Before YarroWCO

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Our first stop on our integrated human practices journey was selecting our project. Based on our interests, we wanted to solve a local problem that involved sustainability – in that way, we could truly deliver a useful contribution. We describe how we chose our project in more detail on our project description page! You will also see there that we mention decision trees that helped us refine our project; here they are!

The decision trees also show the evolution in our team’s thinking. They are all written in hindsight, taking into account feedback we and other teams received. More specifically, they are based on the advice given to us by previous KU Leuven iGEM team leaders as well as our PI. They are also inspired by interactions with stakeholders (both advisors and people from the local community – read the rest of this page for more information), as well as critical questions raised during iGEM meetups (Münster and Utrecht). More details on how specific stakeholders influenced our reasoning are detailed in the menus below.

Our decision trees are sufficiently broad to be applicable to other projects, and we leave this as our biggest contribution to future iGEM teams. Our decision trees can be used as a toolbox to accelerate the development of all project related issues, but especially well-integrated human practices. They provide a structured context that can be followed by other teams and that we expect could raise the quality of IHP across iGEM teams - in good alignment with iGEM's own view of improving the quality of the competition every year.

Stakeholder Map & Summary of Our Human Practices

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When first coming up with our own project, the first and most basic thing we thought about was our own pipeline. This makes sense: we needed to make sure that you’re even able to get the required starting materials and that you are able to sell them to someone. However, we soon realized that we would be integrating into an existing ecosystem with many more stakeholders. This represents the second iteration of our stakeholder analysis. By talking to some of those stakeholders however, we soon realized that we should expand our analysis even further. This is our third and most comprehensive analysis. Because it was largely obtained by talking to stakeholders, we like to call it our “stakeholder map”, rather than just a stakeholder analysis. We use this analogy because a map is progressively filled in and discovered as well, and the same goes for our stakeholder map. The fourth schematic shows everything that we’ve done for integrated human practices apart from the decision trees in the context of the stakeholder analysis. Hence, the reader can consider these schematics as a summary of our integrated human practices page.

Before diving into the matter, we would really like to thank Professor Dirk Stemerding for acting as an advisor in coming up with the stakeholder analyses. He was incredibly helpful in moving from a rudimentary outline of stakeholders to the full picture that we have now. Moreover, he guided us towards creating a well-structured analysis resulting in us clearly identifying the different ​pipelines

General Pipeline

This is more of a basic schematic than a true stakeholder analysis per se. You can see that at this point, our perception of who would actually supply waste cooking oil is very rudimentary. We also don’t have such a clear idea yet of what pipelines exist on the right side (“selling side”) of our pipeline.

Current Ecosystem

This iteration shows a much clearer picture of how this business works currently. There are actually three pipelines shown in this schematic:

1. The hypothetical pipeline relative to our project in green.
2. The current pipeline that uses waste cooking oil as starting material and goes all the way to biofuel production in yellow.
3. The current pipeline that starts from slaughtered cattle and plants, and ends up by delivering steroid medicines to patients in blue.

Please note that some boxes are shared between pipelines and are marked by a colour gradient. On a different note, dotted arrows are hypothetical. Black arrows are arrows that are shared by multiple pipelines. Other arrows are coloured according to the pipeline in which they belong. The line between local population and WCO dealers is dotted because it only exists if a collection point is present in a certain neighbourhood.

A striking element in this analysis is that our project connects two completely unrelated pipelines. We see this as an opportunity as well as a challenge. It is clear that no one has gone here before, but on the other hand, it might be challenging to get all stakeholders on board.

Two boxes were “discovered” by talking to stakeholders, as explained further on the integrated human practices page. Namely, by talking to snack kiosks (“frituurs”), we heard about Quatra and WCO dealers for the first time. It is by talking to Quatra and to authorities that we discovered that most of the WCO that is currently produced is transformed into biofuels.

Stakeholder Map

The third iteration of our analysis shows further understanding of the situation. By talking to the public through our surveys and people we know, we realized that we had to split the general population into two groups. We also realized that the student group couldn’t deposit their oil at EcoWerf.

Furthermore, we soon realized that the world of WCO is heavily regulated. Thus, we added local authorities and also connected them with the pharmaceutical industry, as this is heavily regulated as well. Local authorities are in turn regulated by the EU, which actually promotes and also regulated the transformation of WCO into biofuels. All regulatory interactions are shown in dark green.

The boxes in dark blue are what we call “advisory” relations. Like regulatory bodies, they aren’t inherently part of any pipeline, but they still have an influence on them. They include organizations that care about the environment such as Greenpeace as well as patient organisations. It is important to include them, because they can have an important impact on the market. For example, biofuels are becoming somewhat controversial, which in turn impacts the whole ecosystem. Finally, a more “neutral” advisory group would be experts, that advise both the governments as well as the industry.

