Topic Selection:
Microplastic pollution is a research hotspot in the world. With the development of the investigation on microplastics over these years, more and more literature points out that microplastics is a big threat to both the environment and human body. Our team takes this very seriously and tries to find potential solutions .
The literature states that plastics are used all over the world. Unfortunately, plastics can cause severe environmental pollution due to their limited biodegradation. The smallest ones are called nanoplastics (1 nm to 1 μm) and microplastics (1 μm to 5 mm). These nano and microplastics can enter the body by inhalation through the respiratory system, into the digestive tract by consumption of contaminated food and water, or into the skin through cosmetic and clothing contact. The bioaccumulation of plastic in the body can lead to a range of health problems, including respiratory diseases such as lung cancer, asthma and allergic pneumonia, neurological symptoms such as fatigue and dizziness, inflammatory bowel disease, and even disorders of the gut microbiota[1].
Among the existing concentrated plastic types, the studies on polyethylene glycol terephthalate （PET）and Polyethylene （PE） biodegradation are relatively well developed. After much consideration, we decided to start with the degradation of PET[1].
Background Research: PET contamination and types:
First, when validating the biotoxicity of PET plastic and its degradation products, we found out that the final degradation product TPA is harmful to Aliivibrio fischeri (A. fischeri) ,a bioluminescent Gram-negative Marine bacterium, meaning that TPA monomers can interrupt Marine biodiversity[2]. We found out that microplastic PET itself is a potential threat in the environment, and TPA will still cause harm to the water microenvironment. Therefore, Both of the PET degradation and recycle of TPA are needed.
This is followed by a investigation on the distribution and shape of PET plastic pollution. In order to have a deeper understanding of the pollution distribution and severity of PET microplastics in the environment, we first investigated the relevant literature, and then interviewed professor Xiong XIONG from the Institute of Hydrobiology, Chinese Academy of Sciences. His research direction is the environmental convergence and ecological effect of new pollutants (microplastics). Under the introduction of Mr.Xiong, we have a deeper understanding on the severity of PET microplastic pollution. The microplastics in the water environment are mainly PE, PP, and PET. Here, he pointed out that the main form of PET microplastics in the environment is in the form of fiber, which means that most of the source of PET microplastics is polyester fiber as the material of textile, rather than the macro plastic water bottle, although the latter is the most widely use of PET, so we face PET pollution with small particle size , from nanometer diameter to micrometer diameter.
After selecting the project’s objectives and determining the background through research, we decided to use the engineered E. coli to degrade the PET microplastics present in the environment. At the same time, the most widespread application of C. elegans as a common model organism is to detect toxicity or to explore neural function. Our team has been trying to do synthetic biology with C. elegans since last year, in this year, we want it can be enginnered to be able to further enrich TPA.
Experimental Design:
First we expressed TurboPETase in E. coli BL21, it is an enzyme that can degrade PET to monomer TPA. At the same time, we realized that E. coli itself does not have the ability to transport PET, so PETase needs to be released into the liquid environment outside the bacteria to function. A common method is to purify the enzyme after breaking the bacteria. However, this approach is flawed in terms of cost and practicality, so we tried to secrete the enzyme using four signal peptides commonly used by E. coli , and select the most effective signal peptides for subsequent use.
Subsequently, in order to process the treated toxic monomer TPA, we transferred TphC, TpiA and TpiB to E. coli. Considering that the existing techniques for detecting TPA are relatively complicated (such as HPLC), we characterized the treatment process by PtphC, tphR and eGFP. When TPA is transported into E. coli, TPA will activate PtphC to express fluorescent protein, and the fluorescence intensity is proportional to the amount of the TPA transported.
In addition, we hoped that C. elegans can also participate in this reaction process, but considering that the physiochemical properties of turboPETase itself are not suitable for the intestinal environment of nematodes, we finally chose to let nematodes undertake the task of transporting and enriching TPA, and tried to transfer tphC, TpiA, TpiB, Ptpc, tphR genes and eGFP to achieve similar effects proposed in E. coli part.
Experiment Improvement:
For our preliminary experimental design, we asked professor Janek HYZEWICZ from University of Evry Paris-Saclay, he agreed with our experiment design, but he pointed out that we should determine the size of the materials PET , because C. elegans cannot swallow part of the larger size microplastic particles, and the large particles may produce physical wear and other effects that affect its survival. He suggested that we use microplastics from below 5 micrometer in diameter as our experimental material. Finally, we chose to use nano-diameter microplastics as experimental materials.
In addition, he also pointed out that if we want to judge the degradation effect of PET plastic, we had better use pure PET microplastic samples, because most of the existing finished plastic has added many other organic substances to improve the physical and chemical properties of the plastic. Some additives can affect the accuracy of our nematode detection, and we adopted this idea and used pure microplastics.
For the experiment of synthetic biology, we consulted professor Shangxian XIE from the School of Life Science and Technology, Huazhong University of Science and Technology. He agreed our protocol and suggested that we add MHETase to E. coli, because despite the ability to produce TPA, this deep degradation is incomplete, with a larger part of PET being degraded to MHET and producing large biotoxicity, requiring MHETase to participate in the process of degradation and improve the degradation efficiency. We took his precious suggestion and introduced MHETase during the subsequent experiments.
In terms of C. elegans, our PI professor Lei CHUN pointed out that before the formal use of engineering E. coli, we need to do a pre-experiment in advance to explore whether C. elegans can live normally in a liquid environment containing microplastic PET, and whether it can live normally in a liquid environment containing TPA. See our results section for more details.
Conclusion:
After topic selection, background investigation, basic design and design improvement , we finally obtained a kind of engineering E. coli which could degrade PET and achieve the goal of primary enrichment of TPA, this is our first guardian.At the same time we try to use C. elegans to reach the goal of secondary enrichment of TPA by digesting the E. coli which contains TPA, meanwhile trying to solve the biological safety problems caused by the usage of engineering E. coli, this is our second guardian.
Reference:
1. Winiarska E, Jutel M, Zemelka-Wiacek M. The potential impact of nano- and microplastics on human health: Understanding human health risks. Environ Res. 2024 Jun 15;251(Pt 2):118535. doi: 10.1016/j.envres.2024.118535. Epub 2024 Mar 7. PMID: 38460665.
2. Djapovic M, Milivojevic D, Ilic-Tomic T, Lješević M, Nikolaivits E, Topakas E, Maslak V, Nikodinovic-Runic J. Synthesis and characterization of polyethylene terephthalate (PET) precursors and potential degradation products: Toxicity study and application in discovery of novel PETases. Chemosphere. 2021 Jul;275:130005. doi: 10.1016/j.chemosphere.2021.130005. Epub 2021 Feb 17. PMID: 33640747.