Team:Lund/Description
Project description
Background
Do you ever stop to think about the small choices you make in your everyday life and what consequences it might bring to your surroundings?
Many of the chores and tasks we perform daily may contribute significantly to the ever growing microplastic littering in marine habitats. In fact, a single basket of laundry may release as much as 700 000 microplastic particles with each wash [1] and practicing sports on an artificial grass field contributes to the cumulative release of several tonnes of microplastic debris yearly [2]. Indeed, making a conscious effort in your daily life will have a direct impact on the future of Earth.
Microplastics are tiny fragments of plastics, less than five millimeters long, that tend to accumulate in the environment due to their limited degradability [3]. The origin of these fragments can be divided into two larger categories; primary and secondary sources [4]. Primary sources corresponds to beauty products and withering of larger plastic objects. Examples include shower gels, wear of car tyres and synthetic textiles [5]. Secondary sources originate from slower mechanical or chemical weathering processes on plastics already disposed in nature, as observed in long-term UV degradation of larger plastic bodies [3].
While there exists only limited evidence supporting the toxicity of direct exposure to microplastics, such as shedding from fleece clothing, the potential risk to biota and humans increases drastically when the fragments reach larger bodies of water, such as lakes and oceans [6]. To the primary consumers, the small plastic pieces can look indistinguishable from food stuff, allowing microplastics to enter the food chain. As they are not digestible, they accumulate upwards, causing blockage of the gastrointestinal tract and interference with reproduction of smaller consumers [7]. Another concern of many water-bound microplastics lies in their affinity for hydrophobic compounds. Both heavy metals and persistent organic pollutants, two priority pollutants according to the EPA Clean Water Act, end up accumulating around microplastics [8] [6]. Since these compounds often have a high bioavailability and are only bound to the plastic through weak association, they readily leach off the plastic and can cause adverse effects on the endocrine system and central nervous system [9] [10]. Similarly, plasticizers, or low-weight phthalates, which are common additives in commercial plastics that offer improved flexibility and ductility, has been known to cause hormonal disruption in humans and wildlife [11].
Surely, with extensive research corroborating the complications of microplastics, stringent laws and regulations ought to have been put in place to prohibit microplastic pollution - to protect the marine biodiversity and human health?
Well, not quite.
According to the United Nations’ Environment Programme, microplastics started to appear in the aforementioned products about fifty years ago and up until a few years ago the consumer awareness of the potential ramifications was close to nonexistent. However, in the past few years, the consumer apathy regarding the subject started to cease and political actions were finally put in place. In 2015, former President Obama signed the Microbead-Free Waters Act of 2015, consequently banning plastic additives in cosmetics and personal care products in the US [12] [13]. Some countries have since then followed suit, but far from the majority. Perhaps most notably, the European Union has not made any strides toward the banning of microplastic products [11].
Recent discussions regarding the realization of the UN development goal number 14 concerning the conservation and sustainable use of marine resources [14] once again shed light on the matter and triggered a demand from consumers that retailers limit the amount of products containing microplastics. This caused a domino-effect in the entire supply chain that forced businesses to act to not lose their competitive edge on the market. Consequently, several retailers with significant market shares chose to stop handing out free plastic bags and selling cosmetics containing microplastics [15] [16].
While the issue is being addressed and regulations are moving in the right direction, the process is slow and without a realistic end in sight. We, team iGEM Lund, have therefore chosen to devote this year to participate in the world-wide engagement against microplastic accumulation in the ocean, in line with the United Nations new framework for sustainable development.
How can synthetic biology aid?
With some clever insight into the dynamics of synthetic biology, it can be harnessed to combat various aspect of marine debris. This has been recognized by previous iGEM teams throughout the years and the problem of microplastics in particular has been targeted multiple times, mainly through different methods of biotic degradation [17] [18] [19]. However, while bio-degradation would certainly be the prefered approach, as it eliminates the plastic once and for all, the process is very time-consuming and it does not compete with the abiotic degradation methods that exists today [20] [21]. With this in mind, we set out to apply our knowledge of synthetic biology to alleviate the current microplastic situation. After dialogues with water sanitation experts [link to integrated practices], it was made apparent that not only does the Swedish government allocate very little resources to the sanitation of microplastics in water treatment plants, but that there exists no quick and easy way of even detecting whether sanitation might be necessary or not without the use of expensive laboratory equipment and arduous filtration processes. Currently, the most efficient method of assessing microplastic content is through sieving and several subsequent filtration and separation steps [22]. A simple tool to evaluate the presence of microplastics would therefore be very helpful in that regard. Consequently, μSense was born.
