Part:BBa_K5236025

BhrPETase

The sequence of BhrPETase 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 [1]. This basic part encoding the BhrPETase, which has been predicted and optimized by Wu et al. And was constructed and modified as WT BhrPETase in our project.

Usage and Biology

We trained a Transformer model on 1007 homologous PETase protein sequences obtained from the UniProt Database using the masked language model (MLM) training method. This approach allows the model to learn contextual information about amino acid sequences and predict masked residues accurately [2]. The BhrPETase mutants that scored in the top four in the trained model were used in the construction and tested.

To construct plasmids, we’ve designed primers and performed PCRs. Then, our genes were recombined into plasmids and transformed into chassis. By conducting colony PCR, we are able to test if our parts have been transformed into E.coli 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 798 base pairs and the results are in the right location.

Fig.1 The DNA gel electrophoresis result

When we had completed the plasmid construction and transformation. We need to construct and test the BhrPETase activity. .

Fig.3 Mutated BhrPETase Dynamic Curve
Fig.4 Protein electrophoresis result

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]