iGEM Uppsala 2022 - Integrated Practices

Meaningful change doesn't happen overnight. Whether tackling plastic pollution at local or global scale, groundbreaking advancements for eradicating plastic waste take time. The PETerminator initiative sought to build on previously documented successes in recombinant biology for plastic degrading enzymes. In particular we sought to take the successes of recombinant expression in multiple organisms to a single strain of E. coli.

We additionally took inspiration from previous work of iGEM Teams at Yale, UCL, and even from the 2021 Uppsala iGEMers in FGFuture. Our work within the Human Practices portion of the 2022 iGEM competition continually drew inspiration and feedback from iGEM groups and experts in academia and industry. We compiled and reflected on the broader implications of the PETerminator pathway based on feedback from expert interviews and our own questions about the good our work could do outside of the lab.

Infinite growth in finite space is impossible. Similarly, unbridled production in a closed system inevitably generates pollution due to waste. A glance at a satellite image of the Pacific Ocean offers a grim portrait of the consequences of plastic pollution on earth. The collection of plastic garbage washed out to sea from coastlines (Lebreton et al. 2018) now occupies a surface area roughly 2 million km². Plastics, nonetheless, have solved problems and enabled the advancement of myriad industries since their inception in 1907. Given the many essential roles filled by plastics in daily life, it is difficult to imagine a fast transition back to a plastic free world. How could synthetic biology address the seemingly paradoxical scenario we find ourselves in as of 2022?

In view of the ubiquity of plastic waste worldwide, the Uppsala PETerminator project targeted the communities most upset by plastic pollution. We wanted to establish a metabolic pathway within E. coli that could convert plastic waste, or at least components of plastic waste, to valuable chemicals. These included protocatechuic acid and vanillic acid.
Inspiration for the PETerminator project came from several groundbreaking publications of the last several decades. Among these, two papers highlighted the star enzymes of the PETErminator pathway: PET hydrolase (PETase) and (MHETase). Our team also drew inspiration from previous work by iGEM teams at Yale and UCL.

We concentrated seriously on our wet lab work throughout the summer. Alongside long days spent plating, electrophoresing, and troubleshooting experiments, we had the opportunity to connect with the local community here in Uppsala over our project. We gathered feedback about scaling up and implementing our PETerminator pathway at industrial level in work at Testa Center. We also conducted expert interviews with experts specialising in plastic waste management across a range of industries in August.

In order to best implement human practices methods within our work, we consulted with experts at global and local levels. We conducted a tour of the Uppsala Vatten biogas plant to gain an idea of what kind of scale our PETerminator pathway would need to achieve in order to be viable. We also gathered valuable insight into how to regulate our pathway: no one would want to find essential plastic degraded by rogue PETerminator bacteria! It was very inspiring seeing the huge bioreactors hosting a variety of naturally occurring bacteria producing the gas that makes us able to travel by bus in Uppsala.

We also conducted interviews with several experts from around the world on approaches to managing the paradox of plastic. The topics varied from PET-hydrolysing enzymes and biodegradable plastics to the preservation of art and fashion made out of plastics for future generations, an aspect not many think about in these types of research. All in all, we gained insight into several aspects of our project and received valuable feedback regarding our project idea.

Additionally, we sought to foster enthusiasm for and interest in synthetic biology by reaching out to high school students. The Uppsala iGEM team held lectures and labs with 50 high school students from two different high schools here in Uppsala. As part of our final week in the lab, we conducted a basic transformation experiment using chromoproteins. Lectures from prof., dr., and md., Anthony Forster and prof. dr. Krabbe of Uppsala university gave introductory lectures beforehand about their work in synthetic biology and safety. We had a blast sharing our enthusiasm for synthetic biology with the high school students, and based on their feedback they did too!

From Budding Idea to A Blooming Project

Our very first steps into our iGEM journey this year began with discussing synthetic biology from a general point of view, and how it can be applied to solve many of the world’s most pressing problems. Diving headfirst into literature, previous iGEM projects and looking at global issues through the lens of synthetic biology, our team came up with a diverse pool of initial concepts. We then pitched and discussed each of these ideas amongst ourselves, in addition to seeking out feedback from our PIs and other experts before voting for five ideas that stood out to us. Divvying ourselves up into five groups assigned to each idea, every group devoted themselves to more in-depth research into the concepts and logistics of their respective concept before presenting their results and fleshed out idea to the rest of the team, in addition to our PIs, TAs, previous iGEM alumni and other invited staff members of Uppsala University. With five excellent ideas presented (we regretted that we could choose but one) and a second round of voting, we landed on a single exciting idea, turn plastic into PCA using synthetic biology!

