Part:BBa_K3997006
pGEX-IsPETase-GST
Profile
Name: pGEX-IsPETase-GST
Base Pairs: 2800+bp
Origin: Ideonella sakaiensis 201-F6, synthesis
Properties: Polyethylene terephthalate degradation enzyme
Usage and Biology
Polyethylene terephthalate (PET) is the most widely produced polyester plastic and its accumulation in the environment has become a global concern. At the same time, the daily intake of microplastics by humans is gradually increasing, which damages human health. Therefore, researchers believe that it is important to develop an environmental-friendly plastic degradation method by using microorganisms. Recently, a novel bacterial strain called Ideonella sakaiensis 201-F6 has been discovered that produces a couple of unique enzymes, IsPETase and MHETase, enabling the bacteria to utilize PET as their sole carbon source.
Construct design
The enzyme IsPETaseis a hydrolase, and it is crucial for hydrolysis of PET. To verify this property, we use E. coli as the starting strain and construct an engineered strain of IsPETaseto explore its biological activity of the hydrolysis of PET. To purify the protein, we also transfer the plasmid expressing IsPETaseinto BL21(DE3). We use pGEX as backbone and add a GST tag at its N-terminal. The enzyme is under the regulation of T7 promoter and can be induced by adding IPTG.
The T7 promoter is often used for protein overexpression. It is powerful and specific. It is completely controlled by T7 RNAP. When T7 RNAP is present in the cell, the T7 expression system occupies an absolute advantage compared to the host expression system. Its expression speed is 5 times that of the former.
BBa_K3997003
Name: GST tag
Base Pairs: 693 bp
Origin: Origin: synthetic
Properties: affinity tag
Usage and Biology
Glutathione-S-transferase (GST), a 26 kDa sequence of 211 amino acids, is another widely used affinity tag that increases solubility of the desired protein.
BBa_K3997000
Name: IsPETase
Base Pairs: 2107 bp
Origin: Ideonella sakaiensis 201-F6
Properties: hydrolysis of PET
Experimental approach
In order to present the function of the part, the IsPETase gene was expressed in E. coli under the control of T7 promoter. Then the bacterial cells are collected and crushed. The samples of whole expression cell lysate, supernatant and pellet of cell lysate were analyzed using SDS-PAGE, which is found in the corresponding protein band of approximately 35 kDa (Figure 3).
Lane 6: IsPETase Cell lysate without induction for 20 h at 16oC
Lane 7: IsPETase Cell lysate with induction for 20 h at 16oC
Lane 8,9,10: GST elution fractions of purification of lane 7 by GST-affinity chromatography
References
1. Shosuke Yoshida et al. A bacterium that degrades and assimilates poly(ethylene terephthalate), Science (2016).
2. Harry P Austin. et al. Characterization and engineering of a plastic-degrading aromatic polyesterase, PNAS(2018)
3. Chun-Chi Chen et al. General features to enhance enzymatic activity of poly(ethylene terephthalate) hydrolysis, Nature Catalysis(2021).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1014
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 705
Illegal XhoI site found at 1581 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1337
- 1000COMPATIBLE WITH RFC[1000]
| None |