Human Practices Silver Criterion

The Ethics in Bacto-Aid

In our project’s components there are different ethical concerns. The bacteriocins in the hybrid silk raises questions about health and security: if one is to apply genetically modified organisms (GMO’s) to a natural environment there will be insecurities. The problem is that we can never, with full security, know the consequences - but we can derive an estimated account of what will happen. The Precautionary Principle helps to resolve this, but the interpretation of the Precautionary Principle differentiates according to one’s preferences and intuitions when it comes to emerging technologies and therapeutics. It basically states that it’s sometimes better to not apply a technology or therapeutics, when the results are insecure. In the opposite situation, then the argument is that it is sometimes better to take action, even when one does not have complete certainty on the possible consequences. The Precautionary Principle works as a tool for making decisions that can be rationally justified when uncertainty are present. Sometimes it functions as a rhetorical or epistemological tool, but in the field of bioethics it is mostly a tool for decisions KAPPEL, K. 2011. Bioetik. In: CHRISTENSEN, A.-M. S. (ed.) Filosofisk Etik. Århus: Aarhus Universitetsforlag..

Since bacteriocins have no effect on the humane cells, that we know of, we have decided to work with them. The different trials that a new form of therapeutics must withstand are still necessary for determining if it’s safe enough to apply in a natural environment. A comprehensive risk assessment can be seen at our Safety page.

Justification and happiness

Working with GMO’s leads to thoughts about justification: how can one morally justify the methods and the results? Bioethics handles the questions by supplying the foundation for the discussion. It derives from theories about normative ethics and medical concerns and has then specialized in the questions which rises in working with modifying organisms. In the field of bioethics there is different ways to justify the work and different types of arguments, depending on what area one works with and what problem one tries to solve.

In our project we have focused a lot on antibiotic resistance and plastic pollution as a problem in the community and environment. The general argument for justification we use, is an appeal to utility. This means that the work we are doing aims to increase the overall happiness and utility, which are seen by utilitarians as the most important factor to value actions by. This also makes us ‘proactivists’ which means that one looks upon new technologies with a positive mind KAPPEL, K. 2011. Bioetik. In: CHRISTENSEN, A.-M. S. (ed.) Filosofisk Etik. Århus: Aarhus Universitetsforlag..

For more information on the Precautionary Principle, click here. For more information about bioethics and our justification, then click here.

Theory and Bioethics

Bioethics are defined in different ways according to the subject one is applying it to. A definition that suits that view is Battin’s BATTIN, M. P. 2003. Bioethics. In: WELLMAN, R. G. F. C. H. (ed.) A Companion to Applied Ethics. Blackwell Publishing Ltd. who sees bioethics as a hybrid consisting of philosophy, medicine and law. That definition is now 13 years old and it still fits if there is added a long list of subjects to go with it. Bioethics are ethics about flora and fauna – and the hard questions that emerge in the humane world. Questions e.g. when - or if - a fetus is a person or whether it is wrong to use the nature – bacteria for example – to help the human race thrive.

Theories in normative ethics

One of the interesting aspects of bioethics, as a field, is that there is no dominating strain of normative ethics. The arguments are both deontological in nature as well as utilitarian. When an argument is deontological it focuses on duties. Deon comes from the ancient Greek word for duty or obligation. Simply put: There are some things that one just has to do and things one never does. In deontology there is a principle called Universalizability Principle which states, that for an action to be morally acceptable the maxim which the action is based on has to be universalizable NIELSEN, C. F. 2014. Deontologi In: CHRISTENSEN, A.-M. S. (ed.) Filosofisk Etik. Århus: Århus Universitetsforlag..

An example of a deontological argument would be “it is wrong to play God”, that falls under the category with religious arguments. Some group those arguments with teleological-, religious - and nature arguments KAPPEL, K. 2011. Bioetik. In: CHRISTENSEN, A.-M. S. (ed.) Filosofisk Etik. Århus: Århus Universitetsforlag. Aarhus Universitetsforlag..

The utilitarian arguments are focused on the consequences of our actions. The consequences are all that matters when it comes to the decision of whether something is morally just. The consequences which utilitarianism focuses on are utility and it’s often the most positive arguments in bioethics. The arguments are simple in outline: if the action that is discussed produces, or results in, more utility then one is morally obliged to apply or accept that action HOLTUG, N. 2014. Konsekventialisme. In: CHRISTENSEN, A.-M. S. (ed.) Filosofisk Etik. Århus: Århus Universitetsforlag..

Sometimes the arguments are based on virtue ethics. Where deontology and utilitarianism are based on rules independent of individuals, meaning that the rules apply no matter what, virtue ethics are based on the individuals. The perfect virtuous person is what determines the morally just action. In natural science virtues like integrity, curiosity and intelligence are often highly praised and the idea behind virtue ethics are that the virtuous person’s actions are the morally praiseworthy actions CHRISTENSEN, A.-M. S. 2014. Dydsetik. In: CHRISTENSEN, A.-M. S. (ed.) Filosofisk Etik. Århus: Århus Universitetsforlag.. In relation to bioethics most arguments focus on; that for an action or technology to be morally acceptable it should be so that a virtuous person would accept it. Problems arise when it comes to determine the perfect virtuous person.

Precautionary vs. proactionary

In the discussions of different emerging technologies or therapeutics one can either assume a precautionary or a proactionary stand. The precautionary point of view tends to be a more sceptical view, where one takes a stand against just accepting and embracing new technologies or therapeutics: their motto is better safe than sorry, based on the fear of unknown consequences or that the technology in question has the possibility of changing our life as we know it.

