Team:WFLA YK PAO/Results
Results
ATLATL
1. PCR amplification results of individual gene segments
In both pictures, we see a distinctive expression of IsPETase on the left and
MHETase on the right. From
the marker, we could judge that the band is IsPETase as it’s molecular weight is 897bp and MHETase is
2082bp.
2. PCR amplification results of connected IsPETase-MHETase gene
The recombinant gene segment should be approximately 3000bp. Judging from the
marker, we could see the highlighted band is the recombinant sequence. There is also a left-over of MHETase
and IsPETase that haven’t been completely connected.
3. Gel electrophoresis results on enzyme-cut IsPETase-MHETase gene and
pET28a
Again, judging from the marker, we could see the Is-MH segment that has been cut
and also the vector DNA
that has been cut. There is also a remaining of uncut Is-MH segment.
4. 1st SDS-PAGE results
A wide band between 50 to 70 on the left gel standards matches the weight of
MHETase (61). This suggests
MHETase was successfully induced and expressed. However, at the 7th track, the pattern for purified MHETase
is very unclear, suggesting low solubility of MHETase.
A clear band between 25 and 35 on the right gel standards matches the weight of
IsPETase (31.5). This
suggests IsPETase was successfully induced and expressed. However, the location of the band is higher than
expected. This could be due to the small number of proteins added in the gel electrophoresis. Similar to
MHETase, there are very weak indications of IsPETase on the 3rd and 7th track, suggesting low solubility of
IsPETase.
5. Microscopic image results
It was difficult to tell from the two photos how successful these enzymes were in
decomposing microplastic.
Comparing this microplastic to the control group, we can see that it appears to have some damage on its
edges, and even a few small holes. The decomposition impact, on the other hand, cannot be shown by this
metric. Our capacity to observe minute changes on the PET surface is limited by the modest magnification of
the light microscope.
Xuhui
1. Result analysis of gel electrophoresis-Identifying target DNA
Two groups of recombinant IS-MH genes, separate IS and MH is examined in the gel
electrophoresis. The
actual molecular weight of separate IsPETase is about 790bp, which is different from the expected value of
897bp. The band of MH which is supposed to be 2082bp or the other two recombinant genes which are supposed
to be 3000bp could not be observed. This is because of the experimental conditions and operational problems.
2. Result analysis of SDS-PAGE-Identifying purified protein
Following the order of precipitation, flow through, wash through eluted after 25 mM
imidazole, and 3
separate wash through after 250 mM imidazole to identify proteins for each IS and MH group. The standard
band of IS is about 31. At the 5th and 6th track, there is a sign that suggests the existence of protein,
but the indication of IS is about 27-28, which is still different from the standard molecular weight as it
is supposed to be in the middle of 25 and 35. Reasons include operation problems and experimental conditions
problems, such as the concentration of protein is too low. Also, during the first SDS Page, it is hard to
observe the MH protein, which shows the low solubility of MHPETase. After the second SDS-PAGE analysis and
added the precipitation of MH, this suggests that it formed the inclusion.
3. Result analysis of the microscope picture- comparing enzyme activity of
IsPETase and MHETase to decompose micro plastic molecules.
left: blank
right: IsPETase+MHETase
The two pictures did not provide a clear indication of the true effectiveness of these two enzymes combined to decompose microplastic. From the picture, we can observe that compared to the control group, the microplastic in the group of two enzymes combined seems to have some damage on the edge, and even have some small holes. However, this cannot be a significant indicator to prove the decomposition effect. One problem with qualitative analysis was with the irregular shape of an individual microplastic molecule. As we did not take microscope pictures of each individual molecule before they were put into the enzyme solution, we cannot conduct a meaningful comparison because the comparison between molecules is not valid. Therefore, this study requires more careful quantitative analysis to determine the actual enzyme activity and decomposition effectiveness.
right: IsPETase+MHETase
The two pictures did not provide a clear indication of the true effectiveness of these two enzymes combined to decompose microplastic. From the picture, we can observe that compared to the control group, the microplastic in the group of two enzymes combined seems to have some damage on the edge, and even have some small holes. However, this cannot be a significant indicator to prove the decomposition effect. One problem with qualitative analysis was with the irregular shape of an individual microplastic molecule. As we did not take microscope pictures of each individual molecule before they were put into the enzyme solution, we cannot conduct a meaningful comparison because the comparison between molecules is not valid. Therefore, this study requires more careful quantitative analysis to determine the actual enzyme activity and decomposition effectiveness.
4. Result analysis of the microscope picture- comparing the bacterial activity
of IsPETase and MHETase
to decompose micro plastic molecules.
leftmost: blank
middle: bacterial activity group 1
rightmost: bacterial activity group 2
Previously, we have added microplastic molecules into two solutions containing bacteria designed to synthesize IsPETase and MHETase. After one day on the shaker, we want to compare the bacterial activity of these bacteria on decomposing the microplastic. However, just by observation, it was hard to determine whether the irregular edges can be attributed to the addition of these bacteria specifically. This study requires quantitative analysis, such as using the liquid chromatogram, to compare the effectiveness of decomposition in numerical terms.
middle: bacterial activity group 1
rightmost: bacterial activity group 2
Previously, we have added microplastic molecules into two solutions containing bacteria designed to synthesize IsPETase and MHETase. After one day on the shaker, we want to compare the bacterial activity of these bacteria on decomposing the microplastic. However, just by observation, it was hard to determine whether the irregular edges can be attributed to the addition of these bacteria specifically. This study requires quantitative analysis, such as using the liquid chromatogram, to compare the effectiveness of decomposition in numerical terms.
ATLATL
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
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
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
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
4. 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 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
Future Plan
1. We have difficulties in extracting the protein IsPETase which seems insoluble.
In this case, we are thinking about to re-construct 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.
2. In future, we will consider the requirements of beverage in product preparation,
test the solubility in different common drinks, and further make sample beverage formula. It is very
important for the product development to consider the safety, stability and the storage problems.
3. As our product is designed for human beings, it would be necessary to simulate
the human gastrointestinal environment to test the effect.