Team:Chalmers-Gothenburg/Description

Project Inspiration

When coming up with our project, we wanted to work with something we cared about. For us, this meant dealing with an environmental issue that was both a worldwide problem, but also present locally in Gothenburg, Sweden. While researching with this scope in mind, we came across the issue of PCB pollution. This immediately grabbed our attention, and we set out to do something about it. We also quickly found that the iGEM team from Paris-Saclay, 2013, had based their project around the same issue [1], and we took inspiration from their work when designing our project.

History of PCB

Polychlorinated biphenyls (PCBs) are a group of chemicals that were used for a variety of applications during the 20th century [2]. First described by German scientists in 1881, by the 1930s the chemicals were manufactured commercially and used industrially in dielectric fluids, hydraulic fluids or as additives in adhesives and plastics, to name a few applications [3].

During the time of their widespread use, PCBs were valued for their stability and variable characteristics, which are related to the amount of chlorines attached to the biphenyl skeleton, and the positions at which these are attached [2]. In total, there are 209 possible different structures that PCB can have, called congeners, and examples of such are illustrated in Figure 1 [4].

Figure 1. Examples of PCB congeners.

While the use of PCBs continued for several decades, in 1966 the Swedish scientist Sören Jensen discovered the adverse effects PCB has as an environmental pollutant [5]. It was shown that PCBs and their products have severe toxic effects on animals that ingest them, and that the chemicals bioaccumulate in both humans and marine life, increasing in concentration higher up in the food chain [6, 7]. This discovery eventually led to PCBs being banned in 1978 [8]. However, the same stability that was one of the reasons why the chemicals were so attractive also meant that they are remarkably persistent, and although the levels of PCB in the environment have decreased since they were prohibited, there is still a noticeable amount remaining in the environment [9].

Effects of PCB Today

As mentioned previously, PCBs are very dangerous chemicals. Exposure to PCB can lead to developmental abnormalities, neurological defects, negative effects on the endocrine system and even development of diseases such as type 2 diabetes [7]. The harmful effects of the PCBs that were released into the environment during the 20th century can still be observed all over the world. The orca populations in certain waters are still accumulating PCB, which might cause these populations to completely collapse if nothing is done about it [10].

Figure 2. Orcas and common seals are two animals that are heavily affected by PCBs in the environment.

In Sweden, where our team is located, the effects of PCB can be observed in many places. In the Stockholm area, it is recommended that children and women of fertile age do not eat locally caught herring more than twice a year, to avoid the toxic effects of the PCBs and dioxins present in the fish [11]. In Gothenburg, our home town, there are lakes where the concentrations of PCB are exceptionally high, likely due to old treatment plants that were located close by in the past [12]. The coastal fauna in Sweden has suffered severe negative effects of the contamination. Many birds, such as the white-tailed eagle and the common guillemot, almost went extinct due to poor egg shell quality, as a result of PCB poisoning [13, 14]. Environmental pollutions, and specifically PCB, are still listed as the greatest threat to the Swedish population of common seal, which is one of the apex predators in Swedish waters, and therefore especially sensitive to the bioaccumulating properties of PCBs [15].

Recognizing that this is an issue that affects the wildlife and environment on a global scale, and also one that hits close to home for our team, we set out to find a solution to this problem, using Synthetic Biology.

Project Description

The aim of our project is to engineer a strain of the yeast Saccharomyces cerevisiae, with genes from various bacteria that are able to degrade PCBs. After extensively researching the bioremediating properties of various organisms, the team found a set of genes that would, in theory, be able to break down even highly chlorinated PCBs, which are the most persistent ones [16]. The gene pcbA5, from Dehalococcoides mcartyi, will enable reductive dehalogenation of a wide range of PCBs [17], removing the attached chlorine atoms from the biphenyl skeleton, such that it becomes susceptible towards degradation by the enzymes found in the bph-pathway, displayed in Figure 2 [18].

Figure 2. Illustration of the PCB degradation pathway. A. The enzyme PcbA5 dechlorinates highly chlorinated PCBs, converting them to lower chlorinated congeners. Adapted from Bedard 2014 [19]. B. The bph-pathway breaks down the lowly chlorinated PCBs, converting them to benzoate, pyruvate and acetyl-CoA. Adapted from Agulló et al. [18].

In total, the bph-pathway consists of eleven enzymes. However, to make the project more manageable, it was decided to omit the final three genes, after modeling the system and observing that it could theoretically still produce satisfactory results without these genes. For the 8 remaining genes, the best characterized homologs were selected to construct our pathway. These homologs were found in Pseudomonas pseudoalcaligenes, Rhodococcus jostii and Paraburkholderia xenovorans. With pcbA5 added before the pathway, it becomes theoretically possible to break down most PCB congeners, forming benzoate and 2-hydroxypenta-2,4-dienoate in the process. If this would be successful, a future prospect would also be the inclusion of the final three genes, which are able to further convert the 2-hydroxy-2,4-dienoate to pyruvate and acetyl-CoA.