As a proactionist the fear is not unforeseen consequences, but that the public skepticism will hold evolution and progress back. There is a tendency to see an emerging technology or therapeutics as safe, unless there is evidence that suggests otherwise KAPPEL, K. 2011. Bioetik. In: CHRISTENSEN, A.-M. S. (ed.) Filosofisk Etik. Århus: Århus Universitetsforlag. Aarhus Universitetsforlag..

Arguments in Bioethics

There are different arguments in the discussions. The subject of discussion changes depending on what technology or therapeutics is in question. But behind the different-sounding arguments there is a structure or a system. Most of the arguments falls under different categories:

1. Autonomy and utility
2. Dignity and integrity
3. Teleological, natural and religious
4. Slippery Slope

An argument in category A is an argument that appeals to either the autonomy of an individual or that it will promote utility in the society. It’s a type of argument that one often hears from a proactionist, who normally will focus on the good aspects of an emerging technology or therapeutics. Appeal to autonomy is often motivated from a deontological point of view, but the appeal to utility is often utilitarian in nature KAPPEL, K. 2011. Bioetik. In: CHRISTENSEN, A.-M. S. (ed.) Filosofisk Etik. Århus: Århus Universitetsforlag. Aarhus Universitetsforlag..

Dignity and integrity is mostly seen as a reply to an argument from category A. It’s therefore often used by a precautionist, as their way of saying “slow down - somethings are more important than utility”. The core idea is that living matter has a dignity and integrity that we as a society should respect, but living matter can be defined in many ways allowing room for a versatile argumentKAPPEL, K. 2011. Bioetik. In: CHRISTENSEN, A.-M. S. (ed.) Filosofisk Etik. Århus: Århus Universitetsforlag. Aarhus Universitetsforlag..

The teleological, natural and religious arguments are grouped together, due to their habit of being intertwined in an argument. Teleos is the ancient greek word for ‘goal’ or ‘striving’, meaning that all things have a form of ‘striving’ in their essence - the world included. So the argument normally goes: “There is a bigger purpose with the world, that we shouldn’t try to change or interfere with”. Sometimes it’s intertwined with religion, stating that one shouldn’t try to “create”, because that is only meant for the divine KAPPEL, K. 2011. Bioetik. In: CHRISTENSEN, A.-M. S. (ed.) Filosofisk Etik. Århus: Århus Universitetsforlag. Aarhus Universitetsforlag..

The different types of arguments aren’t always neatly sorted out as they have been presented, but often work in relation to each other or consists of different appeals, all to make a stronger argument.

Principles in bioethics

To guide the appliance and decision-making process there are some principles that apply in all arguments:

1. The No-Harm principle
2. The principle of Personhood
3. The Precautionary Principle

The No-Harm principle is perhaps the oldest principle in applied ethics and dates back to Hippocrates and his thoughts about practicing medicine. It simply states that one should not cause harm to another person. It’s intuitively simple in outline but lacks better definition of what ‘harm’ is - is it only physical harm or does mental harm count as well? The answer to that question normally depends on whom one is asking. For our purpose and understanding we’ll consider it to be intuitive that one shouldn’t cause physical harm or severe mental harm KAPPEL, K. 2011. Bioetik. In: CHRISTENSEN, A.-M. S. (ed.) Filosofisk Etik. Århus: Århus Universitetsforlag. Aarhus Universitetsforlag..

The principle of Personhood tries to state what defines a person. Some believe a person to be someone who is able to have a reflective view over one’s past, future and present. But this definition is far too narrow, excluding babies and people suffering from dementia. Other definitions are based on a contra factual understanding: a person is someone, who under normal circumstances can be expected to reflect over one’s past, future and present. This seems broad and lacks a good definition on what counts as normal circumstances KAPPEL, K. 2011. Bioetik. In: CHRISTENSEN, A.-M. S. (ed.) Filosofisk Etik. Århus: Århus Universitetsforlag. Aarhus Universitetsforlag..

The Precautionary Principle has many different definitions, outlines and functions. It is said to be: “[...] the most innovative, pervasive, and significant new concept in environmental policy over the past quarter century [...]” MARCHANT, G. E. & MOSSMAN, K. L. 2004. Arbitrary and capricious: The precautionary principle in the European Union courts, American Enterprise Institute.. It is meant to be a principle which can help to determine which actions must be taken or which technologies that must be accepted. But as the quote continues one sees a different side of the principle: “[...] It may also be the most reckless, arbitrary, and ill-advised” MARCHANT, G. E. & MOSSMAN, K. L. 2004. Arbitrary and capricious: The precautionary principle in the European Union courts, American Enterprise Institute..

True to the quote the Precautionary Principle seems to have many roles in the discussion about bioethics - it’s both the motto for the precautionary point of view as well as an epistemic principle that is used to explain why it’s sometimes more rational not to act, instead of acting STEEL, D. 2014. Philosophy and the precautionary principle, Cambridge University Press.. It also functions as a rhetorical tool when politicians discuss policies.

The Precautionary Principle’s complexity warrants a more detailed account and discussion, which are both found in the Parts & Procedures chapter, on the safety page.

Studies on the Precautionary Principle

The problem with precaution and risk is that one never seems to have a certain knowledge about the outcome of an event. On everyday basis it does not seem to be a big issue: “maybe I will get wet if I leave the umbrella at home”. But when it comes to the decisions we have to make as a society then it is a different story: e.g. what will the consequences be if one was to allow more use of pesticides? When the consequences can not be known for certain then there is room for potentially big catastrophes - maybe all our water supply will be contaminated, which could lead to hormonal imbalances or something worse and unforeseen.

When considering the options, one should ask at least three questions: “What possible outcomes could the action have? How good (or bad) are these different outcomes? At what likelihood would these different outcomes occur?” The problem is that especially the last question possesses unknown factors. What we, as a society, seem to lack is a way of rationally deciding which action to take when factors are unknown. One also needs to be able to distinguish between which possible risks are important to take under consideration and which are not. It is not rational to take all possible risks under equal consideration - there seems to be some possible risks that are so unlikely that one should not take them too seriously, e.g. when transforming E. coli to synthesize spider silk, the impact on global warming should not be a problem to take into consideration.

