Team:TU Darmstadt/Human Practices

Human Practices

Introduction

Thinking about how a project could potentially affect the world is something every iGEM team should consider and explore. During the entirety of the year, we did just that. We talked to experienced scientists, visited industry giant Evonik, made sure to educate society by teaching at schools, and entered conversations on other occasions as well. To us, it was extremely important to listen to concerns society might have about our project, as well as synthetic biology in general, and address them accordingly. Furthermore, we wanted to make sure our approach was something that could be viewed as a realistic and plausible idea for monomer and polymer production in the future. As complex as Human Practices is, as complex was our approach to tackle it.

Education and Reaching Out

iGEM projects do not work in a void. Most of the time, they tackle real life problems and try to give solutions to difficult dilemmas. For any project, it is important to investigate safety aspects and give an answer to the question, what good it will do. This year, our project focused on the manufacturing of PLGA and PLGC. Both of these polymers consist of monomers that are currently derived from petrochemicals. These chemicals rely on limited resources like petroleum. It is important to establish sustainable alternatives better for the environment. Our approach uses microorganisms as one possible option to generate the needed monomers. The good thing is, microorganisms are an endless resource and their potential for the manufacturing of many other substances is something becoming more and more important as time passes and as limited resources are growing scarce. We, as scientists working on this subject, see a lot of potential and good in what we do, others around us might think more critically than us. Through Human Practices we wanted to get in touch with as many different people as possible and explore problems we might not have considered before. We also wanted to see if our project has potential and if it is something useful for the future.

Our Human Practices approach can be separated into two main categories: the aspect of education and the outreach towards scientists and industries.

Education

Synthetic biology tends to be an often scary and not well understood subject. As scientists working in this field, we recognize its risks but are also aware of its undoubtedly high potential. To us, it was important to show the less scary side and take away some fears the general public might have, while also teaching the importance of safety. Striking a good balance between these aspects is absolutely necessary, so that those we taught are able to spot risks but do not shy away from something potentially helpful.

This year, we went to several schools in our area, visiting their biology courses. We designed our presentation to be very interactive; and were very pleased to see the students' eagerness to participate and share their opinion. While we talked about biology and iGEM in general, we focused heavily on our project and synthetic biology. A good amount of time was spent discussing the students’ concerns. With regards to our application as nanospheres, they feared micro-plastics and medication leaking into the groundwater. Another issue was the controlled dose of medication released from the nanospheres into the bloodstream. Though, in general they seemed very open towards synthetic biology and its application in the production of materials. We were able to ease some of their worries regarding leftover bacteria in the produced monomers.

Through our connection with CompuGene, we were invited by ProLOEWE to spent a few days at the Hessentag 2018 in Korbach, Germany. The Hessentag is a week long, annual event arranged by the german State of Hesse. Its main focus is the representation of the different scientific fields, institutions and regions of the state. Each year, the Hessentag gathers thousands of people from many different backgrounds. We took this opportunity to talk about iGEM and our project with the general public and get their opinion on it. Something we made sure of, was to help them understand synthetic biology and its approaches better, as well as the benefit our project might eventually have. Most visitors seemed very open to our ideas and described our plan as sustainable and helpful.

A less interactive, but just as important experience was our appearance on Radio Weinwelle. Two of our members went on air for an hour and answered questions regarding our project, who we are, and what iGEM is. Our goal was to break down our points to the most important aspects while still conveying everything we deemed important. We greatly appreciated the opportunity and had lots of fun during the show. The hosts even told us, they had never heard of synthetic biology in such a positive and informative way before. We genuinely hope we could fulfill our mission to bring light to a topic generally misunderstood.

Reaching Out

Exploring whether or not our project is plausible quickly became another focus in our Human Practices work. For this cause, we reached out to several companies knowledgeable in polymer production and scientists from all over the world that had worked with certain organisms or were experienced with the expression of our specific genes for monomer production.

One of the biggest helpers came in the form of our contact with Evonik Industries, a company focused on the production of speciality chemicals. Since they are experienced in PLGA production, we took the chance to strike up a conversation about the procedure and what qualities monomers need to have for industrial purposes. They invited us to their location in Darmstadt. Their input caused us to focus and address certain aspects we previously would not have. We briefly touch on these topics down below in our Integrated HP part.

