Integrated Human Practice
Competing in the Foundational Advance track this year meant working on a project with a vast variety of future applications. Initially, it seemed as though there would be little to do for our integrated human practices, because working in foundational advance does not directly affect society. However, since our project has tremendous potential to be utilized in varying scientific and industrial fields, i.e. by simplifying production cycles for biofuels, pharmaceutical, etc., it will finally affect the population as end users. It is for the same reason that we had to consider a lot of different aspects when brainstorming for our human practice activities. Integrating our human practices into our work meant considering its ethical, social and environmental dimensions. In order to fully understand the potential applications, implications and issues of our project, we decided to build our human practices on three pillars:
Project Plan Discussion with Ralf Erdmann
In order to gain fundamental insights into yeast peroxisomes we contacted Prof. Dr. Ralf Erdmann and his research fellows Prof. Dr. Wolfgang Schliebs and Dr. Wolfgang Girzalsky from the University of Bochum who are experts in peroxisomal research. The group´s research topics comprise biogenesis of the peroxisomal membrane, peroxisomal metabolism and metabolite transport, proliferation of peroxisomes and peroxisomal import. During our first encounter on the 6th of April, we introduced them to the essentials of our artificial cell compartment developed from yeast peroxisomes and our blueprint to create and control it, discussing protein import, peroxisomal membrane proteins and peroxisomal metabolism lasted roughly four hours. On the one hand we received great feedback for our idea and additional interesting knowledge about the lab work with peroxisomes. On the other hand we were able to convince all leaders of the working group of our project’s great potential and they appeared to be very interested in the iGEM competition itself.
To maintain contact to the working group from Bochum and gather further useful hints, we invited them to a private meeting with our team in Duesseldorf. In the meantime, we developed our sub projects and achieved first results which we discussed together on the 1st of June. We talked about our latest results and further possible experimental designs for controlling our compartment more precisely. They pointed out one potential weakness of our project regarding the orthogonal peroxisomal protein import. They mentioned that the cargo release inside the peroxisomal lumen could be a possible flaw since the natural PTS1 sequence is typically recognized by Pex8 at the luminal side of the peroxisomal membrane. Subsequently, Pex8 binds to the PTS1 sequence facilitating cargo release of the protein of interest and PTS1 by competitive inhibition. Since we planned to create an artificial PTS1 peptide, the process of competitive binding could be affected by decreased binding affinity of Pex8.
Considering that, we started an alternative project for creating an orthogonal import machinery targeting the second protein import mechanism via Pex7 and the N-terminal peroxisomal targeting signal 2 (PTS2) with a random mutagenesis approach. Interestingly, the peroxisomal protein import facilitated by Pex7 does not include Pex8. Moreover, the exact mechanism of cargo release of PTS2 from Pex7 could not be clarified yet and remains to be elucidated. Nevertheless, Prof. Dr. Erdmann advised us to try the PTS2 mutagenesis approach since the mechanism works under countless conditions although its precise function is still unclear.
Furthermore, we discussed whether the precursor for the desired nootkatone pathway farnesyl pyrophosphate (FPP) is already present in the peroxisome. There is evidence that FPP is imported into plant and mammalian peroxisomes, but the import mechanism in yeast peroxisomes itself remains unrevealed. However, no measurements for FFP in yeast were performed yet, but other experts working on similar fields of metabolic engineering suggest that FPP is abundant in yeast peroxisomes. Moreover, we gathered important clues about the cultivation of yeast strains which are deficient for the Pex11 protein as proliferation factor, learned how to design truncated peroxisomal membrane protein anchors in order to localize several fluorescent marker proteins or enzymes at the luminal peroxisomal membrane and obtained the opportunity to conduct a peroxisomal purification method performed by the working group of Prof. Dr. Erdmann, which is not trivial since the membrane properties are very similar to other cell organelles.