Summary Visualized in the Stakeholder Analysis

The first thing you’ll notice here is that a large portion of the stakeholder map has been removed. This is for readability purposes – please picture the rest of the map to still be there in your head.

Something that should be clear from this schematic is that we mostly interacted with the left side of our stakeholder map. In fact, we did try to get in touch with the pharmaceutical industry, but this turned out to be very hard and we soon decided to shift our attention to different matters. Starting from the top, the summary goes as follows:

1. Experts: gave us advice on how to accomplish our goals in wet lab, dry lab, and team management.
2. Pharmaceutical industry: we made attempts to interact with different corporations in the industry, but nothing worth reporting on in more detail came out of this.
3. Local authorities: we got some information from Valorfrit, an organisation connected to the Belgium government, on exactly how much WCO is produced and collected in Belgium yearly. On the other hand, we realized that what we wanted to do in our project would be regulated (because it would be an API precursor) and decided to think a little more about it.
4. Small businesses: they gave us some descriptive statistics that we report on, and we discovered the existence of Quatra thanks to them.
5. Local community: people domiciled in the municipality where they’re staying: we created awareness about the fact that WCO is dangerous waste that should be disposed of properly. In return, they gave us information on how they dispose of WCO through our survey. By setting up the oil collection point, we also made them supplying WCO to Quatra much easier, even if our main target group were students.
6. Quatra: the European market leader in the delivery, collection and recycling of used cooking oil; we interacted with them, and they told us much more about the current ecosystem surrounding WCO. We collaborated with them to set up the WCO collection point.
7. Local community: people that aren’t domiciled in the municipality where they’re staying: this is very similar to the previous description about local community. However, the oil collection point that we put up is much more important for this community, as they otherwise didn’t have the possibility to get rid of their WCO responsibly.

Interaction with the local community

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What kind of waste cooking oil is generated in Belgium and how do local businesses look at it?

Moreover, 85% of Flemish people (the Northern region of Belgium, where our university is located) eat fries at least once a month while 60% do so at least weekly. Interestingly, over half of the fries aren’t made at home but are either bought as take away or consumed outside the home. It is easy to see that that this generates a lot of waste cooking oil (WCO), which is the flip side of all of these delights. This truly makes our problem local.

Since so many fries are eaten outside of home, we decided to talk to small businesses (the abovementioned snack kiosks) to gather data on how much WCO they generated, how often they had to change the oil, how and if they recycled it, and so forth.

Very importantly, we also wanted to know what type of cooking oil they used, since there are several types with different origins (animal or vegetal being the main distinction). This would help us finetune our wet lab project if we wanted to work with actual WCO.

What we learned from those interviews was the following:

WCO Handling in Belgium

WCO, or oil in general, is a dangerous waste. It is well-documented that one litre of oil can pollute up to one million liters of water (2,3). Additionally, if people simply wash it down the sink, it can lead to fatbergs. Fatbergs are large piles of debris that clog up sewers and that cost hundreds of thousands of dollars each to remove. Moreover, the frequency with which fatbergs are developing is increasing in the UK ⁴ and probably in all of Western Europe.

Based on this, it should be clear that WCO needs to be handled and recycled properly. We decided to investigate how much of it is produced yearly, how it is normally disposed of, and whether we can contribute in this regard.

WCO Disposal Logistics in Belgium

The smallest administrative subdivision in Belgium is called a municipality. For example, the municipality of Leuven includes Leuven and several other small towns right next to it, such as Heverlee. Inhabitants of a certain municipality can only go to an EcoWerf attached to their municipality. They will also need to pay a small fee to enter, based on the amount of trash they’re bringing. Keep this in mind for the discussion in the next section, as this will be very important later. For now, let’s focus on our search for information and data via the authorities.

How could this be a problem to certain demographics, such as students?

Before we evaluate how exactly we promoted awareness, let’s look at another problem hidden in Belgium’s administrative complexity.

As mentioned above, access to EcoWerfs is reserved to those living in the municipality where the EcoWerf being visited is located. Now, by “live”, we actually mean domiciled. The problem here is that in Belgium, students generally aren’t domiciled in the city where they study in. They do “live” there, but only rent a room and thus aren’t considered inhabitants of the municipality. This means that they don’t have access to their local EcoWerf.

