secrete toxins to patients? We thus researched this issue and consulted several experts on the topic.

In principle, we do not think that the risk of infiltration is high, as ‘our’ E. coli bacteria secrete toxins. This does not make the material an attractive environment for foreign bacteria, or at least the targeted ones. Once we will have incorporated a broad spectrum of toxins to be secreted on demand, the risk of infiltration by malignant bacteria will be minimal.

What remains, is the risk of non-targeted bacteria infiltrating the gel. Think of benign bacteria present on our skin, which will be attracted by the nutrient-rich environment of the gel. Although this will not pose a safety-hazard, it would be problematic when they outcompete E. coli and consume the nutrients. It is, however, more likely that E. coli remain the dominant species, as they are present in large amounts. As foreign bacteria lack the binding affinity for Dextran that our E. coli have, this will only happen when the gel forms a sufficiently attractive environment and when they can live together symbiotically with E. coli. This is not necessarily a bad thing, such as in the case of mutualism or commensalism. Whether this happens, will need to be tested experimentally. In case it happens, we researched some preventive measures.

Changing the plaster

A simple measure may be frequent replacement of the plaster. This would ensure fresh nutrients and fresh E. coli bacteria. A disadvantage of this is inconvenience for the patient, which was highlighted by doctors from the Maasstad Hospital. Clinical studies will need to show the optimal treatment duration and replacement frequency.

Quorum Sensing

Bacteria use quorum sensing to regulate activities on a larger scale. This is mostly intraspecies but the molecule AI-2 (Autoinducer 2) has been suggested to be a universal quorum sensing molecule, allowing inter-species communication. Production of this molecule has been found in a wide range of both gram-positive and gram-negative species, by means of the protein LuxS (11). By implementing a luxS knockout in our own bacteria, combined with an AI-sensing gene we can induce production of bactericides upon invasion of unwanted bacteria into our living material. A similar mechanism has also been attempted by NTU-Singapore 2008 iGEM team.

Silver coating

Many metals have antibacterial properties (oligodynamic effect), but silver is the most effective and the least toxic to humans (16). It is commonly used in medical treatments. Silver ion release from silver composites has also been presented as an effect antimicrobial method (17).

However, silver has both an effect on S. aureus and E. coli. Gram-negative bacteria such as E.coli suffer more structural damage from silver than Gram-positive bacteria like S. aureus (18).

Overcrowding

Problem: We should prevent the material from E. coli overcrowding.

Experts: iGEM Düsseldorf 2018 (see collaborations page )

Solution: The risk of overcrowding is low, as E. coli uses most of its resources for protein production. However, we could use a quorum sensing system such as the construct developed by iGEM Düsseldorf 2018.

More information

During the European meetup in Munich we explained iGEM Düsseldorf how we want to make a living material. They recognized the problem of bacterial overcrowding within our construct. Since our bacteria will keep dividing and cannot escape the hydrogel, at some point bacteria will be over-saturated which can lead to problems in protein expression or even dead. They offered to help us, and we had a meeting with them over skype. They were making a construct that uses the Lux Quorum sensing. Their construct contained 3 plasmids: Luxl (synthase for AHL), LuxR (molecule where AHL binds and can then bind to promotor) and pLux (promotor to which LuxR +AHL can bind). When the promoter gets activated, a phage lysis gene will be synthesized. In our case they offered to send us the plasmids without the Luxl in the construct, so when we add AHL to the hydrogel medium, cell growth will be inhibited. This is a great solution to keep our bacteria in control, as we want them to stay in steady numbers within our hydrogel, with steady lysostaphin production.

Conclusion

The concept of Safe-by-Design helped us to prioritize safety during all stages of the project. Thanks to many interactions with stakeholders, we recognized potential problems early on and found creative solutions. We believe that this resulted in a living material and wound healing bandage which will be safe for scientists, patients, medical professionals and the environment. Extensive (clinical) tests will need to verify this. We want to thank all stakeholders that helped us during the process. Without your help, we could not have considered as many aspects of safety!

