Basic Parts
Our project leverages a suite of engineered enzymes to address both bioproduction and environmental sustainability. At the core of this effort are three key coding sequences we have established as BioBricks. We have characterized two variants of Beta-Cyclodextrin Glucanotransferase (β-CGTase), BBa_25RWG8YC and the longer BBa_25IDU15U, which are pivotal for converting starch into valuable β-cyclodextrins. Alongside these, we have also built BBa_25X9DRYP, an optimized and highly efficient TurboPETase that enables the rapid depolymerization of polyethylene terephthalate (PET) plastic. Together, these basic parts provide a foundational toolkit for applications in industrial biocatalysis and plastic biodegradation.
| Part Name | Registry Name | Type | Length |
|---|---|---|---|
| β-CGTase-1011 | BBa_25IDU15U | Coding | 2219 bp |
| β-CGTase | BBa_25RWG8YC | Coding | 2048 bp |
| TurboPETase | BBa_25X9DRYP | Coding | 779 bp |
Composite Parts
Building upon our foundational Basic Parts, we have constructed and characterized a series of functional plasmid systems ready for high-level protein expression in E. coli. Our composite parts include BBa_255BPN6X and BBa_25UNQS1F, which are pET20b(+)-based vectors for expressing the wild-type Beta-CGTase-1011 and a site-directed mutant Beta-CGTase_K647E, respectively. Additionally, we assembled BBa_25GWNXEL in a pET21a backbone for efficient production of the TurboPETase enzyme. These ready-to-use plasmids represent the complete and functional modules of our project, enabling the production of key enzymes for advanced applications in cyclodextrin synthesis and plastic biodegradation.
1. pET21a-TurboPETase
| Part Name | Registry Name | Type | Length |
|---|---|---|---|
| pET21a-TurboPETase | BBa_25GWNXEL | Plasmid | 6134 bp |
Key Features:
- Protein Expression: Designed for the high-level expression of TurboPETase, an engineered enzyme for enhanced polyethylene terephthalate (PET) plastic degradation.
- Promoter/Terminator: Utilizes a strong, inducible T7 promoter and an associated terminator for precise control of expression in appropriate E. coli strains.
- Purification Tag: Encodes a C-terminal 6xHis-tag to facilitate rapid purification via immobilized metal affinity chromatography (IMAC).
- Antibiotic Resistance: Contains an Ampicillin resistance (AmpR) gene for selection and maintenance in bacterial cultures.
- Cloning Flexibility: Features a comprehensive multiple cloning site (MCS) with numerous unique restriction enzyme cut sites (e.g., NdeI, XhoI, EcoRV, BamHI), enabling easy gene insertion, subcloning, and compatibility with standard assembly methods like Golden Gate and Gibson Assembly.
- Cloning Notes: The TurboPETase gene is positioned downstream of the T7 promoter and lac operator. The C-terminal 6xHis-tag allows for straightforward detection and purification of the recombinant protein.
2. pET20b(+) Beta-CGTase_K647E
| Part Name | Registry Name | Type | Length |
|---|---|---|---|
| pET20b(+) Beta-CGTase_K647E | BBa_25UNQS1F | Plasmid | 7413 bp |
Key Features:
- Protein Expression: Designed for the high-level expression of Beta-CGTase-K647E protein in appropriate host systems (e.g., T7 expression strains of E. coli).
- Promoter/Terminator: T7 promoter and corresponding terminator drive the transcription of the target gene.
- Purification Tag: Encodes a 6xHis tag fused to the target protein, enabling convenient purification using Immobilized Metal Affinity Chromatography (IMAC), such as Ni-NTA resin.
- Antibiotic Resistance: Confers Kanamycin resistance (KanR) for selective pressure during plasmid maintenance and protein expression.
- Cloning Flexibility: Offers high cloning flexibility with multiple unique restriction sites. Key sites include XbaI, and a set of compatible/isoschizomer sites (XhoI, PspXI, PaeR7I) which can be used interchangeably for fragment insertion or vector linearization.
- Cloning Notes: The XbaI site is unique and can be used in combination with any of the compatible XhoI/PspXI/PaeR7I sites for directional cloning of the target gene.
3. pET20b(+) Beta-CGTase-1011
| Part Name | Registry Name | Type | Length |
|---|---|---|---|
| pET20b(+) Beta-CGTase-1011 | BBa_255BPN6X | Plasmid | 7362 bp |
Key Features:
- Protein Expression: Optimized for the high-yield expression of Beta-CGTase-1101 in appropriate expression hosts, such as E. coli strains with T7 RNA polymerase.
- Promoter/Terminator: Target gene transcription is controlled by the T7 promoter and its cognate terminator.
- Purification Tag: The vector harbors a 6xHis tag sequence, facilitating one-step protein purification via Immobilized Metal Affinity Chromatography (IMAC) on Ni-NTA resin.
- Antibiotic Resistance: The plasmid confers kanamycin resistance (KanR) for selection in both plasmid maintenance and protein expression cultures.
- Cloning Flexibility: The multiple cloning site provides high flexibility, featuring unique restriction enzymes like XbaI and a group of compatible sites (XhoI, PspXI, PaeR7I) suitable for various cloning strategies.
- Cloning Notes: A common directional cloning strategy involves using the unique XbaI site together with any of the compatible XhoI, PspXI, or PaeR7I sites.