This poses a problem that concerns our project, because this means that students really have nowhere to take their WCO. In theory, they could take it home for disposal, but this is of course very unpleasant as well as unpractical. Moreover, taking it home is, of course, not an option for international students.​​ Additionally, the WCO disposal problem is even bigger for student organisations that organize events on a regular basis! Sometimes, they’ll want to provide snacks and since this is Belgium, those snacks are very likely to be deep fried. This produces significant volumes of WCO that students can’t responsibly dispose of because they don’t have access to EcoWerf.

It was clear to us that WCO was a problem to be addressed. Nonetheless, we needed more data to determine the true magnitude of the problem.

Surveys

We conducted a survey among students and residents from Leuven. We gathered insights from 62 respondents, with 44 of them being students. The goal of our survey was to understand the production and disposal methods of WCO mainly among students.

25% of students responding to our survey expressed being highly concerned regarding the impact of WCO on the environment, and 45% indicated being moderately concerned. At first glance, this seems like good news, but the next questions showed a much more negative image. On average, students were found to generate 0.72 liters of WCO monthly. Alarmingly, 43.3% admitted to disposing of it down the drain and 36.4% indicated they throw it in the regular trash. When questioned about their current disposal habits, most students indicated convenience, lack of proper information on disposal methods, and absence of nearby collection points as key driving factors. This confirmed our suspicion that the awareness of students regarding proper WCO needs improvement.

Crucially, when asked about whether if they would be interested in a WCO collection point nearby, a staggering 86.4% responded yes. This overwhelmingly positive response clearly indicates the need for the establishment of a WCO point in Leuven for students. Addressing this need does not only aligns with the students' preferences but also paves the way for a more sustainable city of Leuven.

In addition, we also wanted to get an idea of awareness concerning ​​WCO among the general population. Among non-students (limited to only 18 respondents), almost 40% indicated to have never even heard of recycling WCO. 50% admitted to throwing it down the trash and 33% admitted to throwing it down the drain. Given that non-students were likely Leuven residents, their response was alarming. It showed a significant lack of awareness and another significant source of WCO pollution in Leuven.

Conclusion 1: Awareness Promotion

For more information on (social) media presence and our efforts at raising awareness, check out our Outreach page.

Conclusion 2: Oil Collection Point for Students

A second important conclusion for us was that we needed to help out students. Before we really go into this however, we should introduce another stakeholder that was loosely mentioned before.

Quatra is a family company based in Belgium that specialises in the collection of WCO from businesses. However, during our introductory meeting, they also mentioned that they organize collection points. These consists of a container where anyone can deposit their WCO. Quatra sells their WCO to the biofuel industry and thus only specializes in collecting WCO.

Here, something clicked. Perhaps there was a possibility to help students through Quatra, right? If they could set up a collection point in the centre of Leuven, the issue would be solved and we would have delivered a tangible contribution to WCO collection in Leuven.

Experts

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How should we handle wet lab?

Most of our students might already be doing their master’s degree, but we certainly did not have the expertise to come up with all aspects of our wet lab project on our own. Thus, we contacted many experts in the field that acted as advisors for our project and helped us figure out the details of wet lab work. They also contributed to novel perspectives and asked additional critical questions that further helped us develop our idea into a solid project.

Wet Lab Experts

Expertise from within and from outside KU Leuven was crucial to the planning and success of our scientific journey throughout the iGEM competition.

Vitor Pinheiro

As our PI, Prof. Pinheiro was instrumental to our wet lab plan from the beginning of our journey. He suggested that we take our strategy one step forward and not only try to leverage Yarrowia lipolytica’s growth on oil, but try to improve it by producing molecules with biosurfactant properties. He shared valuable literature with us that led the wet​ ​lab team propose our pilot library of proteins with biosurfactant properties. Moreover, he put us in contact with a wide network of experts in genetic engineering and yeast biology, whom we later contacted for specific advice on how to carry our project. Prof. Pinheiro provided contin​u​ous feedback on our progress in the wet lab and helped us overcome the obstacles we encountered throughout the summer.

Kevin Verstrepen, Vasileios Vangalis, Seppe Dockxs

Professor Kevin Verstrepen validated our initial approach of growing Yarrowia on a waste stream and pushed us to find a novel angle to our project that would differentiate it from other academic works that work in a similar direction. He put us in contact with two members of his team, Dr. Vasileios Vangalis and PhD student Seppe Dockxs, with whom we had a meeting and discussed our strategy of growing Yarrowia lipolytica on oil, as well as the need for producing biosurfactant molecules. They advised us on which Yarrowia strains would be best to acquire and suggested that we test the growth of the yeast on a glucose and oil substrates for comparison. They later offered practical advice on how to conduct measurements of Yarrowia growth on different substrates. Very importantly, they were able to help us identify other Yarrowia experts from Europe, who then provided valuable material for our project.

