Team:WFLA YK PAO/Proof Of Concept

WFLA_YK_PAO

Proof of Concept

Recently, at the European Society of Gastroenterology, research confirmed for the first time that up to nine types of microplastics have been monitored in human feces. Moreover, in some animal observation experiments, scientists have found that these particles can cause blood clots and that airborne plastic particles can reside deep in the lungs, leading to a variety of diseases, including cancer.

Thereafter it is very meaningful to develop a powerful tool for human beings to help with wiping out microplastics. We have come up with an idea by developing an engineered probiotic functional in degrading microplastics.

PET degradation related enzyme (PETase) was first reported in Science in 2016. In 2018, PNAS reported that MHETase can further degrade MHET into TPA (mono (2-hydroxyethyl) terephthalic acid) and EG (ethylene glycol). An efficient enzyme for the degradation of PET (IsPETase) was reported in Nature Catalysis in 2021. Thus, plastic products can be efficiently degraded into recyclable monomers. Therefore, we decide to express IsPETase and MHETase (PET degradation enzyme) in the cells of probiotics to reduce the content of human microplastics and improve human health.

II. General concept

This is the general engineering flow of our probiotic.
We hope that once our experiments succeed, we will try developing probiotic drinks, which will be distributed to our end users, ranging from white-collar staff, to primary school students and to toddlers.

Supporting Experiment Results;

1. Pilot expression

· IsPETase( ~35.13 kDa)_BL21(DE3)

Fig.1 SDS-PAGE (left) and western blot (right) analysis for IsPETase 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 15 oC
Lane 2: Cell lysate with induction for 4 h at 37 oC
Lane NC1: Supernatant of cell lysate without induction
Lane 3: Supernatant of cell lysate with induction for 16h at 15 oC
Lane 4: Supernatant of cell lysate with induction for 4 h at 37 oC
Lane NC2: Pellet of cell lysate without induction
Lane 5: Pellet of cell lysate with induction for 16h at 15 oC
Lane 6: Pellet of cell lysate with induction for 4 h at 37 oC
The primary antibody for western blot is anti-His antibody

· MHETase( ~65.17 kDa)_BL21(DE3)

Fig.2 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

2. Protein production fusion with 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
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: GSH elution fractions of purification of lane 7 by GST-affinity chromatography

3. Conclusion and suggestion about protein express

The above experiment results indicated that we successfully obtained the protein of MHETase, whereas, we failed to acquire IsPETase protein.

4. Enzyme Activity Tests

Since MHETase protein has been obtained, the following enzyme activity tests have been done. 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 2, 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

This enzyme activity tests also indicate the the MHETase protein is functional, which also provided our with quite a lot of confidence to proceed along with our project since it partially proved the feasibility of our experiment design.

Since we have had difficulties in extracting the protein IsPETase which seems insoluble, we consider re-constructing the plasmid which could be transformed as well as expressed in other types of bacteria, like pseudomonas putida to further test the protein expression. In addtion to this, once we could obtain the purified IsPETase would we further conduct the enzyme activity tests to analyze the performance.