Team:ASTWS-China/Description
Summary
Our world is overwhelmed in plastics pollutions. Concerns about environmental issues and news related to microplastic pollution inspired us to start this project. our mission is to degrade micro/nano-plastics (PET) in the water body by genetically modified E.Coli biodegradation system that produces PETase and strengthens the biofilm system. This combined biodegradable system can not only degrade PET plastics but also attach degraded microplastics, so it will not flow away, thereby preventing it from further pollution. Our product would be environmentally safe and biodegradable. We surveyed the general public as well as professional workers so that we can have the best understanding of our project. By understanding our parts and going through experiments, we created the best outcome of the project, and our mission is on the way.
1. Background
Plastic‘s production increased drastically from the 1950s’ and reached 322 million tons of production in 2015. 1.4 billion tons of waste is produced annually, roughly 10% of which is plastic[1]. Globally, only 18% of plastics waste is recycled, and 24% is incinerated. The remaining 58% are either landfilled or enter the natural environment, where plastics accumulate and persist for a long period (Figure 1) [2]. It is usually broken down from larger plastic pieces and could be broken down again to become microplastics, which are further degraded into nanoplastics [3].
Figure 1 Global production, use, and fate of polymer resins, synthetic fibers, and additives (1950 to 2015; in million metric tons)[2].
1.1 What is nano-/microplastics?
Microplastic refers to any plastic particles that have a diameter less than 5mm [3]. The shortest definition of nanoparticles only concerns about their size, which is limited conventionally to about 100 nm in any direction [3]. Studies have shown that the sources of nano-/ microplastics can be divided into two categories, primary and secondary sources. As shown in Figure 2, primary sources, the original microplastics were initially synthesized at the microscopic level and widely used in various manufacturing industries, such as therapeutic agents, food science, chemical agent, and personal care products and cosmetics. Secondary plastics refer to those produced by the decomposition of large plastic materials. The decomposition products enter the environment due to the environmental weathering of plastic objects. It is worthy that secondary microplastics are considering the major source of microplastic pollution in the marine environment [4].
Figure 2 A conceptual diagram showing the sources of microplastic pollution and their environmental, ecological and health related impacts[4].
1.2 Why we must degrade nano-/microplastics?
With the help of agricultural runoff, wastewater treatment plants, floods, river systems, etc., nano/ microplastics are transported to freshwater systems or into the ocean and further diffused under the action of water currents, wind, and turbulence. Moreover, due to the extremely long shelf life, the nano/microplastics are finally returning to humans (Figure 2). Generally, nano-/microplastics could act as a reservoir of heavy metals, microorganisms, pathogens, and other harmful substances. Due to their unique physicochemical characteristics, microplastics offer a distinct surface for chemical acquisition, pollutant accumulation, and microbial communities. Because of their low degradation rates, microplastics can persist in the environment for decades or even centuries. The long-lasting presence of microplastics in the aquatic environment is considered a threat to many aquatic animals as well as the human body.
1.2.1 Potential impacts on the human body
It is a potential threat to the digestive system, the liver, and the brain. Here are some ways that it could reach humans.
1.2.2 Potential impacts on ecosystem
The coastal environment, which can absorb persistent organic pollutants, are threatened by
microplastics. These particles, microplastics, are dangerous to marine species by exposing
risk on the food chain. It is acknowledged that it is simple for marine animals to absorb
microplastics since they are so small, which would cause terrible health problems. For
example, oyster’s thriving and reproducing abilities are harmed, due to the consumption of
microplastics. Therefore, after a long time, disruption of marine animals’ populations will
eventually break the delicate balance of the marine ecosystem. Now, “Over 700 species of the
marine organism are known to encounter plastics in the environment with clear evidence of
physical harm from entanglement and ingestion”, and people also worry about microplastics
would bring toxicological hazard to marine animals if marine animals digest those tiny
particles with food. Besides, it also impacts the ability to absorb the nutrients in a
negative way [6-9].
Microplastics have done a great deal in harming the environment due to its ability to resist
degradation and its level of fragmentation. It is ubiquitous and can be found everywhere.
Many species, marine animals the most severe, have been threatened. Even humans could be put
at risk. Thus, we conclude that immediate action is needed.
1.3 Current solutions and their drawbacks
In response to the seriousness of the plastic pollution problem, plastic pollution has always been one of the
hot issues. In general, the solutions to plastic pollution can be divided into three categories: containment,
mitigation, and separation[8].
Containment: is primarily through recycling and landfilling. Landfills provide proper physical segregation to
prevent plastic waste from entering the environment. However, it is suggested that landfill runoff may further
spread microplastic pollution.
Mitigation: is mainly a prevention strategy in-laws and regulations. For example, banning the use of plastic
microbeads in cosmetics and encouraging good waste management practices. Although mitigation measures can help
minimize the accumulation of microplastic pollution caused by human actions, they do not affect existing
plastic pollution.
Separation: refers to the separation of microplastics from the sewage matrix to prevent them from entering the
ecosystem. In wastewater treatment plants, there are usually many processes, including thermal degradation,
mechanical action, digestion, and sedimentation, etc. However, in fact, microplastics can easily pollute
wastewater and settled sludge, and will re-enter the ecosystem through further irrigation and agricultural
fertilization.
Consequently, it seems separation and further treatment is a better solution for now and the future.
1.4 Previous iGEM Projects
Plastic pollution treatment is one of the hot issues of iGEM environment track. We reviewed the related projects over the years and summarized the successful project results as follows (Table 1).