Giovanni Maresca Di Serracapriola

Giovanni, a PhD student from Imperial College London, had meetings with our team members Tom and Vlad in which he provided feedback on our broad strategy in the wet​ ​lab, while suggesting appropriate strains of yeast and strategies on how to procure them. His previous participation in the iGEM competition allowed Giovanni to provide advice tailored to the specifics of the competition, which greatly improved both our strategy in the wet​ ​lab, as well as general principles of team and project management.

Ilse Van de Voorde, Jeroen Vereman

Professor Van de Voorde and Dr. Jeroen Vereman, a postdoc in the Van de Voorde lab, validated our approach of expressing hydrophobins and advised us to focus on one sequence representative of each class of protein we are studying, narrowing down our efforts of cloning and expression in the lab. They also provided practical pointers on working with hydrophobins. Moreover, Dr. Vereman was in contact with our drylab team regarding identifying hydrophobin properties which could be improved through de novo design.

Abram Aersten, Yorben Casters

Professor Abram Aertsen from the Food and Microbial Technology (CLMT) group at KU Leuven validated our approach in the wet lab and suggested that we investigate the potentially toxic role that hydrophobin expression could have in our E. coli expression systems. He suggested a live imaging timelapse microscopy experiment and put us in contact with his PhD student, Yorben Casters. Yorben was in close contact with our wet lab team captain, Vlad, to derive the experimental design of the microscopy experiment. Yorben hosted Vlad in the lab for two days and created the experimental set-up for the microscopy experiment. Yorben was also most helpful in the process of data analysis, as well as providing advice on future directions of improving our expression system.

Patrick Van Dijck, Paul Vandecruys

Professor Van Dijck joined our team as secondary PI in August and generously provided lab space for our team, as well as a S. cerevisiae strain and expression plasmids. He consulted us on the feasibility of expressing hydrophobin proteins in yeast. Dr. Vandecruys from the Van Dijck lab worked together with our team to generate in silico assemblies of cloning vectors in S. cerevisiae and derive a secretion strategy for our proteins of interest, based on the endogenous yest secretion signal from the Mating-type MAT alpha 1 protein. His training for members in the lab, sharing of materials and technical expertise greatly contributed to broadening the scope of our project.

How should we handle dry lab?

The reason why we contacted several experts concerning dry lab is very similar to why we did it for wet lab. Building good models, getting the right data, and interpreting said data is just as important to a good iGEM project as having a good understanding of wet lab work. This is why we contacted several people that helped us build our models and complete our dry lab work.

Dry Lab Experts

Vitor Pinheiro

Vitor, as our PI, was involved in all dry lab sub projects on different levels. He provided us with insights on all of them whenever we needed it and along with Alex Fedorec, gave us very helpful suggestions concerning our genetic switch. Vitor also guided us through designing our own hydrophobin. He advised us on which residues to substitute when mutating the hydrophobin sequence in silico and suggested suitable approach.

Jeremy Harvey

Professor Jeremy Harvey was immensely helpful, particularly with our molecular docking. He provided us with a docking protocol and advised on the validation of the protocol using a reference enzyme with its native ligand, the structure of which was determined experimentally. Prof. Harvey approved all of the steps of our molecular docking strategy and clarified many questions we had along the way.

Mathy Froeyen

Professor Matheus Froeyen granted us very helpful input on molecular docking along with prof. Harvey and helped us see some other aspects of our strategy. He advised us to run molecular docking ten times in order to be able to get clusters of the docked conformations. This part was necessary since AutoDock Vina is a statistical tool and every time it is executed, it returns slightly different results but after a few runs, the results converge to the best one. We were really hesitant when selecting the best docked conformations because we knew that Vina does not explore all biological aspects and mostly focuses on minimising the score that reflects the binding energy. Prof. Froeyen advised us to perform double checks on our docking results that concerned analysing interactions between the ligand and receptor. Using this information, we were able to pick conformations for further analysis. Prof. Mathy Froeyen helped us immensely with performing Molecular Dynamics simulations as well. He provided us with all resources, scripts, and guided us through our first MD simulation. He was very open to all of our questions and helped us resolve any problems that occurred on the way.