References

Laros FJM, Steenkamp JEM. Importance of Fear in the Case of Genetically Modified Food. 21(November 2004):889–908.
Lövei GL. Ecological Impacts of GMOs ECOLOGICAL RISKS AND BENEFITS OF TRANSGENIC PLANTS. 2001;1999(James):93–100.
Minnesota Department of Health. Staphylococcus aureus: Fact sheet. 2010.
Dutch National Institute for Public Health and the Environment. Staphylococcus aureus-infecties inclusief MRSA [Internet]. 2014. Available from: https://lci.rivm.nl/richtlijnen/staphylococcus-aureus-infecties-inclusief-mrsa#preventie
Franssen O, Talsma H, Steenbergen MJ Van, Bosch JJK Den, Hennink WE. Synthesis , Characterization , and Polymerization of Glycidyl Methacrylate Derivatized Dextran Results and Discussion Synthesis of Glycidyl Methacrylate Derivatized Dextran . The synthesis of dextran derivatized with Vh-h. 1995;6317–22.
Baidya M, Kobayashi S, Brotzel F, Schmidhammer U, Riedle E, Mayr H. DABCO and DMAP — Why Are They Different in Organocatalysis ?. 2007;6176–9.
Szafulera K, Wach RA, Olejnik AK, Rosiak JM, Ulański P. Radiation synthesis of biocompatible hydrogels of dextran methacrylate. Radiat Phys Chem. 2018;142(October 2016):115–20.
Plieva F, Oknianska A, Degerman E, Galaev IY, Mattiasson B. Journal of Biomaterials Science , Novel supermacroporous dextran gels. 2012;(November):37–41.
Johnson CT, Wroe JA, Agarwal R, Martin KE, Guldberg RE, Donlan RM, et al. Hydrogel delivery of lysostaphin eliminates orthopedic implant infection by Staphylococcus aureus and supports fracture healing. Proc Natl Acad Sci U S A [Internet]. 2018;(12):201801013. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29760099
Kokai-kun JF, Walsh SM, Chanturiya T, Mond JJ. Lysostaphin Cream Eradicates Staphylococcus aureus Nasal Colonization. Microbiology. 2003;47(5):1589–97.
Bastos M do C de F, Coutinho BG, Coelho MLV. Lysostaphin: A staphylococcal bacteriolysin with potential clinical applications. Pharmaceuticals. 2010;3(4):1139–61.
McCaughey LC, Ritchie ND, Douce GR, Evans TJ, Walker D. Efficacy of species-specific protein antibiotics in a murine model of acute Pseudomonas aeruginosa lung infection. Sci Rep [Internet]. 2016;6(June):1–8. ,Available from: http://dx.doi.org/10.1038/srep30201
New England BioLabs. Safety Data Sheet: BL21(DE3) Competent E.coli. 2016. p. 1–9.
Ministry of Social Affairs and Employment. Wetgeving biologische agentia [Internet]. [cited 2018 Oct 3]. Available from: https://www.arboportaal.nl/onderwerpen/wetgeving-biologische-agentia
Merck. BLR(DE3) Competent Cells - Novagen [Internet]. Available from: https://www.merckmillipore.com/NL/en/product/BLRDE3-Competent-Cells-Novagen,EMD_BIO-69053
Guggenbichler JP, Böswald M, Lugauer S, Krall T. A new technology of microdispersed silver in polyurethane induces antimicrobial activity in central venous catheters. Infection. 1999;27(SUPPL. 1).
Kumar R, Helmut M. Silver ion release from antimicrobial polyamide / silver composites. 2005;26:2081–8.
Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO. A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J Biomed Mater Res. 2000;52(4):662–8.

Stakeholder-value analysis

The best that most of us can hope to achieve in physics is simply to misunderstand at a deeper level.

▲

Introduction

As we wanted to improve our project for the people who would be working, handling or get in contact with our product, we performed a stakeholder-value analysis. This analysis was inspired by the iGEM'ers Guide to the Future. We started with a brainstorm session during which we settled on the relevant stakeholders and values. This gave us a good overview of who we wanted to talk to and what we wanted to discuss with them. Inspired by interactions with stakeholders and readings, team members filled the matrix with post-it notes over the course of the summer. Afterwards, we analyzed the findings. We will shortly discuss the most important beneficial aspects of our project to the stakeholders and the main problems to overcome in the stakeholders’ interests.

The matrix

What our matrix looked like after the summer:

Stakeholder matrix on the wall of our office after summer.

Stakeholder matrix
	Safety	Security	Responsibility	User-friendliness	Cost-efficiency	sustainability	Health	Practicality
Patients	Chemical toxicity		Use patch as intended	Painless comfort. Patch stays on wound (reduced pain)	Efficiency	Local treatment	Lysostaphin has no known side effects. Better healing. Bacterial infiltration	How often it is replaced
Scientists/iGEM			Go over all safety conditions		Cheap and easy	Washable. Reusable	Safe handling of chemicals.
Physicians	Specific treatment (prevents resistance)		Right treatment for certain patients	Integrate sensor for tailored treatment			New treatment much needed. Fighting resistant bacteria	Availability of patch through freeze drying
Hospital	Bacterial invasion/infiltration			Freeze drying	Cheaper than existing solutions	Reusable	Reduced risk of bacterial infection
Nurses	chemical toxicity			Easy to apply			Bacterial container ➔ safe handling	Easy application ➔ fewer changes
Insurance companies	Damage costs				Cheap compared to other treatments
Society		Prevent antibiotic resistance	Accepting the use of gmo’s in environment			Reduced use of specific systemic antibiotics
Government	Make safe regulations regarding gmo’s		Legislation		Reduced health care costs		Prevention of resistance against antibiotics
Manufacturers	GMP up to date		Test functionality of manufactured device	Freeze drying	Reusable catalyst dialysis time and volume scalable	Non-recyclable product	Safe production process	Scalable
Environment	No bacterial escape. Used bacteria are barely antibiotic resistant	Kill switch				No toxic chemicals in the final gel

