Shaping our Project

When designing our project, we aimed to create something that could truly be appreciative in the field. We have therefore extensively explored the needs and concerns of the various stakeholders in multiple ways. Each of these interactions has shaped our rather vague initial idea, a smart bacteria for diagnostics, into a well-rounded solution to a known problem in the are of veterinary diagnostics.

Vidilab

Vidilab (a veterinary diagnostics company) was consulted at multiple points in our project, from the early beginning when we didn’t have a project to the very end of our lab period in August We talked both with management (the CEO, Bitte Ljungström) and researchers at the company (Alice Anlind, Sara Hägglund Ljungström). Upon these discussions, we learned what the major issues of the field were, and how large the need was for a new diagnostic tool for small strongyles. This was in the end the reason why we decided to take up the fight against these parasites.

Initially, our project was focusing mainly on small strongyles and in vitro diagnosis of the encysted larvae. From literature we understood that small strongyles formed cysts containing hundreds of individual worms in the intestinal tissues of horses. It was these cysts we initially planned to detect - isolate them from horse tissue and co-culture with bacteria.

Vidilab were kind enough to provide access to horse tissue with encyctated larvae. The cysts we received did not correspond with the literature . Due to these discoveries which we uncovered during the collaboration with Vidilab, we decided to focus on detection of the worms rather than the cysts. But as we knew, free floating small strongyles do not put the horse into significant danger per se, instead we decided only to focus on increasing the possibility to give horses more accurate treatments. Due to Vidilab we also knew that the use of anthelmintics had lead to resistance developing among the strongyles making them into a threat to the equine industry and horses physical health. By giving more accurate treatments, the resistance development rate among strongyle could be decreased.

After a while, during our second visit to Vidilab they also informed us about the difficulties detecting large strongyles since all current techniques we’re based on egg counting in grass samples. After some consideration we outlined a strategy to battle this problem as well. First of all we wanted to investigate the need for a better preventing tool for large strongyle infections as well. In order to gather the data we worked on market analysis based on a survey that we spread among horse owners in Sweden. Secondly, we wanted to try if a similar system as we outlined for the small strongyles could work with large strongyles present in grass, hence we made analyses of the visibility of fluorescent proteins in grass sampling.

Interviews

Horse owners

We have also utilized all the opportunities we had to discuss our project with actual horse owners. From these discussions we learned for example about best ways of isolating samples from horses. We learned about the difficulty of isolating urine from a horse and therefore moved on to investigating detection methods in the feaces. These interviews also first gave us the idea to start investigating on a larger scale to what extent are the horse owners aware of issues related to strongyles infections and the idea of conducting market analysis was born.

Veterinarians

We contacted different veterinarians in the beginning of the project to see how their own experience was of the problematics with strongyles. The overall view was that they did not know about horses being ill from large amount of small strongyle, but they talked about the negative effects with large strongyle. This results pushed us to investigate more on large strongyle infections.

Multinational market research

Before we could start with our project we first needed to look into whether there is a market for our diagnostic tooI and whether we could shape the potential product to an existing nisch. We were interested in the actual state of urgency of the disease along with potential concerns for the future, though as from the above mentioned communication we have been receiving some conflicting information. Some sources would state that strongyle infections are of relatively small concern whether others would see it as a large issue. To analyse this further we conducted a market analysis based on a survey that was distributed not only in Sweden but also abroad. This confirmed that the needs of the horse owners regarding large and small strongyles was consistent to Vidilabs needs, indicating that the majority of each stakeholder had the same view of the current situation. This was partly seen in the large concern regarding small strongyles, contradicting some of our sceptical sources. In addition, we used the opportunity to gather data about how a GMO based diagnostic tool will be perceived among horse owners.

To advocate our stand that amount of treatments can be optimized, we needed to know the mean amount of treatments performed on each horse per year. We received this data from the market analysis. Our predictive model simulating the extension of strongyle infection depending on the amount of anthelmintic treatments suggests that the current situation can indeed be optimized. Fewer treatments than the average today would decrease risk of developing anthelmintic resistance and still keep the horses healthy.

The market analysis gave us insight of the horse-owners perspective and the objective data that we needed to draw any conclusions. We used the data from the survey to confirm our project strategy and to build our different models on. To learn more about Market Analysis read below.

