We plan to design a recombinant B. subtilis to degrade PET in the plastic garbage dumps. There are two enzymes, PETase and MHETase, will be expressed by E.coli. With the aid of earthworm, the recombinant bacterium and proteins can be spread efficiently all over the dump（particularly those in the soil）,which can enhance the degradation process of the dumps.
We plan to use B. subtilis，which are commonly used in lab. As B. subtilis widely exist in nature and have long been used by farmers to improve the quality of soil, it is quite safe to choose B. subtilis as our chassis.
We will construct and amplify the plasmid carrying the genes of PETase、MHETase and florescent protein by B. subtilis. The plasmid will be subsequently introduced into B. subtilis to express those proteins. The proteins all come from natural sources and are non toxic.
Besides, our final products of the process (terephthalate and ethylene glycol) are bio-degradable and environmentally friendly.
In case of unintentional environmental spread, some safety risks include escape of the engineered B. subtilis and leak of the functional proteins into the ecosystems, which maybe lead to an undesired degradation of the PET products in agricultural usage. However, as is mentioned above, the bacteria and the functional proteins are quite safe and will not cause damage to the environment.
We choose B. subtilis as the chassis organism, which is widely used in biological fermentation production. By the way, during the experiments, all the wastes will be sterilized before discarding.Specialists will be responsible for lab safety and team members' safety training. Each time the team members conduct experiments, graduate students and teachers will accompany us to ensure safety. Each of those graduate students and teachers are familiar with their labs and many potential risks involved in experiments.
We will autoclave every dishes and containers for sterilization purpose after we dump them into the garbage can or drainage. In this way, all the bacteria we use won't escape from our lab. We have already received safety and security training.
Our laboratory does not have viruses or other dangerous cells that meet igem project standards and there is no potential risk of contamination.
Relevant laws and regulations:
Regarding the legal basis at home and abroad, there is currently the latest Biosecurity Law of the People's Republic of China (Grass) in China(Second Review Draft), Regulations on the Safety management of Agricultural Genetically Modified Organisms and other laws to provide reference. In addition, there is Germany 's law on genetic engineering "Gesetz zur Regelung der Gentechnik" and what the CBD has in common is that genetic engineering is treated in several different safety levels
There are different treatment standards at the same level, and for our project, we can prove the project experimentally. The engineered bacteria used are in safety group 2 Laboratory safety protocols:
Before conducting the experiment, we were trained by our laboratory manager in charge of strict operation training and safety education. During the experiment, the experimenter needs to wear protective glasses, protective clothing, knee-length trousers and closed shoes to ensure the safety of the experiment. In addition, our laboratory, as mentioned above, has no hazardous biological samples and regular disinfection. The office area is separated from the experimental area, which fully meets the requirements of the IGEM project. When we do experiments, we follow the protocol strictly and are supervised by the lab manager.
To ensure biological safety, biological samples are forbidden to leave the laboratory. All samples, including discarded growth mediums, Petri dishes, and even experimental earthworms, will be sterilized after experiments to ensure there will be no leakage of any organism.