Since Japanese researchers discovered a magical bacterium (Ideonella sakaiensis 201-F6) that can "eat
plastic"[9], encouraging advances related to the biodegradation of PET plastic appear frequently. In summary,
the previous iGEM teams are mainly committed to solving the problems related to PET degradation: 1) improving
the degradation efficiency and 2) improving the thermal stability of PETase (2019 Exeter). 3) Improving the
secretion efficiency of PETase if necessary (2018 Yale). Particularly, for improving the efficiency of enzyme
degradation, currently proposed solutions include combined enzyme design (PETase, MHETase), replacement of
chassis organisms, and sequence-directed mutation (eg. 2019 TU Kaiserslautern, 2019 Exeter). Please see more
details in our CONTRIBUTION page.
As a high school team, inspiring by 19 TU Kaiserslautern, we committed us to the solution of the first topic.
2. Our solutions and design
Consequently, our ultimate goal is to improve the degradation efficiency of PETase. To have better contrast, we choose wild-type PETase to construct our genetic circuit. The core of our design idea is the proximity effect of the enzyme and substrate thereby improve the degradation efficiency. Our biofilm systems mainly include three parts: strengthening biofilm, PETase expression system, and double fluorescence reporting systems. Here are the details as follows.
2.1 Strengthen biofilms system (BBa_K3576000)
Considering that the existing microplastic pollution is distributed everywhere in the water body, thus we add the strengthening biofilm to trap nano-/microplastics that is inspired by the 2017 iGEM TAS-Taipei team. E. coli produces biofilms through the curli operons, which is regulated by two proteins, OmpR and CsgD. It turns out that overexpression of OmpR and CsgD could increase more curli monomers yield and successfully trap the nanoparticles[10]. In our project, we choose the OmpR to regulate biofilm production that would allow curli monomers to be exported and form curli fibers and biofilm (Figure 3).
Figure 3 Schematic diagram of E. coli biofilm yield regulated by OmpR234 and CsgD.
In our strengthening biofilms system, as shown in Figure 4, a strong T7 promoter (BBa_I712074) and strong RBS combination (BBa_B0034) to maximize protein production, and a double terminator (BBa_B0015) to end transcription. Therefore, by overexpressing OmpR234 (BBa_K342003), biofilms could become much more sticky and easy for capturing nano-/micro-plastics.
Figure 4 BBa_K3576000: OmpR234 Expression.
2.2 PETase expression system (BBa_K3576001)
The detailed genetic circuit of PETase expression system is showed in Figure 5. Here, we simply introduce PETase (BBa_K1582000) to impart biodegradation ability to E. coli. Besides, we also add PelB signal peptide (BBa_K2302005) for releasing enzymes out of the membrane. In addition, the fusion GFP protein will provide a visible report to confirm whether the PETase protein is successfully expressed. The main function of this biobrick is overexpressing PETase and transporting out of the membrane to degrade nano-/microplastics.
Figure 5 BBa_K3576001: PETase Expression.
2.3 Double fluorescence reporting systems
To ensure the proximity effect between PETase and the biofilm, the GFP-mCherry double fluorescence reporting system was introduced for the co-expression. GFP refers to the green fluorescent protein (GFP) that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range., and mCherry refers to a member of the mFruits family of monomeric red fluorescent proteins (mRFPs). As shown in Figure 6 is the genetic circuit design of double fluorescence reporting systems. We add the mCherry reporter to the end of the OmpR234 strengthening biofilm circuit and add the eGFP to the PETase expression circuit. Upon co-expression, the two form a bright yellow color, so as to indicate co-expression or co-localization of proteins
Through the sticky effect of biofilm to microplastics, the distance between the co-expressed PETase and substances could be shortened, boosting degradation efficiency.
Figure 6 Schematic diagram of double fluorescence reporting systems.
2.4 Future plan
However, according to the research, the thermal stability is another concern we need to consider. Normally,
enzymes like PETase cannot survive in high-temperature surrounding and therefore not being able to degrade PET
efficiently. Besides, to ensure the biosafety and convenience to use in real world, we also plan to purify the
protein to better demonstrate its degradability (See more details in our Implementation page).
By collaborating with another team (TJUSLS_China 2020), we know that the thermal stability improvement of
PETase is on the way, sometimes, the stability can be improved to be stable even above 75 ˚C. If the
construction of the above system is successful, our next goal is to build a PET degradation system with higher
degradation efficiency and higher thermal stability.
Due to the COVID-19 epidemic, our experimental time is relatively limited this summer. We have sorted out the
references for improving the degradation efficiency and thermal stability of PETase. It is not only as a
reference for our future project design but also we hope to share this survey result publicly with all iGEMers
interested in this topic (See more details in our Contribution page). Hoping our world is better and safe.
References:
[1] Chamas, A., et al., Degradation Rates of Plastics in the Environment. Acs
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[9] Henrik Hamrén，2017，Effects of microplastics on marine life，viewed 29 July
2020，https://balticeye.org/en/pollutants/microplastics-in-marine-animals/
[10] REINALDO126,2020,The Harm Caused By Microplastics in the Marine
Environment,viewed 29 July 2020
https://picnicon.com/microplastics-in-the-marine-environment/
[11] Degradation of Microplastic Residuals in Water by Visible Light Photocatalysis
TAJKIA SYEED TOFA KTH
ROYAL INSTITUTE OF TECHNOLOGY SCHOOL OF ARCHITECTURE AND THE BUILT ENVIRONMENT. (n.d.). Retrieved from
http://www.diva-portal.se/smash/get/diva2:1266570/FULLTEXT01.pdf
[12] https://2017.igem.org/Team:TAS_Taipei/Experimental_Summary#CO