Part:BBa_K5236010

BhrPETase N205G

Since plastic pollution poses a serious global environmental problem, one of the potential solution, enzyme degradation, would be a suitable approach of dealing with plastic wastes. And among all of the plastic pollutions, more than 10% of them are Polyethylene terephthalate (PET). Thus, our team has been looking for possible PET hydrolase to deal with PET. However, according to Nature's publishment on April 27, 2022, traditional PET hydrolases' enzymatic ability of degrading PET are easily affected by the fluctuation of temperature and pH value. Therefore, we decided to--in a synthetic biology way--manually mutate wild-type BhrPETase, which was identified by the Shingo group in a metagenomic study on uncultured thermophiles and was deposited into the NCBI database by the group in 2018 and annotated as a PET hydrolase, to enlarge the acceptable range of temperature and pH level for PET hydrolases to function more efficiently and degrade more PET to solve global plastic pollution as soon as possible. As one of our most-confident mutants, this basic part encodes mutated BhrPETase N205G and was constructed in Escherichia coli in our lab.

Fig.1 The the affinity of the top 19 positions of the BhrPETase enzyme to the microplastic molecules. More negative the affinity is, the better the mutant is.
Fig.2 The BhrPETase-N205G protein structure predicted using Alphafold and ligand binding predicted using Autodock, mutation sites are marked in red in the images.

Usage and Biology

Initially, we've trained a Transformer AI model. This model predicts the top 10 potential mutation sites, which are likely to have significant impacts on the enzyme's structure and function. Next, we analyzed the top 10 potential sites via Meta's ESM-1b model to eliminate the silent mutations, which there are only changes in nucleotides but not in amino acids, functions, or structures, to ensure that our mutants have some postive impacts. For further imformation, please check the model page on our wiki. https://2024.igem.wiki/basis-china/model

To insert our parts into plasmids, we’ve designed primers and performed PCRs. Then, our genes were recombined into plasmids and transformed into chassis. To test if our part codes for the mutated BhrPETase N205G we want and whether the enzyme works, we've completed two large experimental processes. The first step is plasmid construction. And the second is to test the enzymatic activity.

By conducting colony PCR, we are able to test if our parts have been transformed into chassis successfully. The following result of electrophoresis proves that we’ve inserted genes into chassis since the sequence containing our mutated genes has a total of 891 base pairs and the results are in the right location.

Fig.3 The DNA gel electrophoresis result
Fig.4 The illustration of BhrPETase N205G genetic pathway

After proving that our genes existed in chassis, we need to test if the bacteria can survive as usual with our genes. Thus, we’ve coated the bacteria on nutritional petri dish. And after a night, E. coli grew over the plate our plate, justifying that E. coli can survive with the gene of our part.

The result show that chassis carrying our PETase could survive.

Fig.5 BhrPETase-N205G was coated on spectinomycin plates and incubated overnight at 37 ℃

We tested whether the bacteria could translate for our protein, and we examined whether our mutated enzyme (N205G, W229F, M57L, N191S) is more efficient. For this section, we analyzed two results as well. First, the electrophoresis result of our protein proves that our enzyme can be successfully coded by the parts we designed. Second, the dynamic curve of our enzyme shows its high efficiency in degrading rate (x-axis stops at 30min because that's what the professional research teams did).

Fig.7 Protein electrophoresis result

This dynamic curve shows that only N205G's efficiency is able to exceed wild-type after 30 minute, proving that mutated PET hydrolase does have an increase in efficiency.

Fig.6 Mutated BhrPETase Dynamic Curve

After proving that our enzyme are more effiicient, we moved on to test the ultimate and the most essential part of our part examination, which is to test if our mutated enzyme can actually degrade plastics. For this large step of process, we also designed two approaches——scanning electron microscope and high performance liquid chromatography

Different plastic samples were placed in the culture medium of the engineered E. Coli. After two weeks, the samples were observed under an SEM for any alterations in the surface of the plastic by technicians at Shenzhen University. The results demonstrated that plastic with low level of crystallinity were degraded under the exposure to PETase synthesized by our engineered E. coli. Further, the fact that plastic with high crystallinity did not show any significant changes addresses our hypothesis in Cycle 1: PET degradation is affected by the crystallinity of the plastic, which varies depending on its manufacturing process.The SEM allows us to see the changes of plastic pieces with our bare eyes. SEM procedure: 1 Cultivate the bacteria with PET for 14 days. 2 Remove and clean with water 2 Soak in 72% ethanol for 10 minutes (sterilization) 3 Soak in 100% ethanol for 10 minutes 4 Replace the ethanol and soak again in 100% ethanol for half an hour. 5 Dry well in an ultra-clean bench 6 Hand over to engineer

Fig.8 N205G Scanning Elctron Microscope result

However, pure observations are not enough to prove the effectiveness of our enzymes. Thus, we conducted another experiment. Therefore, we tried to use HPLC for compositional identification. HPLC is a method to directly detect the presence of specific compound in a chemical mixture. During the degradation of PET by PETase, TPA is created as a byproduct; hence, the presence of TPA in the final product indicates degradation occurred.

Fig.9 High-performance liquid chromatography analysis. The results show that TPA is present after a 2 week incubation of PET with the engineered bacteria.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 226
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 136
  
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

Lu, Hongyuan, et al. “Machine Learning-Aided Engineering of Hydrolases for Pet Depolymerization.” Nature News, Nature Publishing Group, 27 Apr. 2022, www.nature.com/articles/s41586-022-04599-z.