Our iGEM project focuses on the biodegradation of PET plastics. The core work is developing various genetic elements: basic elements are either existing or newly modified degrading enzymes and mutants, responsible for decomposing PET, processing intermediate products, and optimizing enzyme performance; composite elements are made by combining basic elements with the pET8a vector, allowing these elements to function in the host. The ultimate goal is to build a biological system capable of efficiently degrading PET and find a biological solution to address plastic pollution(Table 1).
Our part collection is not simply a quantitative accumulation of individual parts, but rather a dual-enzyme synergistic reaction.
Step 1: We degrade PET plastic into the monomer MHET. IspETase serves as the positive control to compare the PET degradation efficiency. dsPETase is a plastic-degrading enzyme with high PET degradation activity, which we identified from literature and databases. To enhance the enzymatic activity of dsPETase, we performed site-directed mutagenesis, resulting in eight mutants. We used HPLC to screen these mutants and identified the best one, the Ser mutant.
Step 2: We further degrade MHET into TPA and EG, providing a method for PET recycling. Therefore, we applied MHETase and the Ser mutant in combination, validating the degradation efficiency.
Fig 1. Degradation process of PET plastic and key enzymes [Yoshida etal,2016]
Table1. The part collection
|
Part number |
Part name |
Part type |
Function |
Diagram |
|
IsPETase |
Basic Part |
Derived from Ideonella sakaiensis, PETase degrades poly(ethylene terephthalate) (PET) in natural environments, yielding the monomeric intermediate product mono(2-hydroxyethyl) terephthalic acid (MHET) |
/ |
|
|
dsPETase |
Basic Part |
Derived from deep-sea environment, PETase degrades poly(ethylene terephthalate) (PET) in natural environments, yielding the monomeric intermediate product mono(2-hydroxyethyl) terephthalic acid (MHET) |
/ |
|
|
MHETase |
Basic Part |
Originating from Ideonella sakaiensis, MHETase further hydrolyzes mono(2-hydroxyethyl) terephthalic acid (MHET) into terephthalic acid (TPA) and ethylene glycol (EG). It acts synergistically with PETase to achieve complete degradation of PET |
/ |
|
|
GLY8-GIn |
Basic Part |
The mutant of dsPETase retains the same enzymatic function as PETase. |
/ |
|
|
GLY8-Ser |
Basic Part |
The mutant of dsPETase retains the same enzymatic function as PETase. |
/ |
|
|
GLY8-Thr |
Basic Part |
The mutant of dsPETase retains the same enzymatic function as PETase. |
/ |
|
|
GLY8-Cys |
Basic Part |
The mutant of dsPETase retains the same enzymatic function as PETase. |
/ |
|
|
GLY8-Asn |
Basic Part |
The mutant of dsPETase retains the same enzymatic function as PETase. |
/ |
|
|
GLY8-Tyr |
Basic Part |
The mutant of dsPETase retains the same enzymatic function as PETase. |
/ |
|
|
Arg47-Lys |
Basic Part |
The mutant of dsPETase retains the same enzymatic function as PETase. |
/ |
|
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Arg47-His |
Basic Part |
The mutant of dsPETase retains the same enzymatic function as PETase. |
/ |
|
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pET28a |
Basic Part |
The commonly used E. coli expression vector contains a T7 promoter, antibiotic resistance gene, and replication origin. |
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pET8a-IsPETase |
Composite Part |
The vector for expressing the IsPETase enzyme. |
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pET8a-dsPETase |
Composite Part |
The vector for expressing the dsPETase enzyme. |
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pET8a-MHETase |
Composite Part |
The vector for expressing the MHETase enzyme. |
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pET8a-GLY8-GIn |
Composite Part |
The vector for expressing the GLY8-GIn enzyme. |
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pET8a-GLY8-Ser |
Composite Part |
The vector for expressing the GLY8-Ser enzyme. |
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pET8a-GLY8-Thr |
Composite Part |
The vector for expressing the GLY8-Thr enzyme. |
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pET8a-GLY8-Cys |
Composite Part |
The vector for expressing the GLY8-Cys enzyme. |
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pET8a-GLY8-Asn |
Composite Part |
The vector for expressing the GLY8-Asn enzyme. |
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pET8a-GLY8-Tyr |
Composite Part |
The vector for expressing the GLY8-Tyr enzyme. |
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pET8a-Arg47-Lys |
Composite Part |
The vector for expressing the Arg47-Lys enzyme. |
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pET8a-Arg47-His |
Composite Part |
The vector for expressing the -Arg47-His enzyme. |
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