Market Analysis

The iGEM project started with a brainstorm session to decide in which direction we wanted our project to go. But before starting with our project, we had some crucial questions that needed to be answered. What tools are accessible today and what are their limits? Is there a market for a new diagnostic tool with our approach? Is there a need for a new diagnostic tool for large strongyles as well? How do the potential stakeholder or customer feel about using a diagnostic tool based on GMO?

To answer these questions we chose to proceed by setting up a meeting with the company Vidilab (a Swedish company working with diagnostics of parasites such as strongyles). Vidilab are everyday users of the current diagnostic tools thus are deeply involved and experienced within the area of our project, making them great consultants.

In addition, we conducted a survey to receive greater understanding of the individual perspective of the respondents. The survey was translated into 10 different languages, see table 1 .

Language	Amount of answers from the survey
Swedish	370
Czech	75
Latvian	38

Table 1: The table show the translational versions of the survey, sent in the market analysis, with highest amount of answers.

Ethics

The purpose of our project was to combat the systematic overuse of anthelmintics [1] and thus also address the growing resistance against such compounds. Resistance may arise from multiple factors, like that of mass treatment, underdosing and not varying the drugs used [2] which is not unlike how resistance against antibiotics arise [3]. The resistance against anthelmintics might also be a problem for humans in the future [4] which increases motivation for a new diagnosis and-/or treatment approach. This is “good” news since the organism used in our project, namely the E. coli are inherent to the microbiome of humans and horses alike. We believe that a new, specific and potentially eco-friendly method of diagnosis or treatment would have a positive outcome on society as a whole. Lowering the degree of stress and potential suffering for animals that get infected as well as for their human owners.

There’s however some concerns in public about E. coli as being pathogenic or a transmitter of disease. It’s often the incidence when E. coli contaminated meat for consumption gets coverage in e.g. news media. It’s thus a challenge when conveying a complex topic to the public which demands some scientific knowledge, especially in biology and is something our Human Practise group has been challenged with throughout the project. The risks using E. coli and its given traits for this project are discussed further throughout the text.

GMO, Welcomed by the Public

By authoring and distributing a survey to horse owners we could see that the majority of respondents in Sweden had positive attitudes (83.2 %, 368 respondents) towards a GMO-type of diagnosis method. The survey also showed a public fear of growing anthelmintics resistance against blood worms (77.3% of 370 respondents ranked their fear as 4 or 5 were 5 being the most concerned).

Environmental Impact and Risk

As new biomolecular technologies are presented, having a higher degree of specific interaction against targets and thus a lower ecological impact they may shift public opinion on synthetic biology and allow for other solutions to surface. This would possibly also bring interest back to the natural sciences and thus probably increase solutions that stem from e.g. synthetic biology.

As with most techniques or processes used, whether there be some exercise regimen for increasing athletic performance or treating crops with pesticides to ensure a certain yield, they most often have some sort of risk factor associated with them. No exception is made when organisms are taken from the environment undergoing genetically engineering which may modulate or even add capabilities. Risks associated with engineered organisms may be proliferation in the environment, organism being pathogenic, spreading of capabilities such as antibiotic resistance, synthesis of molecules that might be toxic or harmful to name a few. However, most nations have guidelines and laws regulating which entities operating in their domain must adhere to, whether they are companies, universities or individuals. As for members of the european union there are also an overlaying legislation which its members must follow [5, 6].

The E. coli (BL-21, DH5-alpha) that was used in the lab has a biosafety level of 1, which is the lowest possible on a scale of 4 [7]. That means e.g. that E. coli requires a minimum of safety precautions other than those provided by Uppsala university [8].

Nothing that is considered toxic or harmful was genetically introduced (UnaG, amilGFP) for expression in our E.coli. However, for cultivation and selection purposes used in common synthetic biology practical work, antibiotic resistance was also introduced, which presents a risk in the potential spread of antibiotic resistance (Chloramphenicol, Ampicillin, Tetracycline) if these mechanisms were to spread into the environment. This was avoided to a large degree by following standard lab protocol with specific guidelines regarding waste disposal of GMOs as well as toxic chemicals, antibiotics and their likes.