We also deemed it important to talk to scientists who had previously worked with organisms or enzymes we planned to use. We had a long back and forth email conversation with Dr. Outi Koivistoinen, the author of the paper we based our glycolic acid production in S. cerevisiae on. She helped us by providing her plasmids and strains from said paper. She gave us valuable feedback for this specific part of our project and offered solutions and alternatives to certain approaches. In regards to ε‑caprolactone production in E. coli, we got in contact with the working group of Prof. Dr. Bornscheuer. Their help was much needed and highly valuable for our work with the enzymes CHMO and ADH. We came in touch with them, and Vishnu Srinivasanurthy specifically, through one of our professors from the biology department at TU Darmstadt. Furthermore, we explored the possible implementation of organism Pseudomonas putida by contacting various experts. In what ways all of these experts helped us out, is remarked on down below.

Something that is usually done by iGEM teams at our university is a presentation in front of the departments of biology and chemistry. This year, we had the opportunity to present ourselves twice, once in July and the second time a few days before the Giant Jamboree. The first event allowed us to show our idea and get feedback from professors or other students. It highlighted aspects we had to work on but also validated us in our approach, since the responses were very so positive. The second presentation was a chance for us to show what we had accomplished. It also gave us the chance to practice our presentation skills for the Jamboree itself. In the beginning of October, we also met up with old iGEM members and presented our project in front of them. Our hope was to get feedback from them and prepare for the Jamboree, since they already had experience. We received valuable input, especially for the structure and design of our presentation in Boston, later that month.

Learning and Integrating

Exploring different ways to get in touch with various groups of people is one thing, another is listening to their concerns or feedback and implementing these things into the design of the project. During our Human Practices work, we encountered many different individuals. Their input helped shape our way of thinking and influenced important aspects of our project.
We felt is was best to separate the following into these different aspects:

Society

Even though our education efforts were mostly to teach, we also made sure to establish a two-way conversation. During our visits to various schools, and on other occasions, we received great input that made us investigate other aspects of our project. The public opinion was always an undeniably important factor to us.

Someone pointed out how bizarre it was that we were producing plastic, while everyone else seems eager to find ways to get rid of it. This made us think. Truly, it is quite strange. How can we stand for plastic production, when there is such a big problem surrounding it? Our produced PLGA and PLGC are fast degrading polymers, so the feared threat of them lingering in the environment for decades is not an issue. But still, would it not have been more helpful to design a different project? Finding ways to reduce the amount of plastic, is certainly important. However, an often overlooked factor is, how important and ingrained plastics are in today's society. If we can find a way to produce them in a more sustainable way and make sure they degrade at a rate where they do not pose a threat, can they not be a virtue? When explaining these facts about the process, listeners changed their attitude to a more positive view.

One concern brought up was the possibility of microplastics generated because of our application scenario – the nanospheres. This was something that had not crossed our mind until this point, so we made sure to look into it. What we found out was that this risk does not exist. Our nanospheres consist of the polymer PLGA which degrades completely into the monomers glycolic acid and lactic acid, two compounds harmless to the environment because of further metabolization. This means, our polymer does not pose any risks, neither in the form of microplastics nor in the form of bigger scale polymers.

Application wise, another concern was the possible pollution of groundwater with medical residues. Since our focus was never the production of medically applicable nanospheres, we could not address this problem in depth. Though we did look into it on a very basic level.

Biology

During the year, we established various connections to scientists from around the world who supported us in different aspects of our project.

An important relation came in the form of our contact with Dr. Outi Koivistoinen. We reached out to her after reading her paper on glycolic acid production in yeast, something we intended to do as well. At the time of our initial email, we had already decided to use S. cerevisiae as our production organism. We explained our project to her and asked for support. Her knowledge helped us in many different aspects. For one, she was kind enough to provide us with plasmids and S. cerevisiae strains used in her paper. We were able to perform several experiments with them. See our Gylcolic acid in S. cerevisiae side for further information.
Very early on in our conversation, she recommended the yeast K. lactis over S. cerevisiae, since the amount of produced glycolic acid was higher in this organism. We thought about a switch, but ultimately decided against it. Mostly, because we had already started with S. cerevisiae and were hesitant to change them then. We did, however, look into K. lactis for upscaling purposes. You can find more here.
Another thing she helped us out with was the sequence for the gene of the enzyme AtGLYR1. In this context, we also asked her for her input on purification methods, specifically the decision between Strep- or His-tag, and instructions on assays. She recommended the usage of a Strep-tag, which we did end up using.

