PET Degradation
These parts include the wild-type TfCut2 enzyme from Thermobifida fusca, alongside six engineered TfCut2 variants that are designed to enhance PET degradation efficiency.
| BioBrick ID | Name | Type | Category | Function |
|---|---|---|---|---|
| BBa_25JE6FNZ | TfCut2_5ZOA | Cutinase | B | Wild-type TfCut2 cutinase from Thermobifida fusca that hydrolyzes PET by cleaving ester bonds, producing TPA (Furukawa et al., 2019). |
| BBa_25GGFEQS | TfCut2_Variant 1 | Cutinase | B | Mutant G62A/P193H/S197D; targets catalytic triad (H193, S194, D197), with G62A decreasing MHET binding. |
| BBa_25ZBXUY9 | TfCut2_Variant 2 | Cutinase | B | Mutant H77R/D204C/E253C; H77R enhances binding to PET and PBAT, D204C/E253C forms a stabilizing disulfide bridge. |
| BBa_250E9UVZ | TfCut2_Variant 3 | Cutinase | B | Mutant A65H/L90A/I213S; combines LCC-derived and high-efficiency mutations for enhanced degradation. |
| BBa_25JH17Z6 | TfCut2_Variant 4 | Cutinase | B | D12S mutation selected from MutCompute for enhanced stability in non-conserved region. |
| BBa_25WQ5BX1 | TfCut2_Variant 5 | Cutinase | B | T234L mutation, most stabilizing per MutCompute, though located in a conserved region. |
| BBa_25PC5VWY | TfCut2_Variant 6 | Cutinase | B | Multiple stabilizing mutations (excluding conserved residues) to improve thermostability. |
B: Basic
C: Composite
Cellulose Degradation
This system includes three cellulases, BsEglS (endoglucanase) from B. subtilis and BhBglA (β-glucosidase) from B. halodurans, both selected from TU Dresden’s 2024 project for their high glucose yields, along with CfCbhA (exoglucanase) from C. fimi.
| BioBrick ID | Name | Type | Category | Function |
|---|---|---|---|---|
| BBa_K5117001 | BsEglS | Endoglucanase | B | Cleaves internal β-1,4 glycosidic bonds to generate oligosaccharides, enhancing cellulose accessibility (MacKay et al., 1986). |
| BBa_K5117007 | BhBglA | β-Glucosidase | B | Hydrolyzes terminal β-D-glucosyl residues to release glucose (Naz et al., 2010). |
| BBa_25XGLK8T | CfCbhA | Cellobiohydrolase | B | Hydrolyzes cellulose ends to release cellobiose (Meinke et al., 1994; Liu & Yu, 2012). |
B: Basic
C: Composite
TPA Sensor System
This system uses a TPA-inducible promoter that activates in response to terephthalic acid (TPA), a product of PET degradation, triggering downstream gene expression for biosensing.
| BioBrick ID | Name | Type | Category | Function |
|---|---|---|---|---|
| BBa_J428032 | B0030-m0 | RBS | B | Ribosome binding site assembled via Golden Gate Assembly. |
| BBa_J23100 | J23100 | Constitutive promoter | B | Initiates transcription and regulates expression levels. |
| BBa_J428092 | B0015 | Terminator | B | Stops transcription. |
| BBa_J435100 | CIAP | CDS | B | Reporter enzyme to monitor phosphate-based color change. |
| BBa_J435096 | Beta-lactamase | CDS | B | Detects TPA via nitrocefin color shift from yellow to red. |
| BBa_K4728001 | TphR | Transcriptional Regulator | B | TPA-responsive transcriptional regulator for promoter activation (Kasai et al., 2010). |
| BBa_K4728019 | TPA Transporters | Transporter system | C | Tripartite transporter system enabling TPA uptake (Gautom et al., 2021). |
| BBa_K4728000 | TPA-inducible promoter | Promoter | B | Activated by TphR in presence of TPA (Kasai et al., 2010). |
B: Basic
C: Composite
References
- Furukawa, M., Kawakami, N., Tomizawa, A., & Miyamoto, K. (2019). Efficient Degradation of Poly(ethylene terephthalate) with Thermobifida fusca Cutinase Exhibiting Improved Catalytic Activity Generated using Mutagenesis and Additive-based Approaches. Nature. https://www.nature.com/articles/s41598-019-52379-z
- Gautom, T. et al. (2021). Structural basis of terephthalate recognition by solute binding protein TphC. Nature Communications, 12(1), 6244. https://doi.org/10.1038/s41467-021-26508-0
- Kasai, D. et al. (2010). Transcriptional regulation of the terephthalate catabolism operon in Comamonas sp. strain E6. Applied and Environmental Microbiology, 76(18), 6047–6055. https://doi.org/10.1128/AEM.00742-10
- Liu, M., & Yu, H. (2012). Co-production of a whole cellulase system in Escherichia coli. Biochemical Engineering Journal, 69, 204-210. https://www.sciencedirect.com/science/article/abs/pii/S1369703X12002719
- MacKay, R. M. et al. (1986). Structure of a Bacillus subtilis endo-beta-1,4-glucanase gene. Nucleic Acids Research, 14(22), 9159-70. https://doi.org/10.1093/nar/14.22.9159
- Meinke, A. et al. (1994). Cellobiohydrolase A (CbhA) from the cellulolytic bacterium Cellulomonas fimi. Molecular Microbiology, 12(3), 413–422. https://doi.org/10.1111/j.1365-2958.1994.tb01030.x
- Naz, S. et al. (2010). Enhanced production and characterization of a beta-glucosidase from Bacillus halodurans expressed in Escherichia coli. Biochemistry (Moscow), 75(4), 513–525. https://doi.org/10.1134/S0006297910040164