In our field of synthetic biology we find the possible consequences in the lab and their probability are mathematically decided. But on that note there is a clear difference between the probability for the hypothesis or theory to be correct and the probability threshold we decide by when it comes to risk assessments.

Take for example a hypothesis stating that “the chemical substance X is dangerous and leads to cancer”. For this hypothesis to be accepted into the scientific corpus it needs to fulfill different - and more - requirements than it needs for the society to ban substance X on the base of precaution. Because if they knew it could potentially lead to cancer, they would be scared and why should they risk it? The epistemic values are simply different when it comes to scientific theories compared to the qualities which we value by when it comes to risks. Normally, when it comes to risk assessment it is more fatal to accept a technology or new substance which leads to undesired consequences than to accept a false hypothesis into a scientific theory.

The Precautionary Principle

This is called “The Precautionary Principle”, which has many different definitions but in essence it means: if an activity has potential harms by a certain degree, precautionary measures should be taken even if the likelihood of the harms occurring have not been scientifically confirmed. Other definitions could be: “Where there are threats of serious or irreversible damage, lack of full scientific certainty shall not be used as a reason for postponing cost-effective measures to prevent environmental degradation” Principle 15 of the 1992 Rio Declaration on Environment and Development or “When an activity raises threats of harm to human health or the environment, precautionary measures should be taken even if some cause-and-effect relationships are not fully established scientifically” Wingspread Statement on the Precautionary Principle. One study shows that one bacteria, which produces the bacteriocin leugudin, outcouncours the MRSA in vitro, which are great results, but from these results we do not just treat MRSA patients with the outcouncouring bacteria because we do not know the possible consequences of that action. It could potentially lead to even greater infection or problems for the patient or society.

This leads to the conclusion that even though the essence of the Precautionary Principle is intuitive for most, the precise definition and the influence of the principle is still debated.

The Precautionary Principle can be defined by three parts: The Meta-Precautionary Principle, the “Tripod” and Proportionality STEEL, D. 2014. Philosophy and the precautionary principle, Cambridge University Press.. The combination of these is what makes the Precautionary Principle a good tool for decision-making. The Meta-Precautionary Principle places restrictions on which types of decision-rules should be applied. The decision-rules in question are not to allow inaction because of scientific uncertainty - one should never be paralyzed by a lack of knowledge when facing serious threats STEEL, D. 2014. Philosophy and the precautionary principle, Cambridge University Press.. The “Tripod” part is actually three parts in itself: The epistemological condition, the damage condition and the remedy or action that are recommended CARTER, J. A. & PETERSON, M. 2015. On the Epistemology of the Precautionary Principle. Erkenntnis, 80, 1-13.. The epistemological condition is a proposition that stands in reference to the damage condition. The damage condition is the possible harm(s), which can happen as consequences of the activity in question. The epistemological condition is then the evidence one has for the probability that the damage will occur. The connection between the epistemological condition and the damage condition is most often thought of as a causal connection.

The three conditions in the Tripod are also the case dependent part of the Precautionary Principle. The Tripod is therefore the most flexible part of the Precautionary Principle and the different cases then require different versions of the Precautionary Principle: “[...] any statement according to which satisfying a specific [epistemological condition] and [damage condition] is sufficient to justify a specific [remedy]” STEEL, D. 2014. Philosophy and the precautionary principle, Cambridge University Press.. Which version to choose is specified by the constraints from the Meta-Precautionary Principle.

The last part, proportionality, is a contextualist-based idea. More precise it is the idea that the level of precautionary measures taken corresponds to both the probability and the damage. It is similar to the economic thought of cost and efficiency - if the cost is high, then the efficiency should be as well.

The three elements are the essential parts of the Precautionary Principle when it functions like a decision rule. But it is important to be aware that the Precautionary Principle can function as a procedural requirement, where the Meta-Precautionary Principle is highlighted or as an epistemic rule STEEL, D. 2014. Philosophy and the precautionary principle, Cambridge University Press.. We will here focus on the Precautionary Principle as a decision rule composed of the Meta-Precautionary Principle, the Tripod and Proportionality.

How does it work?

One thing is understanding the essence of the Precautionary Principle, but when it comes to how it should be applied there are different explications of it. Some interpretations focus on catastrophes (Catastrophic Precautionary Principle), for some it is a maximin rule and others again see it as a minimax regret.

The Catastrophic Precautionary Principle is proposed by Hartzell-Nichols HARTZELL-NICHOLS, L. 2012. Precaution and solar radiation management. Ethics, Policy & Environment, 15, 158-171. and states: “Appropriate precautionary measures should be taken against threats of catastrophe” with definitions and restrictions to follow. Some of his requirements are very similar to the parts of the Precautionary Principle that we have already defined: e.g. “A precise probability of a threat of harm is not needed to warrant taking precautionary measures so long as the mechanism by which the threat would be realized is well understood and the conditions for the function of the mechanism are accumulating”. The requirement meets the demand imposed by the Meta-Precautionary Principle, which stated that one should not be paralyzed from taking action by lacking scientific evidence. The demand about consistency is also present in one of his requirements.

The Catastrophic Precautionary Principle then compiles fine with the Precautionary Principle we already have defined and it then seems to be a version of the Precautionary Principle. A version because the damage and epistemological conditions, that are parts of the Tripod, are specified. The damage condition is set to be only “catastrophe” and the epistemological condition to be “the mechanism by which the threat would be realized is well understood and the conditions for the function of the mechanism are accumulating”. As a positive effect, the Catastrophic Precautionary Principle is more well-defined and easier to apply but it is also more limited, where the Precautionary Principle normally is thought of as having a broader range than catastrophes - e.g. genetically modified organisms (GMO’s).