Conclusively, the advice from Prof. Dr. Erdmann boosted our knowledge on yeast peroxisomes and greatly improved our experimental designs. We are very grateful that he and his colleagues took the time for answering our questions and contributing to our discussions.
Brainstorming with John Dueber and Will DeLoache
As we started with our idea of creating an artificial compartment, we came up with different design concepts. When we chose peroxisomes as our chassis we found the work of John Dueber, who characterized the import of proteins and metabolites into the peroxisome. With these tools he set the foundation for utilizing yeast peroxisome in order to build a synthetic organelle. Since we also used his Yeast Toolkit for modular, multi part assembly, we decided that he is the right person to discuss our project designs and contacted him for a Skype call. During the talk we discussed the import efficiency of the Pex7 import mechanism, problems regarding the secretion of the compartments, the importance of membrane permeability and the final goal of controlling the chemical environment of the peroxisomal lumen by integrating new membrane proteins. Due to the low import efficiency of the Pex7 import he expressed concerns using it as the import mechanism of our artificial compartments and instead focuses on the much more stable Pex5 import.
Regarding our secretion project he told us that he realized that the peroxisomes seem to be fixed to the cytoskeleton or at least are very immobile. Prof Dueber proposed that if we can immobilize the peroxisome we would get an increased amount of secreted protein or metabolite. Therefore we went back to design and constructed an Inp1 knockout yeast strain with the Cas9. Inp1 is a protein that tethers the peroxisome to the ER by binding to Pex3 and the knockout leads to immobilized peroxisomes.
Furthermore we discussed that one of the biggest bottlenecks of engineering artificial compartments, especially for metabolic engineering, is the control of the peroxisomal lumen properties, such as pH, redox state or cofactor balance. To overcome this problem we aimed at integrating new membrane proteins into the peroxisomes. We chose the proton pump bacteriorhodopsin and measured the change of pH in the artificial compartment with a pH dependent GFP. Additionally we designed and integrated different plant transporters for several metabolites in our toolbox. Long-term goals like these could be achieved with our toolbox and will provide unprecedented control over the chemistry and physiology of the cell.
Discussing common results with Alison Baker
After the first modeled receptors and peptides were tested negative, we started a second design cycle for an orthogonal import of proteins in our artificial compartment. Therefore we had a Skype call with Professor Alison Baker who is working on basic research aspects of the membrane transport processes in plant cells with a focus on transport of proteins and metabolites into peroxisomes.
She gave us a more detailed view of the protein-protein interactions between the pex5 and pts1 in P. patens and A. thaliana. Based on the new knowledge we modeled the interactions between the modified receptor and pts1 sequence for our artificial compartment. After the second design phase, the new receptor PTS1 pair was tested and could be validated to be fully orthogonal.
Interviewing Dr. Florian David – Biopetrolia
Having acquired an enlightening amount of foundational knowledge about peroxisome basics, compartmentation, and synthetic biology toolboxes, we set out to speak to experts from companies who are already using synthetic biology applications to replace inefficient production cycles by overcoming natural pathway complexity.
During our research, we came across Dr. Florian David from Biopetrolia, a Swedish company that works on yeast strain engineering to find resource-efficient ways to produce valuable compounds such as cosmetic bio ingredients, specialty chemicals, nutraceuticals, pharmaceuticals, biofuels, etc. [1]. Florian kindly agreed to a Skype meeting to listen to our project plans and discuss opportunities and issues.
So far, controlling the size and number of our compartment via Pex11 knockout or overexpression was considered a mere additional feature of our toolbox. Pex11 knockouts were used to create a reduced amount of giant peroxisomes, whereas Pex11 overexpression should provide a large amount of smaller ones. Since Florian and his team are constantly working on finding ways to create resource-efficient and highly productive pathways, he suggested working on the peroxisome's morphology in order to positively influence the yield of metabolic reactions. One of their research groups had previously worked on optimizing the production fatty-acid-derived fatty alcohols, alkanes and olefins in peroxisomes, while at the same time decreasing the accumulation of byproducts. In order to achieve their goal, they worked with both, a Pex31, 32 knockout, as well as Pex34 overexpression, both of which positively affect the size and number of peroxisomes while at the same time lead to higher metabolic yield. For our purpose he recommended working with a Pex34 overexpression, since they found out that a knockout of Pex31, 32 leads to higher permeability of the peroxisomal membrane. After the conversation, we successfully implemented his suggestions into our project.