Throughout our ideation process, we emphasized the human practice element of iGEM by performing a SWOT (strengths, weaknesses, opportunities, and threats) analysis to every idea we considered, not only considering the technical aspect of our project, but also the societal aspect. This would turn out to be incredibly valuable moving forwards with the project, as it served as an excellent guide for us to address not only the advantages our project idea presented, but also its inherent drawbacks and challenges.

The field of plastics research, both in terms of dealing with the current problems caused by plastics and researching new materials that can replace plastics, is broad and multidisciplinary. Throughout our project, we have had a lot of material and research to go through and take inspiration from, whether it comes from an academic institution, an innovative company, or a non-profit organization working to clean our environments from pollution. We reached out to stakeholders in different fields and with different ambitions in order to discuss our idea and possibly get some feedback. Our discussions touched on several topics, but ultimately could be boiled down to: Would our PETerminator be a realistic solution to such a big and complicated problem?

In order to get a better view of these issues, we invited several speakers to present their research to us. The goal for our series of online expert lectures was to get a better view of the problems of plastic and its role in our lives. We also searched out other views on plastic, for example how to make current plastic better in terms of the environment, since we also recognize that it is an important material in for example the scientific and medical field. Therefore, we invited speakers from different fields, who had varying perspectives on this subject.

Expert Lecture with Wolfgang Streit

Prof. Dr. Wolfgang Streit

The very first expert lecture we organized was held by Prof. Dr. Wolfgang Streit from the department of microbiology and biotechnology at the University of Hamburg. We contacted him because of his team’s extensive work with plastic degrading enzymes and the article “New Insights into the Function and Global Distribution of Polyethylene Terephthalate (PET)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes” (2018) written by him and his team, which brought up many interesting aspects that are relevant to our project. Wolfgang’s presentation focused on his research in finding the best “plastic-eating” bacteria and proved to be not only very intriguing, but also relevant and valuable for our project. After a short introduction to the problematic of plastic pollution and an overview of currently known plastic-degrading enzymes, which included both LCC and PETase studied in our project, professor Streit focused on his search for the new and better enzymes, which could degrade plastic more efficiently. In his metagenomic studies, environmental samples are sequenced and potential candidates for plastic-degrading enzymes are identified using hidden Markov models. These candidate genes are expressed through in vitro transcription and translation and screened for their activity. Professor Streit also mentioned a database set up by his colleagues Plastics-Active Enzymes Database (PAZy), which accumulates all knowledge concerning plastic-degrading enzymes.

After the lecture we discussed our project and gained some expert input on several aspects of our design, such as the advantages and disadvantages of our chosen bacteria and our proposed single-cell model. Even though in nature plastic-degrading bacteria always work together with other strains and species, he agreed that for industrial application, which was our ultimate goal, it would be favorable to comprise the pathway into a single organism, for easier maintenance and monitoring. The lecture and following discussion gave us great insight into actual scientific workflow for searching for new potential industrially applicable enzymes and was beneficial and appreciated by the whole team.

Expert Lecture with Wael Abdelmoez

Prof. Dr. Wael Abdelmoez

As our next speaker we invited Wael Abdelmoez, a Professor of Environment & Energy at Mlnia University, Faculty of Engineering, who managed to find time in his busy schedule to talk with us about novel types of plastics and biodegradable plastics. His research focusing on bio- and oxo-degradable plastics was very attractive for us, since it showed yet another perspective on the problems of plastic in the world. Oxo-degradable plastics were mentioned to be currently available, but not providing a solution to the plastic pollution problem, which can be only achieved with truly biodegradable plastics. These plastics would need to be made of currently known biopolymers, such as polysaccharides or proteins, which have existing enzymes for their degradation. This however presents several challenges to make these biodegradable plastics durable enough for actual use and to make the production economically viable. The cheap production of plastics derived from fossil fuels is a major drawback, making development of alternatives extremely difficult, since they cannot compete with the price of traditional plastics. His lecture was truly interesting and professor Abdelmoez’s enthusiasm for the field was contagious and deeply appreciated by our team.