The ability for proliferation of GMOs and organisms in general is determined by their fitness. As a rule of thumb regarding spread of GMOs in the environment, fitness is greatly reduced for most lab strains of microorganisms (model organisms) because they have been modified in numerous ways to make them easy to work with. This is not an adaptation that is suitable for their natural environment from which they originated. Add to this the metabolically expensive process in expressing proteins (UnaG, amilGFP) which can only be deemed as a negative fitness contribution [9]. Regarding risks associated with nematodes see separate safety form and select Uppsala from the meny.

Noting that this was the first time one sought to see if this is a applicable solution to treat parasitic infections caused by nematodes in ungulates, it thus is a long way from a potentially finalized product. If in the future a product would surface, further risk assessments must be evaluated to see if an engineered product would be deemed safe to be orally ingested by a living host. Since enterio-diagnosis-systems based on bacteria is a novel approach there is little to be found in the litterature about its applied effects. However, this sort of assessment is out of the scope of our project and could not be, as well as, was never intended to be experimentally verifiable and only mention to give context and reasoning to future research if implementation of such product is to be realized. In addition to internal reviews, it is common for an external part (like governments) to perform reviews on the product, especially if the product is GMO classified. This is necessary to ensure that the product is safe before letting it hit the market.

Technologies can be used for good and bad. This well reversed statement implies that it’s not the technology itself that is either good or bad, rather its the use and implementation of technology that determines its consequences. Implying that its the use and the intentions of the user that sets the outcome, the user (including companies) should thus be held accountable for any unethical use. Bearing this in mind, that the operators behind a technology must be held responsible for his or her actions, saying no to upcoming technologies or research (such as those of enterio-diagnostic systems) would imply that people are not able to make the right decisions, with the emphasis on a hypothetical misconduct by a few. If this was the case we would have stopped before tied sharpened pieces of rock to a wooden shaft. We should instead treat people as responsible human beings and the development of a new technology as a bridge between an initial state and potential end state, whatever that end state might represent.

Disrupting Commerce

Just as we’ve seen unprecedented advancements in technology in the recent years or in the last century there is no reason to think the future holds a different promise. Research is done both by academia and in the private sector. Their approach and funding often differs in the sense that a company have to make a profit from a product in order to be able to reinvest and stay competitive whilst academia are more self-sustainable in the means of grants and-/or other tax funded solutions as well as donations from various contributors. It might be that a new product developed through iGEM would put a business in a disadvantage, this is of course possible, but we feel this shouldn’t interfere with or discourage teams in their pursuit of new innovative solutions.

Understanding Your Limits - a Burnout Guidebook

Mental health awareness is gaining appearance in society, yet it is still a tabu in many settings. For instance, in a project that you truly care for, the risks and warning signs might go by unrecognized. It is an art to find balance at work but in our opinion it is an essential part of a success in the long run. Therefore iGEM team of Uppsala, chose to acknowledge the importance of understanding one’s limits by writing a guide-book on the topic.

The premise of the guidebook is that many iGEM teams in retrospect admit that work environment in iGEM oftentimes becomes stressful. Factors such as level of experience, responsibilities, ambitions, deadlines e.g. drive projects forward but at the same time easily lead to feelings of overwhelm and increases risk of burning out. However, even if working with iGEM underlines these factors, the topic of stress at work is recognized universally. Thereby we wanted to address this topic by creating material that anyone who seeks guidance can relate to and use. Thereby we tried to make our guidebook as universal as possible - iGEMers as well for students of any discipline and people in as well as outside of academia.

In this little book we have compiled some general background about stress, exhaustion and burnout in order to explain the issue and show that these topics deserve your attention. This is followed by suggestions of some preventive measures that we have found suitable. Some parts of the book might be a repetition of old truths but we tried to mix it up with e.g. mindfulness exercises.

In the guidebook you can also find some statistics about stress levels in iGEM specifically. These statistics are a preview from iGEM work environment report that iGEM Uppsala association is working on based on the survey described further down.

Also, check out the chapter “The importance of authenticity in Organization” from iGEM Lund guidebook . They have also been focusing on improving work environment but instead of more general depictions their guidebook focuses on hands-on-approaches for increasing efficiency through organization of work.

Our hope is that we will encourage people to not overlook the importance of their well-being and lead to healthier relation to work. As we wish to give back to the iGEM community for all the wonderful experiences we hope that future iGEM teams especially, will take part of the material in “Understanding your limits - a burnout guidebook”. iGEM is giving platform for young, brilliant people to do truly amazing things. However brilliant people don't retire after iGEM - take care of yourselves because the world needs you.