Team:WFLA YK PAO/Contribution

WFLA_YK_PAO

Contribution

BBa_K3997000

== Profile ==

Name: IsPETase
Base Pairs: 2107 bp
Origin: Ideonella sakaiensis 201-F6
Properties: hydrolysis of PET

== 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.

Figure 1. Action and function of IsPETase in PET degradation.

The enzyme IsPETase is 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 IsPETase to explore its biological activity of the hydrolysis of PET. To purify the protein, we also transfer the plasmid expressing IsPETase into 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 The speed is 5 times that of the former.

BBa_K3997001

==Profile==

Name: MHETase
Base Pairs: 1752 bp
Origin: Ideonella sakaiensis 201-F6
Properties: hydrolysis of MHET.

== 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, PETase and MHETase, enabling the bacteria to utilize PET as their sole carbon source.

Figure 1. Action and function of MHETase in MHET degradation.

The enzyme MHETase is a hydrolase, and it represents a key step in the process of microbial PET degradation in I. sakaiensis. It cleaves monoterephthalic acid, the PET degradation product by PETase, to ethylene glycol and terephthalic acid, and it is crucial for hydrolysis of

MHET. We attempt to express the MHETase in E. coli strain to express and purify the protein to test its activity of degradation the MHET (Figure 1). So as to set up a method of environmental-friendly plastic degradation.

BBa_K3997004

== Profile ==

Name: pET28a-IsPETase-His
Base Pairs: 2107 bp
Origin: Ideonella sakaiensis 201-F6, synthetic
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.

PET hydrolase, known as IsPETase, is a recently discovered enzyme that has been found to break down PET, or polyethylene terephthalate-(C10H8O4)n. When the IsPETase-containing bacteria or fungi are administered to PET plastic, they secrete the PETase enzyme, which causes PET polymers to bind to the active site of IsPETase, allowing the reaction to occur. During the reaction, the enzyme breaks ester bonds in PET.

Figure 1. Action and function of PETase and MHETase in PET degradation.

BBa_K3997005

==Profile ==

Name: pET28a-MHETase-His
Base Pairs: 1979 bp
Origin: Ideonella sakaiensis 201-F6, synthetic
Properties: Polyethylene terephthalate degradation enzyme

== Construct design ==

Figure 2. Plasmid diagram

The enzyme MHETase is a hydrolase, and it is crucial for hydrolysis of MHET. To verify this property, we use E. coli as the starting strain and construct an engineered strain of MHETase to explore its biological activity of the hydrolysis of MHET. To purify the protein, we also transfer the plasmid expressing MHETase into BL21(DE3) with a 6×His tag at it’s 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 The speed is 5 times that of the former.

BBa_K3997006

= 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 ==

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_K3997008

== Profile ==

Name: pET28a-IsPETase-MHETase
Base Pairs: 2876 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, PETase and MHETase, enabling the bacteria to utilize PET as their sole carbon source. PET hydrolase, known as PETase, is a recently discovered enzyme that has been found to break down PET, or polyethylene terephthalate-(C10H8O4)n. When the PETase-containing bacteria or fungi are administered to PET plastic, they secrete the PETase enzyme, which causes PET polymers to bind to the active site of PETase, allowing the reaction to occur. During the reaction, the enzyme breaks ester bonds in PET.

== Construct design ==

Figure 2. pET28a-IsPETase-MHETase Plasmids diagram

The genes IsPETase and MHETase were synthesized and inserted into pET28a vector to obtain plasmids pET28a-IsPETase and pET28a-MHETase, respectively. A recombinant plasmid pET28a-IsPETase-MHETase, which contains both genes, was also constructed (Figure 2).

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 The speed is 5 times that of the former.