Expert Lecture with Leanne Tonkin

Doctoral researcher Leanne Tonkin

Our final speaker was Leanne Tonkin, a postgraduate researcher, lecturer and conservator in contemporary textile and fashion from Nottingham Trent University, UK. In her research, Leanne focuses on preserving plastic artefacts rather than degrading them. In her presentation she showed us how fashion artefacts made of plastic degrade in their quality and properties in only a few decades and how difficult it is to find suitable conditions to preserve such objects. It was surprising to learn how fragile plastic is in some circumstances, since plastics are usually presented as indestructible “forever” materials. Towards the end of the lecture, she mentioned the contemporary trend of novel biodegradable materials, which is a great move towards a more environmentally friendly and sustainable fashion, but presents a big challenge for conservators such as Leanne Tonkin, who try to preserve fashion articles for future generations as important cultural heritage. This lecture gave us a very different perspective on plastics, since we are very used to seeing plastic as a problem only. It is an important media used for art and fashion, not only for single use plastic straws as is often the view. Plastics have a major role in almost all aspects of our life, even art, which is a big reason why it is so hard to simply abandon this material.

Aim: Learn about bioreactors within the Uppsala waste-management system. Learn more about plastic recycling.

It was a warm and sunny Monday, the last week of June, when we set out to visit Uppsala Vatten and their biogas facility at Kungsängen. We were met by Stina Bengtsson, who gave us a short introduction to the methane-producing bioreactors they have. In short, Uppsala Vatten collects organic waste from Uppsala municipality and several others, grinds and boils it to finally feed it to methane-producing bacteria. The natural gas is collected and methane is concentrated from it, while the remaining organic waste-slurry is used as fertiliser for local farmers. We were then given a tour of the facility and its machines, followed by a short lecture on plastic waste management. Stina gave us some names of people we could contact in order to get a better understanding of how these bioreactors were designed, so that we could use that knowledge in order to understand how our system could possibly be designed in the future if successful.

These bioreactors worked as a tiny eco-system. Based on bacteria from manure, different types of organic waste have been added and the bacterial flora has been formed over time. We wondered if they had a “starter culture”, or if they sometimes had to supplement with bacteria when production was slow, but the starter culture from the manure provided at the start and the constant adding of new “feed” meant that the bacterial flora was very stable.
We also asked about external factors, such as temperature, pH-levels, moisture, and oxygen levels. They do keep the reactors isolated so they have a very low oxygen level, which is crucial to the bacteria forming methane rather than carbon dioxide. However, besides this, they did not affect temperature (for example cooling in the summer heat or warming during winter), pH-levels nor moisture levels. It truly worked as an ecosystem achieving homeostasis.
We were also curious about possible contaminations. It is well known that organic waste can contain E. coli, Listeria and Salmonella, but the boiling of the organic waste in 70°C for an hour was done to prevent contamination. They have never had problems with unfavourable bacteria growing within their bioreactors.
Another topic of discussion was if their bacteria was engineered in some way, or if they would like them to. Their system uses naturally occurring bacteria that have not been genetically engineered in a lab. This is great, since any leakage of bacteria would not be harmful.

Regarding our project, we would advise the following for designing a bioreactor that could possibly feed a PET slurry to engineered bacteria and extract useful chemicals such as PCA:

It’s always best to use non-GMO in case of leakage, or to design the GMO so that it will die when it is moved outside of the bioreactor. There are several strategies for this already established, but for our system it would mean that another set of genes would have to be inserted in addition to the PET-degrading genes.
The plastic would have to be treated before entering the bioreactor so that it would not contaminate it. Heat would be an option, especially since heat would start the process of loosening the PET-molecules from each other and making it easier for the degrading enzymes to access the PET-chains.
As opposed to the slurry fed to the bacteria of Uppsala vatten, the E. coli would get a PET slurry. Would the culture need supplements? Monitoring oxygen levels and pH would be smart, but how would the bioreactor need to be designed so that for example oxygen could be pumped in? Bioreactors used for pharmaceutical production are large compared to our lab bench systems, but a bioreactor able to take care of plastic waste from a smaller city would need to be of a significantly larger size. It would, no doubt, take a lot of talented engineers to design such a system for keeping GMO happy and the world outside safe from any leakage.