Finally, since speaking to experts should also help us to understand the potential of synthetic biology applications, Florian further discussed the benefits of both, their work and our compartmentation approach. These novel applications foster the introduction of tailor-made properties into molecules, making it easier to synthesize substances that are currently obtained from rare natural resources and mostly in environmentally-unfriendly ways. Economically, working with these novel approaches helps gaining independence from market fluctuation of traditional production processes. This means that products like pharmaceuticals, with a universal benefit could be produced at a low cost and therefore made available to a larger amount of the population. Furthermore, producing through compartmentation could ensure stable, scalable and on-purpose production through selective biological processes, bypassing chemical processing.
The conversation with Florian David was valuable in many ways. Not only did he foster our understanding of compartment applications, he also broadened our horizon regarding the potential of our work and common approaches.
21st century solutions to real world problems - Getting to know Evolva
Right when we started planning and designing our toolbox towards creation of an artificial compartment it goes without saying that for the greater good it should be used for production of beneficial compounds that target global issues. During our research we stumbled upon nootkatone, a flavoring substance in the peels of grapefruits. Nootkatone has been proven to have inhibitory effects to insects like mosquitoes and ticks that can cause severe diseases. On the other hand it is completely nontoxic to humans and other mammals, environmentally friendly and volatile.
Looking for a suitable partner to interview and acquire information about working with nootkatone, we found Evolva. Evolva is a Swiss business-to-business company with offices and laboratories in all over the world. They focus on biotechnological solutions for providing ingredients to other companies, especially in the food and beverage, personal care and consumer health sectors. Thereby, they try to overcome natural constraints by aiming at reproducing rare ingredients. One of their fields of interest is the production of nootkatone, a natural insect repellant, conventionally found in the peel of grapefruits. Shortly after making contact, we were able to schedule a Skype meeting with Mary Bosserman (Strain Engineer, Evolva USA), Harald Heider (PI, Evolva Basel) and Sumire Honda (Senior Scientist, Evolva Basel). We presented them with the methods of our artificial compartment toolbox and how we intended to use it as an alternative production method for nootkatone. Due to their experience with nootkatone, they were able to lead us through some of the major bottlenecks of designing and implementing the pathway. It was pointed out that the intermediates valencene and nootkatol as well as nootkatone are hydrophobic. Consequently the intermediates are stored close to the membrane. To achieve a higher yield it was suggested to fuse the relevant enzymes valencene synthase (ValS), P450 BM3 monooxygenase (BM3) and alcohol dehydrogenase (ADH) to membrane anchors to ensure a close proximity of substrate and enzyme. With this input we were able to design the constructs for the nootkatone production accordingly: We decided to utilize the membrane anchor proteins Pex15 and PEX26, which we previously established in S. cerevisiae. The proper localization of these proteins was proofed via fluorescence microscopy.
The enzymes ValS, BM3 and ADH were fused to each of the two anchor proteins. In the first attempt we combined the proteins without linkers in-between. Hereby, there is a high risk of the proteins misfolding and therefore losing their function. Therefore, in the next step, we tried using different kinds of linkers of varying length to suppress protein misfolding. To this point, we could not test the constructs due to lack of time. But we expect to be able to prevent the protein misfolding with this strategy.
Having provided us with all this valuable input, we were invited to follow up meetings with both Mary Bosserman and Harald Heider. In October, we set off to visit Harald and his colleagues in Basel, Switzerland, where we were given the chance to present out project to the research group.