Our solution
For this years iGEM competition, we have decided to engineer a simple, novel biosensor with the goal of determining the presence of microplastics in freshwater. The sensor will be realized by design and implementation of a genetic circuit into Escherichia coli. A logic AND-gate will be constructed using the expression of two heterologous genes to detect the presence of microplastics.
The design
As plastic particles are highly unreactive molecules, two molecules often associated with microplastics, persistent organic pollutants and low-weight phthalates, will be detected. The project will expand on the groundwork laid out by the 2012 UCL iGEM team that recognized that a viable approach to detection of microplastics was through indirect means [17]. Consequently, they found that the NahR transcriptional regulator from the naphthalene degradation operon NAH7 of Pseudomonas putida had notable affinity for organic pollutants, as they share homologous regions with its intended cofactor salicylate. To improve on their work, low-weight phthalates will be recognized concurrently using the human nuclear receptor NR3A1 - the estrogen receptor alpha (ER-α) - to increase the selectivity of the intended sensor. To alleviate the stress of introducing larger quantities of recombinant DNA, only the ligand binding domain (LBD) of the ER-α receptor will be used. This is made possible by the conformational change that occurs upon antagonist recognition. As the N and C terminal are brought into close proximity, self assembly of split proteins is made possible if fused to the sides of ER-α LBD with appropriate linkers [23]. To incorporate this into the genetic circuit design and to add some depth to the project, the reporter will be expressed as fragments of a tripartite split fluorescent protein. As limited research has been conducted on the applications of tripartite split reporters, we intend to investigate their viability of use as reporter element in synthetic biology.
In summary, an AND-gate will be constructed according to figure 1. Both the NahR transcription factor and the ER-α LBD fusion protein will be repressed by a downstreams lac operator to allow for external induction of protein expression. Upon expression, the transcriptional factor NahR will associate with a Psal promoter and in presence of an inducer, such as a persistent organic pollutant, cause a conformational change that promotes the expression of one third of the tripartite split fluorophore [24]. Concurrently, an ER-α LBD fusion protein with the remaining two fluorophore fragments fused to each respective side will be expressed and upon antagonistic ligand recognition, the two fluorophore fragment will be brought into close proximity and allow for self-assembly with the NahR regulated fragment [25].
E. coli was chosen as the chassis for the system at hand, due to its rapid generation time and well characterized nature. Furthermore, both heterologous proteins have been expressed in E. coli successfully [26] [27].
References
[1] Laura Paddison (2016, September 27). “Single clothes wash may release 700,000 microplastic fibres, study finds”. The Guardian. Retrieved 2017-09-01.
[2] KIMO (2017, February 27). “Microplastic Pollution from Artificial Grass – A Field Guide”. KIMO (Kommunenes Internasjonale Miljøorganisasjon). Retrieved 2017-09-01.
[3] GESAMP (2016). “Sources, fate and effects of microplastics in the marine environment: part two of a global assessment” (Kershaw, P.J., and Rochman, C.M., eds). (IMO/FAO/UNESCO-IOC/UNIDO/WMO/IAEA/UN/UNEP/UNDP Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection). Rep. Stud. GESAMP No. 93, 220 p
[4] Eerkes-Medrano, D., Thompson, R. and Aldridge, D. (2015). Microplastics in freshwater systems: A review of the emerging threats, identification of knowledge gaps and prioritisation of research needs. Water Research, 75, pp.63-82.
[5] Boucher, J. and Friot D. (2017). “Primary Microplastics in the Oceans: A Global Evaluation of Sources”. Gland, Switzerland: IUCN, pp 43.