The maximin rule comes from a different background than the Precautionary Principle, i.e. political philosophy rather than bioethics. It has then been transformed for a use in bioethics. The maximin rule is an expression for choosing the action that has the smallest chances of resulting in ‘worst case’ scenarios. It is almost always very restricted and one of the most central restrictions is ‘pure uncertainty’ STEEL, D. 2014. Philosophy and the precautionary principle, Cambridge University Press.. By pure uncertainty is meant “a total lack of information about the probabilities of the relevant possible scenarios” - then the maximin rule is applied to make sure that one assures the highest minimum of good. The proposal for which conditions has to be satisfied before recommending precaution are STEEL, D. 2014. Philosophy and the precautionary principle, Cambridge University Press.:

1. Pure uncertainty
2. There is no possible scenario wherein catastrophe occurs when the precaution is taken
3. Taking the precaution achieves results that are close to the best case that could have been achieved
4. If the precaution is not taken, then catastrophe will occur

These conditions are quite strict, which limits the use of the maximin rule, e.g. it is rare that we have complete uncertainty (1) when discussing the Precautionary Principle. The problem is more often incomplete knowledge.

The minimax regret is originally an economic principle but has been proposed as an explication of the Precautionary Principle by Chrisholm and Clarke CHISHOLM, A. & CLARKE, H. 1993. Fair principles for sustainable development. Natural Resource Management and the Precautionary Principle (ed. Edward Dommen), Edward Elgar Publishing, 109-122.. The minimax regret helps finding what action (i.e. precaution or no precaution) in a given case that has the least difference between its result and the best possible result that could be obtained in the case. If there is no difference between the first action and its result and the best possible result, then there is no regret and it is therefore the action to choose. It can be clarified by showing in the two tables.

Table one.

If one then supposes that the catastrophe is more severe than the expenses, which then are more than the damage (If one sees it as absolute numbers of costs, then: c>e>d). Then the table with the regrets will look like this:

Table two.

The recommendation will then be to select the action (i.e. precaution or no precaution) that has the smallest amount of regret in the case in question. Then, if we stick to the fixed presumptions (c>e>d) then the actions to choose are precaution in case 1 and no precaution in case 2 and 3. The presumptions are dependent of the specific cases.

The problem with the minimax regret is that it treats the epistemological and the damage condition as quantitative, where the Precautionary Principle has to be applicable to qualitative measures as well. The two conditions have to be thought of as quantitative for the terms of ‘difference’ between them makes sense STEEL, D. 2014. Philosophy and the precautionary principle, Cambridge University Press..

Scientific Reproduction

We have made it our obligation to follow the virtue of scientific reproduction. It is fundamental for the scientific method and is integrated in the scientific community.

Science began when people started wondering about the significance of their observations. Aristotle was wondering why the stone fell down when he dropped it from midair. He therefore developed a hypothesis about the essences in all things, which explained that the stone was moving towards its natural place - that the stone had a teleos (Ancient Greek: purpose, aim). Nowadays we laugh at that type of explanations but the essence of science still stands: explaining our observations KLAUSEN, S. H. 2014. Hvad er videnskabsteori. Akademisk Forlag..

Theories and hypotheses have virtues, which a good scientist should comply with, e.g.: to follow the rules of good reasoning, being coherent and explaining a phenomenon. A hypothesis or theory must always have implications that can be empirically tested for it to be of any use. To accept a hypothesis or theory one needs to show that it satisfies all demands - and then it needs to be tested by an objective scientist. This is called the demand of reproduction HEMPEL, C. G. 1966. Philosophy Of Natural Science. Foundations of Philosophy series. Princeton University..

Confirmation and Bacto-Aid

In our project we tried to follow several other iGEM teams’ protocols and methods for our work. We had success following the protocols from the iGEM teams of Imperial College London 2013, Tokyo Tech 2012 and Standford Brown 2015. We used their protocols in the make of PHB and some of their BioBricks. We had trouble with following 2015 UCLA's protocols for producing spider silk. At first we believed we had falsified their method – we certainly believed that we could not corroborate their hypotheses. In the end of the project we found that the things that had gone wrong was not really related to their protocols, but the components we had used in the work. The enzyme we used were of a different type than the one used by 2015 UCLA and our streptavidin beads had run past its expiration date.

This is a great example of there not being a final falsification – there was clearly something wrong with our auxiliary hypotheses. The auxiliary hypotheses were also the first place we started looking when the results did not arrive as planned – so having an overview of them helped us find that the mistakes perhaps were with them and not UCLA's protocols. We also found a mistake in our preliminary purification of PHB. The hypochlorite we used at the first had been stored wrong and the purification became visibly better when we used a different hypochlorite.

Scientific reproduction

Besides using UCLA's protocols, we also tested their BioBricks. We also tested existing BioBricks in the production of PHB. More information on the BioBricks can be found here. Testing existing BioBricks helps other iGEM teams in the coming years, because they will know that the BioBricks have been tested.

Unfortunately, we ran into problems when we tried their protocols for assembly of the silk genes. To do this the restriction sites are a crucial part of the silk gene sequences because they can overlap each other creating desired gene sequences. The genes were designed so that digesting with one enzyme would result in overhangs which would be able ligate. We tried to follow their protocol on digestion several times, however we did not succeed before we tried to digest for a longer time and at a different temperature. We found out that the enzymes we used were slightly different from the one UCLA used and this was the reason why we were not able to make a successful digestion at first.

