In our project, our team has designed a total of 34 parts, with 24 being the basic parts and the rest being 10 parts. All the parts have been designed around our project, and detailed information about each component can be found on the team part page, or by clicking on the component number below.
In order to enhance the enzymatic activity of IsPETase, we analyzed the protein structure and mechanism of IsPETase. Firstly, we mutated IsPETase to obtain different variants. Due to the hydrophobic nature of the PET substrate, we attached a structural domain to the variant with the highest activity to enhance the hydrophobicity of the IsPETase mutant, promoting the binding of the enzyme to the substrate.
| Part number | Type | Description | Designer | Length |
|---|---|---|---|---|
| BBa_K4119002 | Coding | BS2 | Jiacheng Yao | 411bp |
| BBa_K4724090 | Protein_Domain | CBM3-6xHis Tag | Qingqing Zheng | 471bp |
| BBa_K4724004 | Protein_Domain | CBM4-6xHis Tag | Qingqing Zheng | 318bp |
| BBa_K4724005 | Protein_Domain | CBM11-6xHis Tag | Qingqing Zheng | 318bp |
| BBa_K4724003 | Protein_Domain | LSChi4CBM-6xHis Tag | Qingqing Zheng | 297bp |
| BBa_K4724001 | Protein_Domain | LSChi5CBM-6xHis Tag | Qingqing Zheng | 303bp |
| BBa_K4724002 | Coding | IsPETase-Linker | Qingqing Zheng | 831bp |
| BBa_K4724006 | Coding | IsPETaseS93_I94insE-6xHis Tag | Qingqing Zheng | 816bp |
| BBa_K4724007 | Coding | IsPETaseT116R-6xHis Tag | Qingqing Zheng | 813bp |
| BBa_K4724008 | Coding | IsPETaseQ119F-6xHis Tag | Qingqing Zheng | 813bp |
| BBa_K4724009 | Coding | IsPETaseS121E-6xHis Tag | Qingqing Zheng | 813bp |
| BBa_K4724010 | Coding | IsPETaseQ126L-6xHis Tag | Qingqing Zheng | 813bp |
| BBa_K4724011 | Coding | IsPETaseM157A-6xHis Tag | Qingqing Zheng | 813bp |
| BBa_K4724012 | Coding | IsPETaseM157S-6xHis Tag | Qingqing Zheng | 813bp |
| BBa_K4724013 | Coding | IsPETaseW159H-6xHis Tag | Qingqing Zheng | 813bp |
| BBa_K4724014 | Coding | IsPETaseW185F-6xHis Tag | Qingqing Zheng | 813bp |
| BBa_K4724015 | Coding | IsPETase A240_S242del-6xHis Tag | Qingqing Zheng | 813bp |
| BBa_K4724019 | Coding | IsPETaseS93_I94insE/W159H-6xHis Tag | Qingqing Zheng | 816bp |
| BBa_K4724026 | Coding | IsPETaseQ119F/ W159H-6xHis Tag | Qingqing Zheng | 813bp |
| BBa_K4724027 | Coding | Linker | Qingqing Zheng | 42bp |
| BBa_K4724070 | Coding | NusA | Xinyao Li | 1512bp |
| BBa_K4724071 | Coding | TrxA | Xinyao Li | 354bp |
| BBa_K4724072 | Coding | DsbA | Xinyao Li | 63bp |
| BBa_K4724073 | Coding | ompA | Xinyao Li | 63bp |
| BBa_K4724074 | Coding | LSPETase | Xinyao Li | 858bp |
| Part number | Type | Description | Designer | Length |
|---|---|---|---|---|
| BBa_K4724021 | Composite | IsPETase-Linker-CBM3-6xHis Tag | Qingqing Zheng | 1310bp |
| BBa_K4724022 | Composite | IsPETase-Linker-CBM4-6xHis Tag | Qingqing Zheng | 1157bp |
| BBa_K4724023 | Composite | IsPETase-Linker-CBM11-6xHis Tag | Qingqing Zheng | 1157bp |
| BBa_K4724024 | Composite | IsPETase-Linker- LSChi4CBM -6xHis Tag | Qingqing Zheng | 1136bp |
| BBa_K4724025 | Composite | IsPETase-Linker- LSChi5CBM -6xHis Tag | Qingqing Zheng | 1142bp |
| BBa_K4724028 | Composite | IsPETaseQ119F/W159H-Linker-CBM11-6xHis Tag | Qingqing Zheng | 1354bp |
| BBa_K4724085 | Composite | TrxA-LSPETase | Xinyao Li | 1218bp |
| BBa_K4724083 | Composite | DsbA-LSPETase | Xinyao Li | 1212bp |
| BBa_K4724084 | Composite | OmpA-LSPETase | Xinyao Li | 921bp |
| BBa_K4724075 | Composite | NusA-LSPETase | Xinyao Li | 2370bp |
We aim to improve the efficiency of WT degradation of PET. To screen out the better mutants, we obtained ten single-point mutants. The reaction of mutants with PET powder found that BBa_K4724006, BBa_K4724007, BBa_K4724008, and BBa_K4724013 had improved effects compared with WT, and BBa_K4724013 had the most obvious lifting effect. Then a superimposed mutation was performed, and the second round of BBa_K4724026 with the strongest enzyme activity was obtained. In the screening of hydrophobic domains, the degradation of PET powder by BBa_K4724021, BBa_K4724022, and BBa_K4724023 was improved compared with WT, and experiments showed that the BBa_K4724023 activity was the highest, indicating that the BBa_K4724005 was the most hydrophobic. Finally, we connected the mutant with the highest enzyme activity and the domain with the strongest hydrophobicity to obtain the BBa_K4724028 with the strongest enzyme activity in this experiment.
| Part number | Type | Description | Designer | Length |
|---|---|---|---|---|
| BBa_K4724090 | Protein_Domain | CBM3-6xHis Tag | Qingqing Zheng | 471p |
| BBa_K4724004 | Protein_Domain | CBM4-6xHis Tag | Qingqing Zheng | 318bp |
| BBa_K4724005 | Protein_Domain | CBM11-6xHis Tag | Qingqing Zheng | 318bp |
| BBa_K4724021 | Composite | IsPETase-Linker-CBM3-6xHis Tag | Qingqing Zheng | 1310bp |
| BBa_K4724022 | Composite | IsPETase -Linker-CBM4-6xHis Tag | Qingqing Zheng | 1157bp |
| BBa_K4724023 | Composite | IsPETase -Linker-CBM11-6xHis Tag | Qingqing Zheng | 1157bp |
| BBa_K4724006 | Coding | IsPETase S93_I94insE-6xHis Tag | Qingqing Zheng | 816bp |
| BBa_K4724007 | Coding | IsPETase T116R-6xHis Tag | Qingqing Zheng | 813bp |
| BBa_K4724008 | Coding | IsPETase Q119F-6xHis Tag | Qingqing Zheng | 813bp |
| BBa_K4724013 | Coding | IsPETase Q119F/ W159H-6xHis Tag | Qingqing Zheng | 813bp |
| BBa_K4724028 | Coding | IsPETase Q119F/W159H-Linker-CBM11-6xHis Tag | Qingqing Zheng | 1157bp |