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 | The mutant site of Variant 1 is G62A/ P193H/ S197D. H193, D197 and S194 in wild-type can form a catalytic triad, while G62A is a known mutation that can decrease MHET binding. |
| BBa_25ZBXUY9 | TfCut2 Variant 2 | Cutinase | B | The mutant site of Variant 2 is H77R/D204C/ E253C. H77R has one of the highest overall binding affinity for PET and PBAT, while D204C/ E253C is a known pair of mutations that creates a disulfide bridge, in assistance of ion bonding and thermal stability. |
| BBa_250E9UVZ | TfCut2 Variant 3 | Cutinase | B | The mutant site of Variant 3 is A65H/ L90A/ I213S. A65H is potentially as effective as G62A, and the double mutant G62A/I213S has been shown to increase catalytic efficiency. L90A and I213S are known mutations derived from comparison to LCC, indicating these two mutations are naturally compatible. |
| BBa_25JH17Z6 | TfCut2 Variant 4 | Cutinase | B | The D12S mutation is chosen based on the MutCompute’s top predicted stability mutation in the non-conserved region. |
| BBa_25WQ5BX1 | TfCut2 Variant 5 | Cutinase | B | The T234L mutation is chosen based on the MutCompute’s top predicted stability mutation that can retain its function in the conserved region. |
| BBa_25PC5VWY | TfCut2 Variant 6 | Cutinase | B | Multiple residue mutations are implemented based on all MutCompute-predicted stabilizing mutations except those at conserved residues. |
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. Together, these enzymes act synergistically to break cellulose into glucose.
| BioBrick ID | Name | Type | Category | Function |
|---|---|---|---|---|
| BBa_K5117001 | Bacillus subtilis EglS (BsEglS) | Endoglucanase | B | Breaks down cellulose by cleaving internal β-1,4 glycosidic bonds in cellulose chains to generate oligosaccharides. This allows cellulose to be accessible to other enzymes for further breakdown (MacKay et al., 1986). |
| BBa_K5117007 | halodurans BglA (BhBglA) | β-Glucosidase | B | Hydrolyze terminal, non-reducing β-D-glucosyl residues from various glucosides and oligosaccharides, releasing β-D-glucose (Naz et al., 2010). |
| BBa_25XGLK8T | Cellulomonas fimi CbhA (CfCbhA) | Cellobiohydrolase | B | Hydrolyzes the glycosidic bonds at the ends of cellulose chains, releasing 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 polyethylene terephthalate (PET) degradation. When PET is broken down by TfCut2, TPA is released into the environment. This promoter responds to TPA and triggers downstream gene expression only after degradation has occurred, allowing it to be used as a biosensor.
| BioBrick ID | Name | Type | Category | Function |
|---|---|---|---|---|
| BBa_J428032 | B0030-m0 | RBS | B | We assembled BBa_B0030_m0 into the construct via Golden Gate Assembly as a ribosome binding site. |
| BBa_J23100 | J23100 promoter | constitutive promoter | B | This was used as a promoter to initiate the transcription and regulate gene expression. |
| BBa_J428092 | Terminator B0015 | Terminator | B | BBa_J428092 was integrated to stop transcription. |
| BBa_J435100 | CIAP | CDS | B | Constitutive control reporter enzyme used as an internal standard to normalize the output of the TPA detection by monitoring the colour changes generated through phosphate release. |
| BBa_J435096 | Beta-lactamase | CDS | B | By detecting β-lactamase activity with its substrate nitrocefin, which changes color from yellow to red upon hydrolysis, we are able to determine the TPA level, as the color changes correspond to TPA production. |
| BBa_K4728001 | TphR | Transcriptional Regulator | B | Terephalate regulator (TphR) is suggested to code for a transcriptional regulator involved in TPA catabolism in Comamonas sp. strain E6. Once TPA is bound to TphR, it induces a series of gene clusters responsible for the conversion of TPA into protocatechuate (Kasai et al., 2010). |
| BBa_K4728019 | TPA Transporters | Transporter system | C | Expression of the TPA tripartite transporter proteins (TpiB, TpiA) and the TPA-binding protein (TphC) are required to work for the transportation of TPA, one such key step is the cellular absorption of non-permeable TPA, made possible with TphC (Gautom et al., 2021). |
| BBa_K4728000 | TPA-inducible promoter (tph promoter) | Promoter | B | This part is the sequence of the TPA-inducible promoter (tph promoter) originally from Comamonas testosteroni. The promoter is activated by tphR, the activator protein of P_tph, which is activated by TPA-binding. In other words, when tphR binds to TPA, it will in turn activate tph promoter (Kasai et al., 2010). |
| BBa_25YDEAL1 | TPA-inducible expression system | Inducible reporter system | C | TPA-inducible expression system that drives the transcription of CIAP under control of Ptph promoter. Ptph promoter is activated by the transcriptional regulator TphR when bound to TPA. Upon activation, CIAP is expressed and produces measurable colour change when react with its substrate, allowing detection of TPA production. |
| BBa_25R563SV | Tph operon-β-lactamase reporter | Transport system with constitutive reporter | C | This composite part integrates the TPA transport system with a constitutive β-lactamase reporter into a single module. When TPA is transported into cytoplasm through the TPA transport system (TphC-TpiB-TpiA), it binds and activates transcriptional regulator TphR, which in turn binds to Ptph (TPA-inducible promoter). The β-lactamase reporter gene serves as an internal standard to normalize the CIAP signal induced by TPA through its enzymatic reaction with the substrate, producing measurable colour change. |
B: Basic
C: Composite
- 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. Scientific Reports, 9(1), 16038.
- Gautom, T., et al. (2021). Structural basis of terephthalate recognition by solute binding protein TphC. Nature Communications, 12(1), 6244.
- 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.
- Liu, M., & Yu, H. (2012). Co-production of a whole cellulase system in Escherichia coli. Biochemical Engineering Journal, 69, 204–210.
- MacKay, R. M., et al. (1986). Structure of a Bacillus subtilis endo-β-1,4-glucanase gene. Nucleic Acids Research, 14(22), 9159–9170.
- Meinke, A., et al. (1994). Cellobiohydrolase A (CbhA) from the cellulolytic bacterium Cellulomonas fimi. Molecular Microbiology, 12(3), 413–422.
- Naz, S., et al. (2010). Enhanced production and characterization of a β-glucosidase from Bacillus halodurans expressed in Escherichia coli. Biochemistry (Moscow), 75(4), 513–525.