Difference between revisions of "Part:BBa K3997006"

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	<partinfo>BBa_K3997006 short</partinfo>		<partinfo>BBa_K3997006 short</partinfo>
−	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.
		+
		+
		+
		+	[[File:T--WFLA YK PAO--BBa K3997006-Figure1.png|500px|thumb|center|Figure 1. Action and function of IsPETasein PET degradation...]]
		+
		+
		+
		+	=== Construct design ===
		+
		+
		+
		+	[[File:T--WFLA YK PAO--BBa K3997006-Figure2.png|500px|thumb|center|Figure 2. Plasmid diagram...]]
		+
		+
		+
		+
		+	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).
		+
		+
		+
		+
		+	[[File:T--WFLA YK PAO--BBa K3997006-Figure3.png|500px|thumb|center|Figure3. SDS-PAGE of pGEX-IsPETase-GST...]]
		+
		+
		+
		+
		+	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). ====
		+
	<!-- Add more about the biology of this part here		<!-- Add more about the biology of this part here

---

Latest revision as of 13:47, 20 October 2021

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