Difference between revisions of "Part:BBa K3997005"

Revision as of 07:24, 20 October 2021

pET28a-MHETase-His

promoter

Profile

Name: pET28a-MHETase-His

Base Pairs: 1979 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, 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

Figure 1...

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.

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_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_K3997001

Name: MHETase

Base Pairs: 1752 bp

Origin: Ideonella sakaiensis 201-F6

Properties: hydrolysis of MHET.

Experimental approach

Production, purification, and activity analysis of MHETase

1. Construction of pET28a-MHETase

Figure 3. Gel electrophoresis of MHETase PCR products...

2. Purification of MHETase( ~65.17 kDa)_BL21(DE3) In order to present the function of the part, the MHETase 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 and Western blot (only for his-tag proteins from pET28a vector), which is found in the corresponding protein band of approximately 65kDa (Figure 3).

Figure.4 SDS-PAGE (left) and western blot (right) analysis for MHETase cloned in pET28a and expressed in BL21(DE3) strain...

Lane M1: Protein marker

Lane M2: Western blot marker

Lane PC1: BSA (1μg)

Lane PC2: BSA (2μg)

Lane NC: Cell lysate without induction

Lane 1: Cell lysate with induction for 16h at 15oC

Lane 2: Cell lysate with induction for 4 h at 37oC

Lane NC1: Supernatant of cell lysate without induction

Lane 3: Supernatant of cell lysate with induction for 16h at 15oC

Lane 4: Supernatant of cell lysate with induction for 4 h at 37oC

Lane NC2: Pellet of cell lysate without induction

Lane 5: Pellet of cell lysate with induction for 16h at 15oC

Lane 6: Pellet of cell lysate with induction for 4 h at 37oC

The primary antibody for western blot is anti-His antibody

Figure.5 SDS-PAGE (left) analysis for MHETase cloned in pGEX-6P-1 and expressed in BL21(DE3) strain...

In addition, MHETase genes were also cloned to the expression vector pGEX-6P-1, which produce recombinant protein fusion with Glutathione-S-transferase (GST) tag.

Lane 1: MHETase Cell lysate without induction for 20 h at 16oC

Lane 2: MHETase Cell lysate with induction for 20 h at 16oC

Lane 3,4,5: GSH elution fractions of purification of lane 2 by GST-affinity chromatography

Proof of function

1. Enzyme Activity Tests

The activity of MHETase was indicated by the decline absorbances at a wavelength of 240 nm (Figure 4). The wavelength is the specific absorption of MHET. As shown in figure 2, after 1 d reaction, MHET concentration was dropped by 31.4%.

Figure 6. Detection of residual MHET by measuring the absorbance at 240 nm...

Figure 7. MHETase activity assay...

Improvement of an existing part

Compared to the old part BBa_K2013003, expressing MHETase, we design a new part BBa_K3997005, which is expressing MHETase.

Figure 8. The blast results about the DNA sequence of our new part BBa_K3997005 and the old parts BBa_K2013003...

The BBa_K2013003 part is useful as an example. iGEM16_UESTC-China 2016 has cloned this part into pUC57 and did codon optimization before synthesizing it to encode the target product successfully in E. coli.

The difference is that our goal is to build an environmental-friendly method to achieve plastic degradation, and we set up a reaction system to test its property of hydrolysis of MHET. We chose MHETase, to construct our desired engineering strain.

First of all, we constructed a composite part BBa_K3997005, and transformed it into E.coli BL21(DE3). We successfully expressed and purified MHETase protein, and detected the enzyme activity.

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