[6] Wright, S. and Kelly, F. (2017). Plastic and Human Health: A Micro Issue?. Environmental Science & Technology, 51(12), pp.6634-6647.
[7] Tanaka, K. and Takada, H. (2016). Microplastic fragments and microbeads in digestive tracts of planktivorous fish from urban coastal waters. Scientific Reports, 6(1).
[8] Federal Security Agency (2002). Federal Water Pollution Control Act.
[9] Avio, C., Gorbi, S., Milan, M., Benedetti, M., Fattorini, D., d'Errico, G., Pauletto, M., Bargelloni, L. and Regoli, F. (2015). Pollutants bioavailability and toxicological risk from microplastics to marine mussels. Environmental Pollution, 198, pp.211-222.
[10] Anderson, J., Park, B. and Palace, V. (2016). Microplastics in aquatic environments: Implications for Canadian ecosystems. Environmental Pollution, 218, pp.269-280.
[11] Jansen, J., Veldhuis, F. and Schreuder, M. (2017). Towards a European ban on microbeads - Leiden Law Blog. [online] Leidenlawblog.nl. [Accessed 1 Sep. 2017].
[12] 114th United States Congress (2015). An act to amend the Federal Food, Drug, and Cosmetic Act to prohibit the manufacture and introduction or delivery for introduction into interstate commerce of rinse-off cosmetics containing intentionally-added plastic microbeads.
[13] The National Oceanic and Atmospheric Administration (2017). What are microplastics?. [online] [Accessed 1 Sep. 2017].
[14] United Nations (2015). Transforming our world: the 2030 Agenda for Sustainable Development. [online]
[15] United Nations (2017). Goal 14 : Sustainable Development Knowledge Platform. [online] [Accessed 1 Sep. 2017].
[16] Lagercrantz, S. (2016). Apotea städar bort miljöbov. [online] Dagens Medicin. [Accessed 1 Sep. 2017].
[17] iGEM team University College London 2012 (2012). Welcome to Plastic Republic. [online] [Accessed 1 Sep. 2017].
[18] iGEM team Berlin 2015 (2015). Enzymatic Flagellulose. [online] [Accessed 1 Sep. 2017].
[19] iGEM team Virgin 2014 (2015). NyGone - a microplastic biofilter. [online] [Accessed 1 Sep. 2017].
[20] Ghosh, S., Pal, S. and Ray, S. (2013). Study of microbes having potentiality for biodegradation of plastics. Environmental Science and Pollution Research, 20(7), pp.4339-4355.
[21] Gewert, B., Plassmann, M. and MacLeod, M. (2015). Pathways for degradation of plastic polymers floating in the marine environment. Environ. Sci.: Processes Impacts, 17(9), pp.1513-1521.
[22] Masura, J., et al. (2015). Laboratory methods for the analysis of microplastics in themarineenvironment: recommendations for quantifying synthetic particles in watersand sediments. NOAA Technical Memorandum NOS-OR&R-48
[23] McLachlan, M., Katzenellenbogen, J. and Zhao, H. (2011). A new fluorescence complementation biosensor for detection of estrogenic compounds. Biotechnology and Bioengineering, 108(12), pp.2794-2803.
[24] Park, H., Lim, W. and Shin, H. (2005). In vitro binding of purified NahR regulatory protein with promoter Psal. Biochimica et Biophysica Acta (BBA) - General Subjects, 1725(2), pp.247-255.
[25] Cabantous, S., Nguyen, H., Pedelacq, J., Koraïchi, F., Chaudhary, A., Ganguly, K., Lockard, M., Favre, G., Terwilliger, T. and Waldo, G. (2013). A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association. Scientific Reports, 3(1).
[26] Schell, M. and Wender, P. (1986). Identification of the nahR gene product and nucleotide sequences required for its activation of the sal operon. Journal of Bacteriology, 166(1), pp.9-14.
[27] James L. Wittliff, Laura L. Wenz, Jing Dong, Zafar Nawaz, Tauseef R. Butt (1990). Expression and Characterization of an Active Human Estrogen Receptor as a Ubiquitin Fusion Protein from Escherichia Coli. The Journal of Biological Chemistry, 265 (35), pp 22016-22022.
