Difference between revisions of "Part:BBa K3997004"

Latest revision as of 16:49, 21 October 2021

pET28a-IsPETase-His

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...

Construct design

Figure 2. Plasmid design diagram.

BBa_K3997001

Name: 6×His

Base Pairs: 18bp

Origin: synthetic

Properties: Polyhistidine tag

Usage and Biology

It is an polyhistidine tag, which is used in the purification of recombinant proteins

BBa_K3997000

Name: IsPETase

Base Pairs: 2107 bp

Origin: Ideonella sakaiensis 201-F6

Properties: hydrolysis of PET.

IsPET 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 IsPETase 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.

Experimental approach

In order to present the function of the part, the PETase gene was PCR amplified and cloned into pET28a plasmids backbone in order to express the protein in E. coli under the control of T7 promoter.

Figure 2. Gel electrophoresis of PETase-MHETase PCR products...

Proof of function

1. Enzyme Activity Tests

The activity of MHETase was indicated by the decline absorbances at a wavelength of 240 nm (Figure 3). The wavelength is the specific absorption of MHET. As shown in figure 4, after 1 d reaction, MHET concentration was dropped by 31.4%.

Figure 3. Detection of residual MHET by measuring the absorbance at 240 nm...

Figure 4. MHETase activity assay...

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