Plastics (poly (ethylene terephthalate) (PET)) has greatly facilitated people's life. However, due to the fact that plastics are difficult to degrade, the use of a large number of plastic products not only leads to environmental pollution, but also gradually increases the amount of microplastics consumed by people every day, which damages human health. Through the study of multiple model organisms, microplastics ingested through diet are mainly concentrated in the digestive tract. Therefore, the degradation of ingested microplastics through intestinal probiotics is an effective method to prevent the spread of microplastics to other parts of the body and reduce the content of microplastics in the intestine and human body.
The human gut is an important digestive organ and a good place for microbes to inhabit. Studies have shown that gut of healthy adult is home to about 1,000 to 1,100 different types of microbes, with individual populations up to 100 trillion, 10 times the number of human cells. The complex and diverse metabolic activities of human microorganisms not only assist the digestive function, but also participate in important physiological and pathological activities of various systems. Intestinal flora takes the complex diet of human body as the main carbon source, and also assists the digestive system to degrade various drugs and poisons. Probiotics with special properties can be obtained through genetic means, ultimately maintaining life health and improving the quality of life.
PET degradation related enzyme (PETase) was first reported in Science in 2016. In 2018, PNAS reported that MHETase can further degrade MHET into TPA (mono (2-hydroxyethyl) terephthalic acid) and EG (ethylene glycol). Efficient enzyme for degradation of PET (IsPETase) was reported in Nature Catalysis in 2021. Thus, plastic products can be efficiently degraded into recyclable monomers. Therefore, we decide to express IsPETase and MHETase (PET degradation enzyme) in the cells of probiotics to reduce the content of human microplastics and improve human health.
II. General concept
First of all, we will manipulate PCR on two types of plasmids: pET28a-IsPETase and pET28a-MHETase. After each PCR process, we will manipulate electrophoresis on the samples that we have got, in order to examine if our PCR is successful and to extract the DNA fragments we want. Therefore, from the first step, we will obtain DNA fragments IsPETase and MHETase. We connect IsPETase and MHETase together to form IsPETase-MHETase. Finally, we will get plasmids: pET28a-IsPETase, pET28a-MHETase and pET28a-IsPETase-MHETase.
We will transform these three plasmids into chemically competent cells, E. coli BL21(DE3). Then, we will carry out IPTG induction for E. coli BL21. After this, we will manipulate SDS-PAGE, Coomassie brilliant blue staining, enzyme activity test and probiotics activity test to detect the expression of IsPETase and MHETase proteins.
III. Expected results
1. The full length of the synthesized IsPETase gene is 897bp and MHETase is 2082bp.
2. The plasmid pET28a-IsPETase, pET28a-MHETase and pET28a-IsPETase-MHETase are successfully constructed, which are identified by enzyme digestion.
3. IsPETase and MHETase protein products with biological functions were could be obtained from E. coli BL21. SDS-PAGE and Coomassie brilliant blue staining show that these two proteins are expressed.
4. In vitro experiments (enzyme catalysis) and in vivo experiments (bacteria catalysis) confirm that the protein had PET degradation performance.
1. Shosuke Yoshida et al. A bacterium that degrades and assimilates poly(ethylene terephthalate), Science (2016).
2. Harry P Austin. et al. Characterization and engineering of a plastic-degrading aromatic polyesterase, PNAS(2018)
3. Chun-Chi Chen et al. General features to enhance enzymatic activity of poly(ethylene terephthalate) hydrolysis, Nature Catalysis(2021).