Back to Top

Plant

Placeholder image Placeholder image Placeholder image Placeholder image Placeholder image

Our motivation

The ongoing pollution of all types of water by agricultural, industrial, and medical waste is destroying ecosystems and life around the world1,2 .It is not possible to confine toxic substances locally to their area of use - neither regionally nor nationally nor globally. Herbicides or insecticides used in fields run off into rivers and lakes and can pollute the environment far from where they were originally applied. Anti-fouling chemicals used in marine coatings pollute oceans and marine ecosystems. There are endless examples that could be added to the list. Additionally, every year, millions of new chemicals are synthesized that we can neither adequately characterize nor contain. The question of how long nature can tolerate this poisoning becomes more important every day. Of the nine planetary boundaries that set limits within which humanity can develop and thrive in the future, we have already crossed several, including environmental pollutants (novel entities)3(Fig.1). Crossing such planetary boundaries dramatically increases the risk of irreversible environmental damage. Since the Earth is no longer capable of reversing man-made pollution on its own, we see it as our duty, our obligation, to reverse the pollution we have caused. We must act now to ensure that there is a future in which humanity can flourish.

Placeholder image

Fig.1 Planetary boundaries
The planetray boundaries were designed to describe the impact of human activities and are divided into 9 different categories. Of 6 we have already crossed the safe operating space4.

Chlamydomonas reinhardtii

As we discussed selecting an organism for the implementation of our innovative system, one organism stood out prominently: The well-studied, photosynthetic green microalgae Chlamydomonas reinhardtii. Owing to its minimal nutrient requirements, this alga offers facile, cost-effective and sustainable cultivation, making it a very exiting model organism for biotechnology5 and modern industries6. Its fast growth rate7 coupled with the simplicity of genetic modification, few isoforms of enzymes and the absence of tissues, positions Chlamydomonas reinhardtii as an excellent model organism, an intermediate stage before testing in higher, more complex plants. Moreover, its nuclear and chloroplast genome were sequenced in 2007, enhancing our understanding of its genetic makeup8. Furthermore in 2018 our PI Prof. Schroda co-authored a paper on a newly developed modular cloning toolkit for Chlamydomonas reinhardtii. This on the golden gate cloning based system enables rapid genetic manipulation of the algae9. This very system we worked with during our iGEM time. Its implementation not only elevated our working pace but also empowered us to do experiments beyond our initial imagination. Further we want to shift focus to newly developed vectors enabling the direct cloning of level 0 parts into level 2 vectors, facilitating the cloning time even further.10

Our Project

We have focused our effort on the bioremediation of all kinds of hazardous substances threatening us and the environment. In pursuit of this goal, we created several different genetically modified strains of Chlamydomonas all of which share a common element: The cytochrome P450 (CYP) enzymes. These remarkable enzymes can be found across all kingdoms of life, having diverse function such as the biosynthesis of e.g. steroids and the metabolism of numerous xenobiotics. We have found that the potential of using CYP enzymes for bioremediation is virtually untouched and that there are hundreds, if not thousands of novel, undiscovered and uncharacterized enzymes that have the potential to be used for bioremediation of all types of toxic substances. Envisioned within our minds is a comprehensive library (Fig.2) consisting of diverese strains, each targeting distinct hazardous substances. This concept enables the creation of a cocktail of different strains, perfectly suited to target any specific pollution.
Throughout our igem time we successfully introduced several of these enzymes in Chlamydomonas using the MoClo system. We were able to express 5 out of these 6 enzymes and gained a lot of insight on how they work and how you could messure their activity.

We did it! We were able to show CYP activity! This is a big step!
Placeholder image

Fig.2 Expression of CYPs in the cytosol
(1a-7a) Level 2 MoClo constructs for expression of the enzymes CYP3A4, 9Q3, 2D6, the POR and CYPCamC containing either the FLAG or HA-tag were designed. (1b-7b) The UVM4 strain was transformed with the construct in (a). 30 antibiotic-resistant transformants (depending on the construct) were cultivated in TAP-medium and samples taken after 3 days. Whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting using an anti-HA antibody. In the resultant blot, the black arrow marks our enzymes and the white arrow marks a cross reaction of antibodies. The expression of CYP3A4 (~57 kDa), CYP 9Q3 (~59 kDa), CYP2D6 (~ 56 kDa), the POR (~ 77 kDa) and CYPCamC (~ 47 kDa) is visible. For reference, the UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) or FLAG-tagged VIPP1 were used as a negative and positive control, respectively

Conclusion

During our project we made significant contributions to the iGEM phytobrick registry by introducing several innovative composite parts simultaneously testing a lot of existing parts, all of which are standardized and MoClo compatible. Our aspiration was to inspire and encourage other teams to pursue the path of our project and use Chlamydomonas for bioremediation and thereby contribute to a more ecologically sustainable and cleaner world. Our system itself offers a lot of future work, with the possibility to add numerous new CYPs targeting new substances to broaden our library. Additionally, the potential for measuring enzymatic activity by other usage of other substrates, or co-expression with a regulated reductase offers an untapped potential.

