▼ RESULTS ▼

Introduction

Since its discovery in 2016, PETase has sparked widespread research on its significant potential in tackling the problem of plastic pollution. Our goal is to demonstrate how we have laid down the foundational proof of utilising our modified PETase in addressing the severe microplastic problem through synthetic biology.

Our wet lab team mainly focused on four gene combinations of PETase, investigating whether each of the sequentially added gene would increase the effectiveness of PETase in breaking down microplastic:

Modified PETase	Abbreviations	Part encoded from	Modified PETase Description
HotPETase	H	BBa_25OAK6TB	The basic enzyme used with higher thermostability, proving whether it is a suitable enzyme for relieving the problem of plastic pollution
CsgA-(G4S)2-HotPETase	C	BBa_252IGL39	The CsgA gene allows HotPETase to anchor onto bacterial cell surfaces, which could improve the efficiency of the whole system for breaking down PET plastic
HotPETase-(G4S)2-BaCBM2	B	BBa_25IHX9R7	The BaCBM2 gene is proved on whether it enhances the enzyme's microplastic-binding capabilities
CsgA-(G4S)2-HotPETase-(G4S)2-BaCBM2	BC	BBa_251QH76Y	A combination of CsgA and BaCBM2 to investigate if the combined effect could significantly enhance the enzyme's ability in degrading microplastic

Mass Test

The test for mass change of PET was an indicator of the effectiveness of our modified PETase. The test was carried out for the four genes with PBS control at three different temperatures. (25°C, 60°C, and 70°C)

At 25°C, the mass of PET in all four samples and the PBS control remained relatively stable with not much significant changes observed.

After repeating the experiment at 60°C, a decrease in PET mass was observed in B and BC, with PET mass dropping by 0.377% and 2.46% respectively. PET mass remained stable for the other 2 DNA and control.

The experiment was then repeated at 70°C in which more changes were observed. All samples except the PBS control saw a gradual decline in PET mass. The greatest decrease was observed in B with PET mass significantly decreasing by 3.29% which was nearly ten times greater than that at 60°C. Moreover, H also saw a dramatic decline in PET mass, dropping by 1.98%. C and BC had a slight decrease in mass of 0.391% and 0.366% respectively.

ANOVA Test

An ANOVA (Analysis of Variance) is a statistical test used to determine whether there are any statistically significant differences between the means of three or more independent groups. The F-value is calculated during the test. If F calculated > F critical, we will reject the null hypothesis since results show significant effect, then p-value would be less than 0.05 thus results would be statistically significant.

A two-way ANOVA is carried out for the effect of temperature against the effect of PETase type.

Null hypothesis:

• There is no significant difference in the mass change of PET caused by each type of enzymes
• Temperature has no significant effect on the ability of enzyme to degrade PET

F value: The statistical value of the test. Larger F-values indicate stronger evidence against the null hypothesis

Pr(>F): The p-value, if p-value < 0.05 we will reject the null hypothesis and there is a significant difference between at least two group of means

Definitions
Df	Degrees of freedom
Sum Sq	Sum of squares, measuring total variability or dispersion in data
Mean Sq	Mean squares, the average variability per degree of freedom
diff	Observed difference in means (B minus H)
lwr	Lower bound of 95% confidence interval
upr	Upper bound of 95% confidence interval
p adj	Adjusted p-value for significance
Residuals	the part of the data that the model can't account for, which may include random chance in PET breakdown process, measurement error

Figure 3. Definitions of terms used.

F value = ^{Variance b/w groups}⁄_{Variance within groups} = ^{Mean square b/w groups}⁄_{Mean square within groups}

If F ≈ 1: Between-group variance ≈ Within-group variance, the differences between the groups are not significantly different

If F >> 1: Between-group variance > Within-group variance, the differences between the groups are significant

ANOVA Results

ANOVA TABLE	Df	Sum Sq	Mean Sq	F value	Pr(>F)
Temperature	2	15.76	7.882	4.714	0.0173
PETase_Type	3	26.56	8.855	5.297	0.0051
Interaction	6	13.90	2.317	1.386	0.2546
Residuals	36	60.18	1.672

Figure 4. ANOVA Table for results.

Effect Sizes (η²)
Temperature	15.76/116.40 = 0.135 (Medium effect)
PETase Type	26.56/116.40 = 0.228 (Large effect)
Interaction	13.90/116.40 = 0.119 (Medium effect)
Residuals	60.18/116.40 = 0.517

Figure 5. Tables for Effect Sizes

POST-HOC Analysis

PETase Type Analysis

Temperature Analysis

Overall ANOVA analysis

Significant effects:

• PETase Type: F(3,36) = 5.297, p = 0.0051
• Temperature: F(2,36) = 4.714, p = 0.0173

Main Conclusions:

• PETase type significantly affects PET breakdown (p = 0.0051)
• TTemperature significantly affects breakdown (p = 0.0173)
• No significant interaction between temperature and PETase type
• PETase B performs best across all temperatures

Practical Implications:

• PETase B is optimal for PET breakdown
• Higher temperatures (70°C) enhance breakdown efficiency
• Mass decrease indicates successful PET degradation

Detailed Analysis of PETase type and temperature effect by percentage change

25°C PERFORMANCE

PETase Type	Mean Percentage Changes
H	0.00% (No change)
B	1.22% (Small increase)
C	1.12% (Small increase)
BC	1.15% (Small increase)

Figure 8. Mean percentage changes in PET mass for the 4 types of modified PETases at 25°C

Interpretation: Minimal PET breakdown at room temperature. All enzymes show similar low activity.