According to their wiki, and the uploaded parts (K1763009 & K1763423) in Parts Registry, the genes named MaSp1 AB- and MaSp2 AB core sequence should contain two restriction sites for the enzyme BsaI, but we found, through SnapGene, that the sequences only contained a single restriction site at 108bp. The two genes could not be applied for Iterative Capped Assembly (ICA) as they had suggested.

When following UCLA's protocols, we were not able to assemble more than three genes corresponding to either one MaSp1 gene or MaSp2 gene (see more on the silk page). Even though we made adjustments to the protocols, we were still not able to make the four monomers, consisting of 12 fragments, which we aimed for. This could be due to the streptavidin beads, which are essential when performing ICA. We found out that the beads had passed their expiration date. Unfortunately, we first saw this mistake too late.

These examples show the importance of scientific reproduction. This is also what research ethics is about: presentation of results and methods. There is bound to be mistakes in the ongoing research and scientific reproduction helps determine these and tries to correct them. As stated above, a falsification does not entail that the competing hypothesis is verified - it just is not falsified.

Outreach

We decided early on that we wanted to engage as much as possible with the society and especially high school students and children. The youth of the society are the upcoming in the field of science as well as the ones that will be most affected of the antibiotic resistance and plastic pollution. We believe that the more awareness of these problems the better - the same goes for general knowledge of synthetic biology and GMO’s.

We have raised awareness of iGEM, synthetic biology, bioethics and research ethics. The forms of engagement range from participating in official events at our university to a theme day both in the laboratory and in the classroom at high schools.

Research day

Every year in April the University of Southern Denmark (SDU) arranges a whole day filled with workshops, lectures and talks. This year the faculty of Biochemistry and Molecular Biology had a big workshop area with the theme “The Good, the Bad and the Ugly”: the probiotic bacteria, the pathogenic bacteria and the multiresistant bacteria. With our focus on antibiotic resistance we fitted right in. We spend the entire day discussing, explaining and teaching - we had so much fun and learned a lot both about how the concept of synthetic biology is perceived in the society, but also how we should communicate it.

High School talks

We have talked several times before an audience composed of high school students.

• BioOL

  Our first attempt was the Danish participants for the International Biology Olympiad. They were the perfect audience, asking a lot of critical questions, which made us think twice about how we made our presentation. We altered to make it less focused on what iGEM is and more foused on the community aspect and the problem of antibiotic resistance.

  
  

  

• KOLD College and Odense Tekniske Gymnasium

  We also had the pleasure of presenting our presentation and a talk about bioethics for two technical high schools in Odense: Odense Technical High School (OTG) and KOLD College. At KOLD College we had an entire theme day where we helped the students in the laboratory and entertained them in between with details of our project. This included the preliminary presentation, which we will bring to Boston and theory about the experiments they had to do in the laboratory. We also had our movie, which explains our project with us as well as a collective brainstorm for the prospects of our project.

  
  
  

  Us at KOLD college, teaching their students.

  

  The two classes at OTG.

  
  

• Academy for Talented Youth

  At the end of the summer we had the opportunity to be a part of another event at the university, as SDU was visited by the Academy for Talented Youth. We presented our presentation and had an extra focus on bioethics - and we had a really good discussion with them. We then supervised them in the laboratory.

  
  

  

Other talks

• Symposion

  As a good way of getting more feedback we held a presentation at Symposion - a philosophical association at SDU. The audience were composed of professors in philosophy, students from philosophy and other students who found it interesting. There were an almost hour long discussion with lots of good constructive critique and advices for our human practices. Their ideas and arguments had a big influence on our writings on bioethics and philosophy of science. They gave us ideas to other forms of arguments in bioethics, e.g. the religious arguments. They also gave us the idea of "Track-record" for a way of handeling the Precautionary Principle.

  
  

• OldGEM

  In September we had the amazing opportunity to meet with the previously iGEM teams from SDU. We held our presentation and they gave so much feedback that it filled 3 pages in notes! The feedback was a very welcomed gift that we used for adjusting our presentation. Otherwise than that, it was a lot of fun meeting the old teams and hearing their pointers on our project. They also gave us a little pep talk, which we appreciated!

  
  

  

• Biochemistry and Molecular Biology

  The day before we left for Boston we had the opportunity to present our project for the whole Institute of Biochemistry and Molecular Biology (BMB). This event was our rehearsel of the presentation, which we will bring with us in Boston. We are hoping that our presentation will inspire other students to apply for next year's iGEM competition.

  
  

The media

• SDU made an informational video about us and our project, which got featured at their facebook page and on youtube. It was so much fun recording it and we had a strict focus on showing a sense of humor and making our project understandable and relatable to the public. Hopefully this video will inspire other students to apply for next year’s iGEM team at SDU.

  
  

• We also made our own video, which can be seen at the Home chapter. We made the video a part of our presentation after showing it to the high schools we visited. They thought it was a great way of explaining our project in a short time. We therefore use it both as a different way of explaining as well as a resumé of our presentation.

Prospects

How would we make Bacto-Aid?

Even though we spent hundreds of hours in the lab, we did not end up standing with the Bacto-Aid in our hands in Boston. But do not worry - we got a plan for how you can end up with our bandage. On this page we will enlighten you about the next steps in our project, which we would have followed if the days contained more than 24 hours or if we had a bunch of effective minions available. These steps have to be fulfilled before we can create a full functional patch that can be used in hospitals and in the everyday life.

Making a 4 monomer silk construct

There were some difficulties with producing a longer silk construct, and whether it was due to the reproducibility of UCLA's method or the too old streptavidin beads, we did not have the time to find out. However, the first step would then, of course, be to try the ICA method with new streptavidin beads.

As soon as a 4 monomer silk construct is produced, we would insert it into E. coli and purify it from the bacteria. Here, we would have liked to try a purification method without the use of His-tag, because earlier literature have demonstrated that recombinant silk produced without a His-tag have better mechanical properties compared to the fibres made from silk proteins with a His-tag Tokareva, O., et al. (2013). "Recombinant DNA production of spider silk proteins." Microbial Biotechnology 6(6): 651-663..