After talking to Prof. Dr. Kabisch from our university about production of the monomer ε‑caprolactone in E. coli with the enzymes CHMO and ADH, he referred us to the working group of Prof. Dr. Bornscheuer. We were told that they had already worked with both enzymes in the same way we were intending to use them. Through our conversations with them, we learned that CHMO works at a higher rate than ADH, and to ensure them working together optimally, their expression rates have to be adjusted. They were kind enough to supply us with a plasmid, containing both enzymes with the proper rates. Their input even helped us with assay settings. More information on ε‑caprolactone production can be found here.

Since one of our focuses lay in the upscaling of monomer production, we wanted to explore other possible organisms. Dr. Outi Koivistoinen recommended the yeast K. lactis as one alternative. We decided to look into P. putida as yet another option for glycolic acid production, but had no expertise with this bacterium at all. Therefore, we contacted several scientists and asked about their opinion and input. In general, all of them recommended P. putida for metabolic engineering purposes. The biggest problem seemed to lie in the upscaling, though. In the end, we only got to work with Pseudomonas putida briefly, since time and resources were not enough. For further details on on our work with this organism, see here.

During our contact with these scientists, one quote stuck out to us the most:
“One has to go case by case, there is not just one chassis that fits all needs.”
- Victor de Lorenzo

This sentiment encouraged us in our approach to explore and use a variety of organisms for the production of monomers. Unfortunately, our work could only be realized with E. coli and S. cerevisiae practically, as well as P. putida and K. lactis in theory. There is an abundance of work to be done and an optimal path for an industrial relevant production has yet to be found.

Industry

For us, it was important to find out how reasonable it was to assume that our project could work under real conditions. Getting someone, who works within polymer production everyday, to look at what we tried to do, posed a significant opportunity.

Evonik Industries, a company focused on the production of speciality chemicals, has a branch location in Darmstadt. We found out that they produce PLGA and make nanospheres out of it as well, so we were eager to get in contact with them. Luckily, we got the chance to sit down with a few of their employees to talk about polymer and nanospheres production. They were very helpful and supportive, especially, regarding certain aspects of our own polymerization method. We looked at some of our results with them and they consulted with us in how to optimize the synthesis process. A fact we were not aware of, was that glycolic acid is more reactive than lactic acid and gets integrated into the polymer much quicker. This information influenced our Modeling approach, in which we tried to establish a kinetic model of our polymerization.
Since the viscosity in our polymerization experiments was problematic, we were unable to complete our reactions fully, and not all monomers could be built-in. Combined with the fact that glycolic acid was incorporated faster, we were not able to predict the properties of the synthesized polymer. Evonik told us, under industrial circumstances, polymerization reactions are completed 100% which allows for an accurate prediction of the resulting PLGA properties. Under these conditions, the problem of viscosity is nonexistent, because of the used stirring technique. As we were limited in what we could do in our lab, we were not able to test out other stirring options, like they suggested. In theory, this should however, remove the unpredictability of the reaction. More on the synthesis process can be found here.

We were especially curious what they thought about our way of manufacturing monomers. When we pitched them the approach to use microorganisms, they seemed generally interested, but voiced some of their concerns as well. One of the biggest problems they saw, was the small amount that we would be able to produce this way. The output gained through metabolic engineering is not yet enough to compete with chemical alternatives. And since the monomers should be incorporated into polymers applied in medicine, purity is a vital fact as well. That is something that is currently hard to achieve via this method. In the end, they welcomed the idea to find alternative ways to produce glycolic acid and lactic acid more sustainably, and said they would consider using them, if certain standards would be met in the future.

After our meeting, we stayed in contact. Therefore, we were able to ask more questions to receive further recommendations. Since we could only touch on nanosphere production briefly during our first conversation, we used this opportunity to clarify some aspects. How we approached nanosphere synthesis, can be read here.

Conclusion

Our Human Practices work has led us to various places and while not all that we did seems to fit together neatly, everything tackled specific aspects of our project. We informed society about synthetic biology, taught them to be cautious, but also showed them, how amazing this field could possibly be. We established a dialog with many different people and talked about our project. Through this, we gained valuable feedback, which influenced us greatly. We contacted scientists from a range of biological backgrounds and had them evaluate our approach. They offered support and information to specific aspects. We explored the use of other organisms as monomer producers and engaged in a great and valuable conversation with employees from Evonik, who were experienced in the chemical part of our project. This allowed us to judge whether or not our plan had potential for the future of PLGA/PLGC and monomer production. We would like to thank everybody who took the time to discuss with us. We greatly enjoyed exchanging ideas and talking about the future of synthetic biology.