While discussing possible future implementations of our project, we turned to people working closer to the issue for advice. Since genetically engineered cells cannot be released into the environment without careful considerations and specific precautions taken, our future vision is to have the cells in a closed system, similar to a bioreactor, where PCB contaminated soil and water can be inserted and then returned cleansed of PCB. The general idea is that the best precaution, to prevent irresponsible spread of the organism to sensitive ecosystems, is to take the PCB to the yeast, and not the other way around. Our vision is that one day, biology will be what reverses the damage done to the environment throughout the decades of reckless polluting.

Further Reading

Delve deeper into the details of our Project Design, check out the simulated degradation pathways in our Modeling, or see how we integrated expert opinions into the development of the project in Integrated Human Practices.

References

  1. Team:Paris Saclay - 2013.igem.org [Internet]. 2013.igem.org. 2013 [cited 16 October 2019]. Available from: https://2013.igem.org/Team:Paris_Saclay
  2. Crine JP, editor. Hazards, decontamination, and replacement of PCB: a comprehensive guide. Springer Science & Business Media; 2012 Dec 6.
  3. Erickson MD, Kaley RG. Applications of polychlorinated biphenyls. Environmental Science and Pollution Research. 2011 Feb 1;18(2):135-51.
  4. Mullins MD, Pochini CM, McCrindle S, Romkes M, Safe SH, Safe LM. High-resolution PCB analysis: synthesis and chromatographic properties of all 209 PCB congeners. Environmental science & technology. 1984 Jun;18(6):468-76.
  5. Jensen S. The PCB story. Ambio. 1972 Sep 1:123-31.
  6. Czub G, McLachlan MS. A food chain model to predict the levels of lipophilic organic contaminants in humans. Environmental toxicology and chemistry. 2004 Oct 1;23(10):2356-66.
  7. Gupta P, Thompson BL, Wahlang B, Jordan CT, Hilt JZ, Hennig B, Dziubla T. The environmental pollutant, polychlorinated biphenyls, and cardiovascular disease: a potential target for antioxidant nanotherapeutics. Drug delivery and translational research. 2018 Jun 1;8(3):740-59.
  8. Hesse JL. Polychlorinated biphenyl usage and sources of loss to the environment in Michigan. InNational Conference on Polychlorinated Biphenyls, November 1975, Chicago, Illinois: conference proceedings 1976 Mar (p. 127). Environmental Protection Agency, Office of Toxic Substances.
  9. Alcock RE, Bacon J, Bardget RD, Beck AJ, Haygarth PM, Lee RG, Parker CA, Jones KC. Persistence and fate of polychlorinated biphenyls (PCBs) in sewage sludge-amended agricultural soils. Environmental pollution. 1996 Jan 1;93(1):83-92.
  10. Desforges JP, Hall A, McConnell B, Rosing-Asvid A, Barber JL, Brownlow A, De Guise S, Eulaers I, Jepson PD, Letcher RJ, Levin M. Predicting global killer whale population collapse from PCB pollution. Science. 2018 Sep 28;361(6409):1373-6.
  11. Livsmedelsveket, Dioxiner och PCB. [Internet]. Livsmedelsverket.se. 2019 [cited 16 October 2019]. Available from: https://www.livsmedelsverket.se/livsmedel-och-innehall/oonskade-amnen/miljogifter/ dioxiner-och-pcb
  12. ] Göteborgs-Posten, Totalförbjudet gift i Askim känt i åratal - men ingenting har gjorts. [Internet]. gp.se. 2019 [cited 16 October 2019]. Available from: https://www.gp.se/nyheter/g%C3%B6teborg/totalf%C3%B6rbjudet-gift-i-askim-k%C3%A4nt-i-%C3%A5ratal-men-inget-har-gjorts-1.13891903
  13. Havsörn - Världsnaturfonden WWF [Internet]. Världsnaturfonden WWF. 2019 [cited 16 October 2019]. Available from: https://www.wwf.se/djur/havsorn/#artdata
  14. Sillgrissla - Världsnaturfonden WWF [Internet]. Världsnaturfonden WWF. 2019 [cited 21 October 2019]. Available from: https://www.wwf.se/djur/sillgrissla/#hotfylld-historia
  15. Sälar is Sverige: Hot - Världsnaturfonden WWF [Internet]. Världsnaturfonden WWF. 2019 [cited 16 October 2019]. Available from: https://www.wwf.se/djur/salar/#hot
  16. Food and Agriculture Organization of the United Nations. Assessing soil contamination A reference manual [Internet]. Fao.org. 2019 [cited 13 October 2019]. Available from: http://www.fao.org/3/X2570E08.htm?fbclid=IwAR2kMzvRuwBMAF_ndSCfdRNQ-Bue_b6lx0gcO6fNJtj8b7zd_ExHZ2Qk2BE
  17. He J, Bedard DL. The microbiology of anaerobic PCB dechlorination. InOrganohalide-Respiring Bacteria 2016 (pp. 541-562). Springer, Berlin, Heidelberg.
  18. Agulló L, Pieper DH, Seeger M. Genetics and biochemistry of biphenyl and PCB biodegradation. Aerobic Utilization of Hydrocarbons, Oils, and Lipids. 2019:595-622.
  19. Bedard DL. PCB dechlorinases revealed at last. Proceedings of the National Academy of Sciences. 2014 Aug 19;111(33):11919-20.