For the spinning of silk, we would first have tried to spin our silk by wet spinning, which is a very simple and inexpensive method Teulé, F., et al. (2013). "A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning." Nature Protocols 4(3): 341-355.. In relation to using the recombinant silk for wound healing, it could also be interesting to prepare a three dimensional scaffold of the silk. Here a number of methods, such as salt leaching, gas forming or freeze-drying, have been reported to generate porous three-dimensional matrices Nazarov, R., et al. (2004). "Porous 3-D Scaffolds from Regenerated Silk Fibroin" Biomacromolecules 5: 718-727..

The detailed description of how we want to proceed with a 4 monomer silk construct can be reviewed on this page.

The hybrid silk fibre

The idea of the hybrid silk fibre was to incorporate bacteriocins between silk monomers. From the literature, it is known that silk does not lose its function when other proteins are combined with the silk Gomes, S. C., et al. (2011). "Antimicrobial functionalized genetically engineered spider silk." Biomaterials 32(18): 4255-4266.. We also found that proteins incorporated in silk monomers does not lose their functions either Gomes, S. C., et al. (2012). "Biological responses to spider silk-antibiotic fusion protein." Journal of tissue engineering and regenerative medicine 6(5): 356-68.. This is why we believe that if the bacteriocins were to be incorporated in the silk monomers, then both components would maintain their functions and thereby their effects. We are also interested in creating hybrid silk with more than one bacteriocin incorporated, to see if they have a better effect than single bacteriocins between the silk. Our hypothesis is that silk fiber, which is proven to be immune neutral and promotes wound healing, combined with the antimicrobial effect of the bacteriocins, would create a synergistic effect resulting in decreasing infections and also a decrease in healing time in patients with severe wounds.

We tried to ligate the bacteriocin together with a silk fragment, but we did not succeed despite having the proper overhangs. The next step will then be to make more experiments with the goal of making a successful ligation between the bacteriocins and a silk construct. In theory we should succeed, so it might just take some more trials before we do.

Improving the production of PHB

PHB should be used as the envelope for our Bacto-Aid. To do this, we first need to optimize the production of PHB even further. This can be done by changing the current gene (phaCAP) in the BioBrick from Tokyo Tech 2012 with a stronger promoter and an additional stronger RBS. The use of a weak promoter and an additional weak RBS might also be an aspect in how much PHB there can be produced in the cell. The new device should then be added to our secretion system instead of the old from Tokyo Tech 2012 BioBrick, and hopefully make the E. coli produce even more PHB than we see now.

To produce more rentable PHB, we should first consider which microorganism to use to make the mass production of PHB most efficient. We have considered using Ralstonia eutropha and Bacillus subtilis, which are two commonly used organisms in the industry. Secondly we need to develop our production so it fits with a continuous stirred-tank reactor. We will imply this reactor for large scale production, so we will be able to regulate parameters such as temperature, oxygen level and amount of required nutrients for the chosen organism. It will likewise also be good if we could decrease the use of chemicals to make the production more profitable.

We also need to design a 3D model of our envelope for Bacto-Aid, so we could print the model and produce the envelope through 3D printing. This should be done by creating 3-5 models of an envelope for Bacto-Aid and the best envelope will be selected critically by important criterias for the use of PHB on the skin and how well it fits these criterias.

Producing Bacto-Aid

If we have fully developed our sub elements, we should be able to make Bacto-Aid and test its effect on wound healing. The next step would then be to develop methods for how we could scale our production of the sub elements up to the industrial scale.

Perspectives

Early on the project we realized that there are many interesting applications for our chosen sub elements, and as we got to talk with different experts, we saw even more opportunities. On this page we have highlighted some of the most interesting ideas, to give you a broader perspective of the true potential of each sub element. We have also included some of the ideas the high school students gave us during our workshops. So take a look and be inspired to how Bacto-Aid’s sub elements can be integrated in your future.

Bacteriocins

The promising MIC results of the bacteriocins gives us the opportunity to use them as a supplement to traditional antibiotics, thus facing the evolution of antimicrobial resistance (AMR). Here is some of the ideas for their future applications.

Combination of traditional antibiotics with bacteriocins (Top)

The bacteriocins could serve as a supplement to traditional antibiotics, since our purified bacteriocins were capable of inhibiting the growth of Methicillin Resistant S. aureus strains. It could also be hypothesized that a combination of traditional antibiotics with bacteriocins could elicit a synergistic effect towards resistant bacterial strains. This leaves us with the opportunity to shift the balance of resistance and prevent the evolution of antimicrobial resistance against traditional antibiotics Dan, Y., et al. (2012). "Bacteriocins Produced by Lactic Acid Bacteria a Review Article." APCBEE Procedia 2: 50-56..

Combination with pulsed electric fields (Top)

A combination of bacteriocins and technologies, such as pulsed electric fields, ables us to disrupt the cellular membrane for penetration of the cell and have been suggested to be highly effective Dan, Y., et al. (2012). "Bacteriocins Produced by Lactic Acid Bacteria a Review Article." APCBEE Procedia 2: 50-56.. This method could be a way for us to give the hybrid bacteriocin Laterosporulin-ThuricinS access to cross the cell membrane of P. aeruginosa. This technology can also be a way for us to optimize the effectiveness of the bacteriocins generally.

