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. Si-face binding is the main binding pose of PET in the active site of BhrPETase. And was constructed and modified as WT BhrPETase in our project.[2] The superior activity and thermostability of BhrPETase rendered it one of the most promising PETases for plastic waste recycling and bioremediation applications in the future [3].
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 [4]. 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.
In order to secret WT BhrPETase, we start to construct plasmid for protein expression. As figure.4 shows, we insert several parts to accomplish the expression for WT BhrPETase.
The function of each parts:
T7 promoter: A Strong promoter recognized by T7 RNA polymerase, used to regulate gene expression of recombinant proteins.
Lac operator: Operator that can be activated by IPTG, used to control gene expression by lactose or IPTG.
RBS: Ribosome binding site.
WT BhrPETase:The basic part encoding the BhrPETase who had been mutated.
pelB: The sequence encodes a signal peptide that enables secretory expression of PETase.
6xHis: A label for protein purification
T7 terminator: Terminates transcription.
When we had completed the plasmid construction and transformation. We need to construct and test the BhrPETase activity.
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We successfully submitted BhrPETase as a standardized part and experiments showed that BhrPETase was shown to have higher enzymatic activity than IsPETase at room temperature. After our sequence optimization, the appearance of BhrPETase-N205G is expected to improve the activity of BhrPETase.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Reference
[1] Kato, Shingo, et al. “Long-Term Cultivation and Metagenomics Reveal Ecophysiology of Previously Uncultivated Thermophiles Involved in Biogeochemical Nitrogen Cycle.” Microbes and Environments, vol. 33, no. 1, Jan. 2018, pp. 107–10. https://doi.org/10.1264/jsme2.me17165. [2]Wang, N., Li, Y., Zheng, M., Dong, W., Zhang, Q., & Wang, W. (2024b). BhrPETase catalyzed polyethylene terephthalate depolymerization: A quantum mechanics/molecular mechanics approach. Journal of Hazardous Materials, 477, 135414. https://doi.org/10.1016/j.jhazmat.2024.135414 [3]Xi, X., Ni, K., Hao, H., Shang, Y., Zhao, B., & Qian, Z. (2020). Secretory expression in Bacillus subtilis and biochemical characterization of a highly thermostable polyethylene terephthalate hydrolase from bacterium HR29. Enzyme and Microbial Technology, 143, 109715. https://doi.org/10.1016/j.enzmictec.2020.109715 [4] Lu, Hongyuan, et al. “Machine Learning-aided Engineering of Hydrolases for PET Depolymerization.” Nature, vol. 604, no. 7907, Apr. 2022, pp. 662–67. https://doi.org/10.1038/s41586-022-04599-z.
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