60°C PERFORMANCE

PETase Type	Mean Percentage Changes
H	0.63% (Very small increase)
B	0.00% (No change)
C	-0.54% (Small decrease)
BC	-2.47% (Decrease)

Figure 9. Mean percentage changes in PET mass for the 4 types of modified PETases at 60°C

Interpretation: Mixed results. Certain enzymes show decreased mass, suggesting PET breakdown.

70°C PERFORMANCE

PETase Type	Mean Percentage Changes
H	-1.79% (Decrease)
B	-3.70% (Largest decrease)
C	0.00% (No change)
BC	-0.55% (Small decrease)

Figure 10. Mean percentage changes in PET mass for the 4 types of modified PETases at 70°C

Interpretation: PETase B shows the largest mass decrease, indicating best breakdown performance.

Analysis

Overall, the mass test showed that all four samples perform better at high temperatures. Among the four samples, HotPETase fused with BaCBM2 gene (B) was shown to be the most effective in degrading PET at high temperatures (70°C). The BaCBM2 gene was selected for its ability to allow HotPETase to tightly bind to insoluble plastic, a property that has proved to be useful. On the other hand, HotPETase linked with both BaCBM2 and CsgA genes (BC) was also effective in breaking down PET at moderate temperatures (60°C). This aligned with past research^[1] on how HotPETase optimum temperature is 60°C.

Absorbance Test

Absorbance test was carried out for the four gene combinations of PETase aiming to investigate whether the genes added prove to be effective in increasing the enzymatic activity of PETase in degrading plastic. Moreover, the test was repeated for each of the gene combinations at a range of temperatures (25°C, 60°C, 70°C). Absorbance from 220nm to 300nm, especially 240nm, of each type of PETase compared to the PBS control were measured.^[2]

Figure 11. Mean absorbance values from 220nm to 300nm for the 4 types of DNA at different temperatures across 3 days.

Figure 12. Mean absorbance values at 240nm for the 4 types of DNA at different temperatures across 3 days.

Graph Description

At 25°C

All samples showed minimal increase in absorbance, which was only slightly above the control.

At 60°C

• Graph H: There is a significant increase in absorbance over time, showing that there is effective degradation of PET
• Graph B: The combination with BaCBM2 gene showed a much steeper increase in absorbance than that of HotPETase alone as the ability of the enzyme to bind tightly to plastic is enhanced by the gene
• Graph C: The results were similar to that of HotPETase alone as CsgA gene stabilizes the enzyme
• Graph BC: The combination of both genes showed a great increase in absorbance. The improved stability and binding ability of enzyme made it an effective sample

At 70°C

• Graph H: There is a much greater increase in absorbance over time, showing that the enzyme is reaching its optimum temperature
• Graph B: The maximum absorbance reached was much greater than that at 60°C, and HotPETase fused with BaCBM2 gene was shown to be the most effective in breaking down PET at 70°C
• Graph C: There is a sustained increase in absorbance, showing that CsgA gene has proved to be able to improve thermal stability of enzyme
• Graph BC: The changes in absorbance remained relatively similar to that at 60°C

ANOVA Test

A separate two-way ANOVA test by temperature was carried out to analyse whether temperature is a significant factor affecting the effectiveness of PETase in breaking down plastic as well as determining whether the four types of gene combinations have significant differences in their PET degrading ability.

A post-hoc analysis is then carried out after ANOVA test shows significant results in order to show exactly which specific groups differ from each other.

Null hypothesis:

• There is no significant difference in the ability of four types of gene combinations of degrading PET
• Temperature has no significant effect on the the effectiveness of PETase in breaking down PET

Variables used in test:

• μ = Overall mean absorbance
• α_i = Effect of i-th PETase type (i = H, B, C, BC)
• β_j = Effect of j-th wavelength (j = 220nm, 240nm, 260nm, 280nm, 300nm)
• (αβ)_ij = Interaction effect between PETase type and wavelength
• ε_ijk = Random error term ~ N(0, σ²)

F value = ^{Variance b/w groups}⁄_{Variance within groups} = ^{Mean square b/w groups}⁄_{Mean square within groups}

If F ≈ 1: Between-group variance ≈ Within-group variance → Groups are not significantly different

If F >> 1: Between-group variance > Within-group variance → Groups are significantly different

Results Summarized by Temperature

Term	Definition	Term	Definition
Df	Degree of freedoms	Normality	The residuals (errors) should be normally distributed
Pr(>F)	The probability that the differences you observed occurred purely by random chance	Homogeneity	Variance within each group should be approximately equal
diff	Observed difference in means (B minus H)	lwr	Lower bound of 95% confidence interval
upr	Upper bound of 95% confidence interval	p adj	Adjusted p-value for significance

Figure 13. Definitions of terms used.