After the production of nootkatone in the laboratory, the compatibility of the product is tested. Mosquitoes and ticks are used for the examination; these were previously examined on the transmissible pathogens so that only healthy animals are used for further trials. For this the insects are kept in cages, in which you can put in your fingers for tests. In these examinations, for example, a finger is partially treated with nootkatone and marked. Then the finger was held in the insect cage. Then it is observed how the insects behave, whether they avoid the place with the medium. These tests are repeated several times. Furthermore, nootkatone is being investigated for side effects or whether allergies occur. In the next step, the best composition is found out and the concentration of nootkatone. In addition, ecological effects are tested, for example the effects on bees or other vulnerable pollinators. After these various tests, the product can be launched on the market.
During the intense conversation and discussion we learned, that it is a far away from the lab product to the market finished insect repelled since a lot of regulatory criteria have to be fulfilled. In October we had our second Skype meeting with Mary Bosserman from evolva to present and discuss the latest developments and results. Furthermore Mary Bosserman and several other scientists gave us valuable advices for evaluation and interpretation of our experimental results.
Outside the box - Science is more than just lab work
Science is not only about research and lab work – we were aware of that long before we commenced our work. However, while engaging with the public, at schools during lectures or while walking the streets and talking to people, we realized that criticism towards synthetic life sciences did not only come from older generations. Fear, skepticism and opposing opinions came from young and old, academics and non-academics. So we asked ourselves: Why are people skeptical and where does that fear come from?
As passionate scientists it is clear that we are aware of the limitations and potential issues of synthetic biology; however, our attitude towards science is strongly positive. Determined to broaden our horizons and better understand the opinions and thoughts of the people, who might criticize science, but will in the end be affected by our work, we set off to speak to experts, who are dealing with ethical, religious, and socio-economic aspects of science. So we visited Dr. Johann Ach, Scientific Coordinator at the Reference Centre for Bioethics at the University Münster. He and his colleagues are working on the “theoretical fundamental questions of establishing standards in medical and biological ethics and biopolitics”. He introduced us to a so called “compass of questions” to examine and evaluate scientific project from a societal, non-scientific perspective.
- What is our work about and what is its purpose?
- What is life?
- Does our work really benefit the general public?
- What are ecological and health issues?
- Could our work be misused to cause harm, if it is openly available?
Because we are working on creating artificial cell compartments as a next step towards a completely synthetic cell, Dr. Ach made us aware that the conception of “synthetic life” raises questions in people's’ minds about “the value of life”. The Max-Planck-Institute has dealt with this point. An important property of creatures is the compartmentation that is used to create independent microenvironments for naturally competing reactions. These reactions and the composition of the compartment are controlled and archived by a genetic plan. The preservation of the cell is ensured through metabolism of the environment. These reactions are catalyzed by enzymes. Without these enzymes the cells would not survive, as many reactions would proceed too slowly. In addition, the internal environment of the cell is controlled. Another point which distinguishes life is that all living beings can grow and multiply. Reproduction involves the passing on of genetic information to the offspring. In this distribution of the genetic information, mutations can occur, which cause selection.
One of the most important aspects of our conversation with Dr. Ach was the issue of communication. He pointed out that understanding between the scientific and non-scientific community often fails, because of misdirected communication. Very often, scientists condemn opposing opinions, because for them it seems clear that science benefits society. However, rather than blocking concerns of the general public, they should be listened to and integrated into scientific work.
After an enlightening conversation, we decided to contribute to an improved communication between scientists and nonscientists, so we decided to create a platform where both parties can meet and discuss both, opportunities and issues of synthetic biology. We thought about different ways and decided to organize a Synthetic Biology Meet Up for the public. At the House of University in Duesseldorf, we invited established scientists and iGEM teams to present their work to the general public. During plenty of networking breaks guests of the event were able to approach scientists and ask them about their work. For many of them, this was a first-time opportunity. At the end of our Meet Up, we initiated a debate between speakers and guests. For that purpose we had invited Christopher Coenen, an expert in human practices in science to lead the conversation.