Alexander Fedorec

Alex Fedorec from UCL in the United Kingdom advised us throughout our journey with our genetic switch. We reached out to him having very little experience and only few general ideas. Alex helped us jumpstart our bioswitch project. He suggested that we look into a toggle switch since it’s relatively easy to design and would fit perfectly with the purpose of our switch. He also suggested us to contact a research group (Rodrigo Ledesma-Amaro's Lab) that is developing and looking into synthetic biology tools in Yarrowia lipolytica. During a meeting with Alex and Vitor, we also discussed our problems with the design and talked about our ideas. We discussed multiple possible triggers for our switch such as temperature, metabolic load and the morphological transition from yeast-to-hyphal growth in Yarrowia lipolytica. Alex also advised us not to over focus on all of the details (which we had been doing) and try to have fun with the designs. We received very helpful advice and insights on both designing and simulating parts of developing a genetic switch.

How should we handle team management?

Experience from within KU Leuven

Agile Methodology

Not knowing what Agile really was, we stumbled upon a 2019 paper in Nature Careers on how Agile team project management can work for research projects. On the 12th of May, Julia and Brigitte had a call with the author of the paper, Laura Pirrio. Dr. Pirrio is a chemical engineer and a big advocate for using Agile team management in academic research projects. Considering how popular Agile is, it is surprisingly hard to find good information about what Agile really is. Therefore, we interviewed her to find out how we could use Agile our own team management.

End Result: How We Functioned as a Team

Regulations: Things to Consider for Future Application

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While talking to stakeholders and putting together our stakeholder map, we realized that our product would have to abide to important regulations (also see the arrow between “YarroWCO” and “local authorities” in said map). This is also the feedback that we received from the author of a paper⁵ about current ways of synthesizing steroid medicines, Jose Luis Garcia Lopez.

In essence, our aim is to produce a precursor for an active pharmaceutical ingredient. The easiest place to start when thinking about regulations concerning the manufacturing of medicinal products are the European good manufacturing guidelines. The relevant chapter is “Part II - Basic Requirements for Active Substances used as Starting Materials”.⁶ Before diving into more detail, we need to consider three definitions:

When considering our product, it should be clear that it falls in the third category. It is not an API per se, let alone a medicinal product, meaning that it should be classified as a starting material and thus treated as such. The next question is whether this actually falls under GMP guidelines. The guideline says the following:

“The manufacturer should designate and document the rationale for the point at which production of the active substance begins. For synthetic processes, this is known as the point at which "Active Substance Starting Materials" are entered into the process. For other processes (e.g. fermentation, extraction, purification, etc), this rationale should be established on a case-by-case basis.” ⁶

Because of the nature of our starting material, we think that in our case, we would probably have to follow GMP guidelines, even though we’re not aiming at producing the API itself.

The high temperatures that are used for deep frying (170-190 °C) induce all kinds of chemical reactions (such as oxidation reactions) that lead to degradation of the ingredients. The products generated by those reactions can be toxic to human health above certain concentrations (such as cyclic monomers of fatty acids). We can consider these compounds as impurities when considering purity of our target intermediate, namely campesterol.⁷

What would have to be done is to study whether significant amounts of these impurities are present in our purified intermediate, namely campesterol. As should be clear from the rest of our wiki, our team never got to the point where we could do such characterization, and this would require a lot more work. If the impurity profile is unacceptable, several more iterations of the engineering cycle would have to be applied to see how we could adapt the process to get rid of the impurities. Moreover, we could also tweak our hypothetical filtering protocols to attempt to remove the impurities before even introducing our WCO into our cell culture.

The text above shows how we would deal with regulations, but what we shouldn’t forget here is the public’s view on the matter. It is one thing to reassure the authorities that our product is safe, but we would also want to reassure the public of the same thing. Our past experiences on the side of integrated human practices have clearly shown to us that many people don’t have such a good understanding of science and might be sceptical towards our product. After all, we’re making something vital to the health of many patients from waste.

To address this, we would first collect more data through surveys and more interactions with stakeholders. Based on these results, we could then do an information campaign to inform the public of the safety of our product, all while taking into account feedback from stakeholders.

We know that this point is all very hypothetical, but we wanted to address it. It is one of the first logical questions that seems to come up in the minds of stakeholders, so it only makes sense to answer it.

Conclusions and Steps Forward

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Integrated human practices is about talking to people. These people can be experts or complete laymen, stakeholders from the industry or people from decision making bodies. The one common denominator is that you can always learn something if you listen carefully, and listen we did.

We believe that our project wouldn’t have been complete – let alone the same – if it weren’t for these interesting conversations. Everyone that we talked to concerning our project inspired us to do more, do better, changed our perspectives, and always improved the overall quality of our idea.

It was only when looking back on all of these encounters that we realized that we should go for the best integrated human practices prize. To us, it felt natural to have all these encounters and meet all these people. It didn’t really feel like work, more like enrichment of ourselves as well as our project. It is only in hindsight that we realized that we had done an incredible amount of work on integrated human practices, hence our candidacy for the prize.