Discussion

Patients

The end-users, the patients, should have the most comfortable and easy experience using our patch for their wounds. Since it is a gel-like structure, treatment will be painless and continuous, reducing stress and effort to get the treatment. Placement of the patch can take place at home and replacement should be done less often compared to band aid. This not only reduces stress, inconvenience and pain as we learned from the Maasstad burn wound center , but fewer actions will also reduce costs. As developers of the patch it is also our task to make it chemically safe. The chemicals secreted by the bacteria in the patch are harmless to humans and have no known side-effects, but the chemicals used to produce the gel are very toxic on their own. To reduce the risk of these compounds, our gel is washed several times after synthesizing to remove those chemicals. However the possibility is also there to replace them with less toxic chemicals. Overall, patient safety seems to be guaranteed. The issues of patient safety and chemical toxicity are discussed under safe-by-design.

Scientists/iGEM

We, the scientists working on the wound healing patch, have the main responsibility to make the product as safe as possible for every stakeholder. The safety aspect for us or other scientists working on this patch is again safely handling the chemicals used. This means taking the necessary safety precautions like wearing gloves or work in a fumehood etc. Decreasing toxicity of the chemicals by replacing them with less dangerous compounds will also greatly help with safely working on this patch.

Physicians

Physicians have the general responsibility to get people the best treatment possible. Our wound healing patch helps physicians provide the best treatment, since it our patch is specific for long lasting, infected wounds. The specificity of the bacteria that are killed by the patch has the advantage of reducing chances of developing resistances among a lot of other bacteria on the skin. Resistant bacteria are already a problem in infected wounds, as emphasized to us by the medical doctors we interacted with. Another advantage of the patch for physicians is the fact that it can be freeze-dried and thus stored effectively for a long time and is ready off-the-shelf. This makes prescriptions and availability easier.

Hospitals

Hospitals have similar interests as physicians: fighting resistant bacteria via the reduced use of common antibiotics and easy storage through freeze-drying. At the same time, limiting the treatment cost is a high priority for hospitals.

Nurses

For the people applying the patch, the nurses, ease of application is important. This is ensured by designing the patch like band-aid. This is also important for their skills on using it, as not much has to be changed in their training or surroundings to learn how to apply such a patch. However, as they are the ones handling the wound healing patch, they are concerned by making the patch as safe as possible as well. Reducing chemical toxicity is their main interest along with the fact that the bacteria inside the gel do not pose a risk to the nurses.

Insurance companies

Our contact at Plasmacure, Koen Lim, had been working for several insurance companies. He could thus inform us about the wound care market, as we described on the Applied Design page. Koen Lim expected that our product will drastically decrease health care costs once it is fully functional. Wound care currently costs Dutch society over 3 billion euros (1), so a cost reduction would be welcomed by insurance companies.

Society

As the increasing antibiotic resistance of bacteria is a major problem for society, it is our responsibility to reduce the systemic use of these antibiotics. We are trying to achieve this by using bacteriocins, and not common antibiotics to reduce the increase in antibiotic resistant bacteria. Another thing that we have to take into account however is that there is fear among people about the use of genetically modified organisms (GMOs). People are afraid of potential dangers. As an iGEM team, it is thus importance to increase awareness of the benefits of GMOs as well as of the danger of and preventive measures against antibiotic resistance. On top of that, we should highlight the additional safety layers introduced by our design. We tried to contribute to this through our education and outreach.

Government

The government is responsible for regulations to ensure GMO safety. As Gelcatraz introduces a safer method of working with GMOs outside of the laboratory, it may form an incentive to reconsider the regulations in certain cases. Obviously, this should only happen when the safety is guaranteed and will require a social debate. Furthermore, the government is faced with the problem of antibiotic resistance. Charged with the responsibility of finding a solution, the government would most likely welcome our alternative treatment of wound infections. Lastly, the treatment of infected wounds is an extremely expensive business due to repetitive, costly treatments. Since our patch would speed up wound healing and reduces costs, it would be beneficial for the government.

