Difference between revisions of "Part:BBa K5236010"
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<partinfo>BBa_K5236010 parameters</partinfo>
 
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===Reference===
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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.

Revision as of 03:46, 2 October 2024

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 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 N205G plate coating result


We tested whether the bacteria could translate for our protein, and we examined whether our mutated enzyme is more efficient. For this section, we analyzed two results as well. First, the dynamic curve of our enzyme shows its high efficiency in degrading rate. Second, the electrophoresis result of our protein proves that our enzyme can be successfully coded by the parts we designed.


Fig.6 Mutated BhrPETase Dynamic Curve

Fig.7 Protein electrophoresis result


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

The SEM allows us to see the changes of plastic pieces with our bare eyes. 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.


Fig.8 N205G Electron Scanning Microscope result

Fig.9 N205G HPLC result

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.