Bacteriocins as probiotics (Top)

Probiotics are living microorganisms that when consumed in modest amounts, confer to health benefits for the host. Studies have demonstrated the impact of bacteriocin-producing strains’ ability to compete within complex microbial communities and thereby influence the health of the host. Here it was seen that bacteriocins are able to directly inhibit the invasion of competing strains or pathogens, or modulate the composition of the microbiota and have an influence on the host immune system Dobson, A., et al. (2012). "Bacteriocins Bacteriocin production: a probiotic trait?" Appl Environ Microbiol, 78, 1-6.. However, when using bacteriocins as probiotics it is important to make a thorough evaluation of the ethical aspects of it, as well as a risk assessment.

It is suggested that probiotics producing bacteriocins could lead to positive probiotic functionality in a number of ways:

• Bacteriocins could function as colonizing peptides, facilitating the introduction and dominance of a producer into an already occupied niche, thereby facilitating the competition of probiotic bacteria with the unfavourable resident bacteria.
• Bacteriocins could act as antimicrobial peptides that directly inhibits competing strains or pathogens.
• Bacteriocins could function as signalling peptides that either signals other bacteria through quorum sensing and bacterial cross talk within microbial communities, or through signalling cells of the host’s immune system Dobson, A., et al. (2012). "Bacteriocins Bacteriocin production: a probiotic trait?" Appl Environ Microbiol, 78, 1-6..

Ointment against MRSA (Top)

Purified or partially purified bacteriocins hold great promise in treating an unhealthy bacterial flora by targeting pathogenic bacteria and may be employed as pharmabiotics or novel alternatives to existing antibiotics. Promising results have been shown in fighting MRSA with purified bacteriocins Dobson, A., et al. (2012). "Bacteriocins Bacteriocin production: a probiotic trait?" Appl Environ Microbiol, 78, 1-6.. Hans Jørn Kolmos suggested that we could use pure bacteriocins in a salve, which can be applied at the nostrils to avoid MRSA that typically niches here. Applying the bacteriocins at the nose, will make their effect most effective on carriers.

Bacteriocins for medical equipment (Top)

It has been proven that surgical suture coating with antimicrobial peptides incorporated is more effective than the Vicryl Plus Antimicrobial Suture against S. aureus in time-kill experiments Li, Y., et al. (2012). "New bactericidal surgical suture coating" Langmuir : the ACS journal of surfaces and colloids., 28(33), pp. 12134–9.. There lies therefore great potential in using bacteriocins for medical equipment.

Spider Silk

Recombinant spider silk can be used for many purposes. Today’s goal for wound treatment is a patch that can provide protection, care and minimize the amount of pain the patient has to go through. During our project with creating Bacto-Aid, we found out that the silk’s potential is great. Here is some of the ideas for its future application.

Creation of skin cell culture (Top)

It has been a goal for many years to get a material that is as close to human skin as possible, because skin grafting from other parts of the body or donors is often limited. Given the many properties of silk, there has been tentatives of combining it with keratinocytes and fibroblasts to create a skin cell culturing. It was also shown that spider silk could guide the proliferation of the cells in the proper directions (staying in the frame) Wendt, H., et al. (2011). "Artificial skin--culturing of different skin cell lines for generating an artificial skin substitute on cross-weaved spider silk fibres." PLoS ONE, 6(7), pp. e21833..

Antimicrobial gauze (Top)

As an alternative to our Bacto-Aid, our hybrid silk could be used as an antimicrobial gauze. The gauze could then be used for larger wounds and for wounds producing a lot of liquid. The liquid was a problem that Laura Jakobsen made us aware of, and the gauze could be a way of solving this. The gauze could also be dipped into the analgesic Ibumetin.

Soft implants (Top)

Soft implants have been subject of interest the past couple of years. Here it has been suggested that spider silk could be braided in a structure and grafted to the bone Li, G., et al. (2015). "Silk-based biomaterials in biomedical textiles and fiber-based implants." Adv Healthc Mater, 4, 1134-51.. This would allow quicker healing of the bone and ensure the creation of proper bone structure.

Nerve scaffolds made of spider silk have likewise shown great potential. Usually bigger motor neurons will not regenerate if degraded or cut. Silk as nerves, could lead to the gain of motoric activity in extremities that could otherwise not be regained after major accidents Li, G., et al. (2015). "Silk-based biomaterials in biomedical textiles and fiber-based implants." Adv Healthc Mater, 4, 1134-51..

Some types of silk have great mechanical strengths and they can therefore be used as an artificial ligament or tendon. The combination of silk with stem cells could allow cells to proliferate and differentiate into the proper type of cells, which could potentially make tendons and ligaments heal, a thing that does not occur naturally Li, G., et al. (2015). "Silk-based biomaterials in biomedical textiles and fiber-based implants." Adv Healthc Mater, 4, 1134-51..

Textiles made from silk (Top)

Silk could be used as a textile for surgeons or for patients with skin problems. The spider silk can tolerate today’s sterilization process (autoclaving), making it possible to be used for different forms of medical interventions. Some already existing products made of silk have shown good results towards reducing the reaction of the disease in question Li, G., et al. (2015). "Silk-based biomaterials in biomedical textiles and fiber-based implants." Adv Healthc Mater, 4, 1134-51..

PHB

Previous iGEM teams (Tokyo Tech 2012 and Imperial College team 2013) have been receiving promising results in improving the production of PHB. With our introduction of a secretion system, the PHB production may finally be a financially beneficial solution to the current plastic consumption. Here is some of the ideas for PHB’s future application.

Implants and coating for guided bone regeneration (Top)

PHB is biocompatible and biodegradable, making it well suited for application inside the human body due to the hydrolysis of the polymer that produces a ketone body, commonly found in blood Rodriguez-Contreras, A., et al. (2016). "Modification of titanium surfaces by adding antibiotic-loaded PHB spheres and PEG for biomedical applications." J Mater Sci Mater Med, 27, 124.. These properties gives PHB the capability to become an implant that could be broken down slowly under the healing process of the bone. The slowly degradation of the plastic makes it useful to guide the bone regeneration. Using PHB as implant could allow us to 3D print the structure of a joint and thereby fulfill the medical field’s wish to create treatments adapted to each patient.