  1. Panthi S, Sapkota AR, Raspanti G, Allard SM, Bui A, Craddock HA, Murray R, Zhu L, East C, Handy E, Callahan MT, Haymaker J, Kulkarni P, Anderson B, Craighead S, Gartley S, Vanore A, Betancourt WQ, Duncan R, Foust D, Sharma M, Micallef SA, Gerba C, Parveen S, Hashem F, May E, Kniel K, Pop M, Ravishankar S, Sapkota A. Pharmaceuticals, herbicides, and disinfectants in agricultural water sources. Environ Res. 2019 Jul;174:1-8. doi: 10.1016/j.envres.2019.04.011. Epub 2019 Apr 17. PMID: 31015109.
  2. Ruuskanen S, Fuchs B, Nissinen R, Puigbò P, Rainio M, Saikkonen K, Helander M. Ecosystem consequences of herbicides: the role of microbiome. Trends Ecol Evol. 2023 Jan;38(1):35-43. doi: 10.1016/j.tree.2022.09.009. Epub 2022 Oct 13. PMID: 36243622.
  3. Linn Persson, Bethanie M. Carney Almroth, Christopher D. Collins, Sarah Cornell, Cynthia A. de Wit, Miriam L. Diamond, Peter Fantke, Martin Hassellöv, Matthew MacLeod, Morten W. Ryberg, Peter Søgaard Jørgensen, Patricia Villarrubia-Gómez, Zhanyun Wang, and Michael Zwicky Hauschild Environmental Science & Technology 2022 56 (3), 1510-1521 DOI: 10.1021/acs.est.1c04158
  4. J. Chris Slootweg, Using waste as resource to realize a circular economy: Circular use of C, N and P, Current Opinion in Green and Sustainable Chemistry, Volume 23, 2020, Pages 61-66, ISSN 2452-2236, doi.org/10.1016/j.cogsc.2020.02.007.
  5. Stefan Schmollinger, Dissection of the HSF1-dependent stress response with a special focus on the chloroplast HSP70 system
  6. Scaife MA, Nguyen GTDT, Rico J, Lambert D, Helliwell KE, Smith AG. Establishing Chlamydomonas reinhardtii as an industrial biotechnology host. Plant J. 2015 May;82(3):532-546. doi: 10.1111/tpj.12781. Epub 2015 Mar 8. PMID: 25641561; PMCID: PMC4515103.
  7. Severin Sasso, Herwig Stibor, Maria Mittag, Arthur R Grossman (2018) The Natural History of Model Organisms: From molecular manipulation of domesticated Chlamydomonas reinhardtii to survival in nature eLife 7:e39233 https://doi.org/
  8. Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Maréchal-Drouard L, Marshall WF, Qu LH, Nelson DR, Sanderfoot AA, Spalding MH, Kapitonov VV, Ren Q, Ferris P, Lindquist E, Shapiro H, Lucas SM, Grimwood J, Schmutz J, Cardol P, Cerutti H, Chanfreau G, Chen CL, Cognat V, Croft MT, Dent R, Dutcher S, Fernández E, Fukuzawa H, González-Ballester D, González-Halphen D, Hallmann A, Hanikenne M, Hippler M, Inwood W, Jabbari K, Kalanon M, Kuras R, Lefebvre PA, Lemaire SD, Lobanov AV, Lohr M, Manuell A, Meier I, Mets L, Mittag M, Mittelmeier T, Moroney JV, Moseley J, Napoli C, Nedelcu AM, Niyogi K, Novoselov SV, Paulsen IT, Pazour G, Purton S, Ral JP, Riaño-Pachón DM, Riekhof W, Rymarquis L, Schroda M, Stern D, Umen J, Willows R, Wilson N, Zimmer SL, Allmer J, Balk J, Bisova K, Chen CJ, Elias M, Gendler K, Hauser C, Lamb MR, Ledford H, Long JC, Minagawa J, Page MD, Pan J, Pootakham W, Roje S, Rose A, Stahlberg E, Terauchi AM, Yang P, Ball S, Bowler C, Dieckmann CL, Gladyshev VN, Green P, Jorgensen R, Mayfield S, Mueller-Roeber B, Rajamani S, Sayre RT, Brokstein P, Dubchak I, Goodstein D, Hornick L, Huang YW, Jhaveri J, Luo Y, Martínez D, Ngau WC, Otillar B, Poliakov A, Porter A, Szajkowski L, Werner G, Zhou K, Grigoriev IV, Rokhsar DS, Grossman AR. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science. 2007 Oct 12;318(5848):245-50. doi: 10.1126/science.1143609. PMID: 17932292; PMCID: PMC2875087.
  9. Pierre Crozet, Francisco J. Navarro, Felix Willmund, Payam Mehrshahi, Kamil Bakowski, Kyle J. Lauersen, Maria-Esther Pérez-Pérez, Pascaline Auroy, Aleix Gorchs Rovira, Susana Sauret-Gueto, Justus Niemeyer, Benjamin Spaniol, Jasmine Theis, Raphael Trösch, Lisa-Desiree Westrich, Konstantinos Vavitsas, Thomas Baier, Wolfgang Hübner, Felix de Carpentier, Mathieu Cassarini, Antoine Danon, Julien Henri, Christophe H. Marchand, Marcello de Mia, Kevin Sarkissian, David C. Baulcombe, Gilles Peltier, José-Luis Crespo, Olaf Kruse, Poul-Erik Jensen, Michael Schroda, Alison G. Smith, and Stéphane D. Lemaire ACS Synthetic Biology 2018 7 (9), 2074-2086
  10. Niemeyer J, Schroda M. New destination vectors facilitate Modular Cloning for Chlamydomonas. Curr Genet. 2022 Aug;68(3-4):531-536. doi: 10.1007/s00294-022-01239-x. Epub 2022 Apr 16. PMID: 35429260; PMCID: PMC9279246.
WaveyFooter