Team:United Shanghai HS/Integrated Human Practices


Integrated Human Practices

The initial idea of our project is to build an engineered bacteria that can degrade PET and solve PET pollution. We initially chose Escherichia coli, a common engineering bacteria, as our chassis, hoping to degrade it through extracellular expression of E. coli PET enzyme (by adding secretion signal peptide) to achieve our goal. With this initial simple idea, we visited the Shanghai Academy of Agricultural Sciences, East China Normal University, and East China University of Science and Technology, consulted relevant teachers, and perfected our project through exchanges, information review and thinking.

We first visited Mr. Zheng Xianqing from the Institute of Ecological Environment Protection, Shanghai Academy of Agricultural Sciences. Mr. Zheng acknowledged that we had chosen PET pollution as the subject, but he suggested to us to use pathogenic strains such as Escherichia coli as the chassis. , Putting into the environment may cause pollution problems. In addition, Mr. Zheng also asked us whether we can use earthworms to help achieve our project goals, because it is difficult for engineering bacteria to evenly distribute in the soil and reach deep soil layers, and the ability of engineering bacteria to multiply in nature may be limited. Earthworms can not only distribute engineered bacteria evenly throughout the soil, but the intestinal tract of earthworms is also a good environment for the proliferation of microorganisms, which can be described as killing two birds with one stone.

After this visit, we replaced the bottom plate bacteria with Bacillus subtilis. This strain is naturally present, harmless to the environment and has been widely used by farmers as beneficial bacteria, and its safety is higher. In addition, we checked the literature and found that Bacillus subtilis is the main flora in the intestinal tract of earthworms. Studies have shown that the number of Bacillus subtilis can increase dozens of times after passing through the intestinal tract of earthworms. Therefore, Bacillus subtilis is obviously more suitable than E. coli As a chassis.

Afterwards, regarding the choice of which enzyme can be used for the efficient degradation of PET, we visited Mr. He Defu from East China Normal University. He believed that plastic biodegradation is a scientific and technological problem. Current studies have shown that its degradation efficiency is generally very low. But at present, scientists have discovered efficient enzymes that degrade PET. This is 2016. Japanese scientists isolated PETase from the I.sakaiensis strain. Its hydrolytic activity and selectivity for PET are significantly higher than other PET hydrolases, MHETsae At the same time, it was found in this kind of bacteria, and it was confirmed that it can be coupled with PETase to express and play a role in further decomposition. He suggested that we can introduce the genes ISF6_4831 (PETase encoding gene) and ISF6_0224 (MHETase encoding gene) into the engineered bacteria to realize the function of the engineered bacteria to degrade soil PET.

Finally, we visited Mr. Li Youyuan from East China University of Science and Technology. Mr. Li Youyuan agreed with the design of our project and felt that this plan was more reasonable. However, he believed that our design lacks a regulatory mechanism: if we simply express these two Enzymes may waste the resources of the bottom plate bacteria, which greatly increases the survival pressure of the bottom plate bacteria. At the same time, it also appears that our design is relatively rigid and simple.

A reasonable regulatory mechanism is important, but unlike some soluble small molecules, PET, as a solid, cannot enter cells at all, let alone find the regulatory elements that are directly activated by it, so we choose to start with its degradation products. Looking at the information, we found that PET degradation product ethylene glycol can activate the glycine riboswitch. Using this as a breakthrough, we finally designed our existing regulation method: using the incomplete repression of the sucrose promoter to express a small amount of PETase and MHETase, If PET exists in the soil, it will be degraded to produce ethylene glycol, which activates the glycine riboswitch to express more enzymes, and more enzymes means more ethylene glycol, and more ethylene glycol will further activate The expression of more enzymes realizes the positive feedback of enzyme expression. When there is no PET, a small amount of enzyme protein is expressed, and once PET is present, a large amount of enzyme protein can be expressed to efficiently achieve PET degradation.

In summary, through the communication with teachers during the comprehensive HP process and the post-investigation of our team, we proceeded from the initial simple idea, continued to improve and perfected, and finally produced our current project design. Comprehensive HP has brought us a lot of help, allowing us to learn a lot of knowledge and greatly broaden our thinking. Thank you very much to all the teachers who provided valuable suggestions for our project! !