Manufacturers

For manufacturers different topics are relevant. To guarantee the safe production of the wound healing patch, it has to meet the Good Manufacturing Practices (GMP) every therapeutic has to meet: precise description of production from raw material to fully functional patch. This will establish a safe production process. However, an efficient production process is just as meaningful for manufacturers. The volume in which gel is produced, is scalable and the catalyst used for the gel production is reusable. This is all documented on the safe-by-design page. Unfortunately, in upscaled production, gels are more prone to errors. Testing the gels on the demanded properties is the manufacturers responsibility, but this should be straightforward. Besides being more prone to errors, our gel is not recyclable to make new gels, but the gels can however be stored efficiently using freeze-drying.

Environment

Low toxicity is of interest to the environment as well as to other stakeholders. The same applies to the prevention of bacterial escape from the gel. To reduce survival rates of potentially escaped bacteria, we use a kill switch. As explained under safe-by-design, this means that survival outside of the gel is not possible for our bacteria. Moreover, our bacteria can easily be killed using regular antibiotics. To conclude, Gelcatraz should not pose significant threats to the environments when it is used responsibly.

References

Meerwaldt R, Das F, Fentener van Vlissingen J, de Lange E, Maessen-Visch MB, van Montfrans C, et al. Kwaliteitsstandaard Organisatie van wondzorg in Nederland [Internet]. 2018. Available from: https://www.demedischspecialist.nl/sites/default/files/Kwaliteitsstandaard Organisatie van wondzorg in Nederland.pdf

×

Lorem Ipsum

×

Lorem Ipsum

Team:TU-Eindhoven/Human Practices

Human Practices

Integrated Human Practices

I. Finding a project

February & March

April & May - TU/e contest

April & May - TU/e contest

Insights

Follow-up

II. Finding an application

May 14, 2018. Dutch iGEM meetup RIVM and Rathenau Institute.

May 14, 2018. Dutch iGEM meetup RIVM and Rathenau Institute.

Insights

Follow-up

May 16, 2018. Meeting Prof. Dr. Patricia Dankers

May 16, 2018. Meeting Prof. Dr. Patricia Dankers

Insights

Follow-up

May Brainstorm sessions

May 22, 2018. Netherlands Biotechnology Congress

May 22, 2018. Netherlands Biotechnology Congress

Insights

Follow-up

June 1, 2018. Meeting with an ethicist: Dr. Lily Frank

June 1, 2018. Meeting with an ethicist: dr. Lily Frank

Insights

Follow-up

June 9, 2018. Dutch Technology Week

June 9, 2018. Dutch Technology Week

Insights

Follow-up

June 18, 2018. Kick-off Safe-by-Design Challenge: Skype with Korienke Smit and Niek Savelkoul (RIVM)

June 18, 2018. Kick-off Safe-by-Design Challenge: Skype with Korienke Smit and Niek Savelkoul (RIVM)

Insights

Follow-up

June 22, 2018. Meeting Dr. Sandra Hofmann

June 22, 2018. Meeting dr. Sandra Hofmann

Insights

Follow-up

June 22, 2018. The final decision

June 22, 2018 The final decision

III. Shaping the design

July 3, 2018. Plasmacure

July 3, 2018. Plasmacure

Insights

Follow-up

July 20-22, 2018. European Meetup Munich

July 20-22, 2018. European Meetup Munich

Insights

Follow-up

August 01, 2018. Skype call with Lori Goff

August 01, 2018. Skype call with Lori Goff

Insights

Follow-up

August 03, 2018. Skype with iGEM Düsseldorf: The potential of quorum sensing as a mean to prevent overcrowding and/or infiltration

August 03, 2018. Skype with iGEM Düsseldorf: The potential of quorum sensing as a mean to prevent overcrowding and/or infiltration

Insights

Follow-up

August 07, 2018. Meeting with professor Menno Prins about biosensing

August 07, 2018. Meeting with professor Menno Prins about biosensing

Insights

Follow-up

August 10, 2018. Safe-by-design: Phone call with Cécile van der Vlugt

August 10,2018. Safe-by-design: Phone call with Cécile van der Vlugt

Insights

Follow-up

Augus 13, 2018. Kill switches: Meeting with Prof. dr. Jan van Hest

Augus 13, 2018. Kill switches: Meeting with Prof. dr. Jan van Hest

Insights

Follow-up

July – August 2018 iGEM'ers Guide to the Future – Stakeholder value matrix

July – August 2018 iGEM'ers Guide to the Future – Stakeholder value matrix

Insights

Follow-up

August 21, 2018. Freshmen’s week @ TU/e

August 27, 2018. Burn wound center Maasstad Ziekenhuis

August 27, 2018. Burn wound center Maasstad Ziekenhuis

Insights

Follow-up

Sept 21, 2018. Ethical analysis – update