This type of plastic is also suitable for load-bearing orthopedic implants, as the moduli of elasticity, tensile strength and strain properties of PHB are similar to bone material Meischel, M., et al. (2016). "Adhesive strength of bone-implant interfaces and in-vivo degradation of PHB composites for load-bearing applications." J Mech Behav Biomed Mater, 53, 104-18.. PHB’s strong alkaline ester linkages makes its surface more hydrophilic. This gives human osteoblasts the opportunity to proliferate and inhibit the growth of S. aureus on the surface of the implant Karahaliloglu, Z., et al. (2015). "Antibacterial Nanostructured Polyhydroxybutyrate Membranes for Guided Bone Regeneration" J Biomed Nanotechnol, 11, 2253-63. Karahaliloglu, Z., et al. (2015). "Antibacterial Nanostructured Polyhydroxybutyrate Membranes for Guided Bone Regeneration" Hua Xi Kou Qiang Yi Xue Za Zhi, 18, 215-8.. PHB will therefore be able to create a secluded space, where it is possible to avoid infection and the formation of fibrous connective tissue.

Coating of implants in combination with antimicrobial compounds (Top)

Today’s implants could also be improved by a bactericidal coating of PHB. Implants used today are often made of titanium, which have no bactericidal effect. When inserted into the body, a bacterial contamination could create a biofilm, which could resolve in an infection and a necessity to remove the implant again Connaughton, A., et al. (2014). "Biofilm disrupting technology for orthopedic Implants: What’s on the horizon?" Frontiers in Medicine, 22.. PHB could be loaded on the surface of the implant as spherical micro- or nano particles loaded with antibiotics or bacteriocins, as suggested by Morten Østergaard Andersen. The bacteriocins will be released continuously and thereby prevent infections and biofilms.

Intracellular drug delivery (Top)

One of today’s issues among drug delivery systems in the human body, is when a drug has to be transported intracellularly. PHB nanoparticles can cross the plasmalemma, thereby solving one of our treatment limitations Kapoor, S., et al. (2016). " Intracellular delivery of peptide cargos using polyhydroxybutyrate based biodegradable nanoparticles: Studies on antitumor efficacy of BCL-2 converting peptide, NuBCP-9." FInt J Pharm, 511, 876-89..

There is, likewise, an increasing wish to create a personalized treatment to each patient. To avoid side effects and fulfill today’s wish, an in vivo drug delivery gel based on PHB could provide a non-invasive, local and steady release of medicine. In the example of cancer, the degradability of PHB is taken in advantage by incorporating the chemotherapeutic into the gel. As the gel is degraded, the medicine is released. Adding or changing components of the gel, changes the rate of which the medicine is released, thus allowing customization Wu, L. Y., et al. (2016). " PHB-Based Gels as Delivery Agents of Chemotherapeutics for the Effective Shrinkage of Tumors." Adv Healthc Mater..

Future market for PHB (Top)

Plastic is a big part of many daily routines. Biodegradable plastic, produced by E.coli, has a huge potential, because it can replace conventional plastic. The main thing that withholds this from happening, is the price. PHB is today far too expensive compared to the traditional production. Nevertheless further improvement in mass production of biodegradable plastic offers a wide spectrum of usage, e.g. in the agricultural and horticultural sector, for food, medicine packaging and much more " PlasticsEurope ". Hopefully, this breakthrough will happen within the next 10-20 years and thereby change the plastic market drastically. The price of one kilo Polystyrene (used commonly in today’s tableware) is between 0.50-1.29€ " Plasticker". PHB’s price is however, very high at the moment. A price around 1.5 - 2€ pr/kilo may rise the interest of PHB for future investors.

Our contact with several plastic companies gave us hope for this to happen, but they also pointed out, that it is the customers that should be convinced if they were going to implement PHB in their company.

Ideas from high school students (Top)

We asked the students to imagine how our project would be incorporated in 50, 100 and 150 years in our everyday lives. They were especially very interested in the future prospects of bacteriocins. Some of their ideas are presented here.

50 years

• Bacto-Aid can prevent scar tissue creation
• it will be possible to buy Bacto-Aid at the local pharmacy and grocery stores. Hospitals will use it and it will be a part of people’s first aid kit
• Bacto-Aid will be in spray form
• Bacto-Aid can treat people with psoriasis

100 years

• bacteriocins could be a composite of makeup, shampoos and creams to prevent infections
• bacteriocins could be applied on door handles to prevent bacteria to spread
• disinfection gels will contain bacteriocins. Hans Jørn Kolmos also told us that resistance towards disinfection gels has become more common. We are therefore obliged to invent a new alternative
• Bacto-Aid can now be bought as a cream and in a tablet form. The tablet will be able to cure internal infections
• the bacteriocins will be incorporated in bedclothes
• apply bacteriocins in chewing gum
• Bacto-Aid as a special bandage for people with amputated bodies

150 years

• here we might go back to the antibiotics we know today
• chip in the body which sends out bacteriocins when needed, e.g. for old people
• bacteriocins will make our food last longer
• bacteriocins will be in medical suits and operation gloves

Roger, over and out (Top)

These are just a few examples of the possible applications, that our project contains. We have mainly focused on the medical sector, but other applications can be assumed as well. It is just the beginning and as Thomas Edison said: “There’s a way to do it better - find it”. The foundation for our idea has now been made, but we will continue to improve our idea and hopefully one day we will end up with a sustainable product, which we can commercialize.

We look forward to present our project in Boston, and hear about all the other iGEM projects.

Thank you for reading our interactive lab book, we hope you enjoyed the tour!

Click next if you want to see our attempt on winning the best model award.