Team:United Shanghai HS/Design

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"Patrolmen" for the PET contaminated soil

At present, human society has entered the "plastic era". The production and use of a large number of plastic products has caused a large amount of plastic waste to enter the environment, spreading on land, lakes, coastlines, ocean surfaces, seabeds and even abysses. The problem of plastic degradation needs to be solved urgently.

The composition of plastic:

According to surveys, about 80% of the plastic products on the earth are made of ethylene terephthalate, or PET for short, which is a chemically inert and difficult to degrade aromatic polyester. Its waste The accumulation of environmental and ecological problems has brought tremendous pressure.

The discovery of miraculous bacteria:

In 2016, PETase was separated from I.sakaiensis. The enzyme has high sequence similarity with the PET hydrolyzing activity, but its hydrolytic activity and selectivity for PET are significantly higher than other PET hydrolytic enzymes. The discovery of PETase is a breakthrough in the study of PET biodegradation. The elucidation of the structure and mechanism of PET will provide important clues to the degradation of PET and other plastics. MHETsae was also found in this kind of bacteria, and it was confirmed that it can be expressed in conjunction with PETase to further decompose it. TPA and EG can be directly metabolized by cells, eventually producing CO2 and H2O.

Figure 1：PET degradation metabolic pathway

Experimental design:

In the burying yard where the PET plastic is concentrated, the modified Bacillus subtilis is used to initially degrade the PET to accelerate the degradation of the PET plastic in the burying yard. At the same time, the activities of earthworms are used to improve the distribution of engineering bacteria and enzymes, and solve the difficulty of frequent mixing in the dump site. Plasmids were used in the experiment, the genes ISF6_4831 (PETase encoding gene), ISF6_0224 (MHETase encoding gene), (GFP fluorescent protein), mCherry (fluorescent protein). The function of inserting GFP is to track the distribution of engineering bacteria in the soil. At the same time, by detecting the fluorescence in the body or feces of the earthworm, check whether the engineered bacteria have been swallowed by the earthworm. The expected result is that the engineered bacteria can be carried elsewhere through the activities of earthworms, and promote the distribution of engineered bacteria in the soil. The main function of mCherry reporter gene is to prompt the need to add sucrose to amplify the effect.

1.Obtaining the fusion gene sequence

We found in the literature that Wu Bian’s team from the Institute of Microbiology of the Chinese Academy of Sciences optimized the enzymes required by the Osaka bacteria to degrade PET plastic, and obtained a redesigned enzyme with significantly enhanced stability, which made the Osaka bacteria degrade the PET film efficiently. Improved by 300 times, we use its optimized ISF6_4831 protein sequence. (PETase)

According to the relevant data of NCBI, we found the protein sequence of Osaka bacteria ISF6_0224; the protein sequence of GFP, mCherry fluorescent protein. (MHETase,GFP,mCherry) We fused the gene sequences of ISF6_4831, GFP; ISF6_0224, mCherry fluorescent protein respectively.

Figure 2: Structure model of PETase protein

In the literature, we found that the signal peptide SPLamB has the highest exocrine efficiency. We deleted the signal peptide sequence before the ISF6_4831 (expressing PETase) and ISF6_0224 (expressing MHETase) gene fragments, and added the signal peptide SPLamB gene before the fusion sequence Sequence to increase the exocrine expression of the protein.

Figure 3: The gene sequence of SPLamB, MHETase and PETase

2. Design of control elements

BHET and MHET will accumulate in the process of PET degradation. These intermediate hydrolysates have a high affinity with enzyme substrates and are competitively inhibited, which will reduce the degradation efficiency of cutinase. Carboxylesterase can reduce the inhibitory effect of MHET.

Figure 4:The process of PET hydrolase degrading PET

By evaluating the expression level of the zearalenone (ZEN) degrading enzyme gene ZLHY6 and the activity evaluation of the degrading enzyme, the effects of two constitutive strong promoters P43 and Plaps in regulating the expression of heterologous genes were compared. The results showed that in Bacillus subtilkis (Bacillus subtilkis) Bs 168, the degrading enzyme gene regulated by PlapS was highly expressed, and the degrading enzyme activity reached the highest value at 12 h of fermentation, and the enzyme activity was 219.02 U/mL. The ZEN degrading enzyme gene expression vector pWBZ7 mediated by the promoter PlapS can be stably inherited in Bs 168, laying the foundation for the efficient secretion and expression of degrading enzymes.

Figure 5:Schematic diagram of plasmid pWBZ1 and pWBZ7

The sucrose promoter PsacB is the third commonly used promoter system in Bacillus subtilis. The promoter is the promoter of the sucrosecanase encoded by the SACB gene on the Bacillus subtilis genome. The transcription of the PsacB promoter is not strictly regulated by sucrose, and can be transcribed without an inducer, but the intensity is 100 times lower than that of sucrose induction.

①Xiong Haitao et al. "Research progress on Bacillus subtilis expression system and its promoter." Guangxi Science. 2018.
②Zhou Yanjing et al. "Study on the regulation and expression of two strong promoters in Bacillus subtilis." Cereals, Oils and Foodstuffs Science and Technology. 2015.

Figure 6: Literature report--Hamachi K et al. Biosystems. 2013

The above literature mentions that ethylene glycol can affect the conformation of the glycine riboswitch and enhance transcription. The structure of ethylene glycol is very similar to that of glycine. Although it is not a ligand for the glycine riboswitch, it affects the riboswitch and enhances transcription.

Figure 7: Plasmid construction

When transferred into the transformed plasmid, Bacillus subtilis can express the downstream PETase and MHETase two enzymes under the natural environment due to the loose regulation of the sacB promoter, and will initially degrade some ethylene glycol. A large amount of ethylene glycol, ethylene glycol will hide the terminator contained in the riboswitch, further activate the glycine riboswitch, express more PETase and MHETase, and achieve the regulation of protein expression, that is: no PET is two kinds Enzymes are expressed in a small amount, and PET is expressed in large amounts, so that the amount of enzyme expression is determined according to the amount of environmental PET. As a reporter gene, mcherry can indicate the need to add sucrose to amplify the effect, and more ethylene glycol will increase the transcription of PETase and MHETase. Present a positive feedback effect.

The two fluorescent proteins are for experimental needs, that is, to verify the expression of the protein. If the sucrose promoter is effective, we can theoretically detect a small amount of GFP, and if the subsequent activation of the glycine riboswitch is effective, then the mcherry can be detected. Expression (as a signal to remind us that both components can work normally).