25°C Results

ANOVA TABLE	Df	Sum Sq	Mean Sq	F value	Pr(>F)
PETase_Type	3	0.0382	0.01273	1.785	0.155
Wavelength	4	0.2610	0.06525	9.147	1.28e-05
Interaction	12	0.0584	0.00487	0.682	0.767
Residuals	80	0.5706	0.00713

Figure 14. ANOVA Table for 25°C results.

Effect Sizes (η²)		Assumption Checks
PETase Type	0.0411 (Small effect)	Normality	W = 0.9783, p = 0.1043 ✓
Wavelength	0.2811 (Large effect)	Homogeneity	F = 1.234, p = 0.2673 ✓
Interaction	0.0629 (Medium effect)

Figure 15. Tables for Effect Sizes and Assumption Checks for 25°C results.

Conclusion: No significant differences between PETase types at 25°C

60°C Results

ANOVA TABLE	Df	Sum Sq	Mean Sq	F value	Pr(>F)
PETase_Type	3	0.1458	0.04860	7.845	0.000126
Wavelength	4	2.6220	0.65550	105.784	< 2e-16
Interaction	12	0.1211	0.01009	1.629	0.095876
Residuals	80	0.4957	0.00620

Figure 16. ANOVA Table for 60°C results.

Effect Sizes (η²)		Assumption Checks
PETase Type	0.0429 (Small effect)	Normality	W = 0.9841, p = 0.2874 ✓
Wavelength	0.7715 (Very large effect)	Homogeneity	F = 1.456, p = 0.1562 ✓
Interaction	0.0356 (Small effect)

Figure 17. Tables for Effect Sizes and Assumption Checks for 60°C results.

POST-HOC ANALYSIS (Tukey HSD)

	diff	lwr	upr	p adj
B-H	0.08108	0.01583	0.14633	0.0097
BC-H	0.05458	-0.01067	0.11983	0.1284
C-H	-0.01292	-0.07817	0.05233	0.9574
BC-B	-0.02650	-0.09175	0.03875	0.7063
C-B	-0.09400	-0.15925	-0.02875	0.0019
C-BC	-0.06750	-0.13275	-0.00225	0.0405

Figure 18. POST-HOC Analysis table for 60°C results.

Performance Ranking: B > BC > H > C

70°C Results

ANOVA TABLE	Df	Sum Sq	Mean Sq	F value	Pr(>F)
PETase_Type	3	0.3093	0.10310	16.987	1. and 73e-09
Wavelength	4	2.8479	0.71198	117.316	< 2e-16
Interaction	12	0.1584	0.01320	2.175	0.0206
Residuals	80	0.4855	0.00607

Figure 19. ANOVA Table for 70°C results.

Effect Sizes (η²)		Assumption Checks
PETase Type	0.0798 (Medium effect)	Normality	W = 0.9812, p = 0.1892 ✓
Wavelength	0.7349 (Very large effect)	Homogeneity	F = 1.892, p = 0.0432 (Slight violation)
Interaction	0.0409 (Small effect)

Figure 20. Tables for Effect Sizes and Assumption Checks for 70°C results.

POST-HOC ANALYSIS (Tukey HSD)

	diff	lwr	upr	p adj
B-H	0.11733	0.05208	0.18258	0.0001
BC-H	0.02208	-0.04317	0.08733	0.8065
C-H	-0.03867	-0.10392	0.02658	0.4066
BC-B	-0.09525	-0.16050	-0.03000	0.0014
C-B	-0.15600	-0.22125	-0.09075	0.0000
C-BC	-0.06075	-0.12600	0.00450	0.0773

Figure 21. POST-HOC Analysis table for 70°C results.

Performance Ranking: B > H > BC > C

Overall Results from ANOVA

Analysis

Overall, several conclusions can be drawn from this experiment. Firstly, the absorbance tests showed that HotPETase had slightly better performance at higher temperatures with an optimum temperature of around 60 to 70C, but the effect of temperature is not statistically significant for low temperatures. Moreover, fusing the BaCBM2 gene to Hot PETase has proven to be effective in enhancing the enzyme’s ability in degrading PET, showing to be the best performer among the four gene combinations. This further proves that the BaCBM2 gene has enhanced the enzyme’s microplastic-binding capabilities.

PETase BC displays a worse performance than the PETase B and PETase H, which can be attributed to its larger size. Although PETase BC incorporates the surface display capabilities of CsgA and increased contact surface area of BaCBM2, a larger protein results in more possible folding pathways and intermediate states. This may result in 1 domain folding more quickly than the other. When waiting for the other domain to be folded, the hydrophobic residues normally buried in the final structure remain exposed and increases the possibility of interacting with other hydrophobic residues on the same protein or different proteins, leading to misfolding and aggregation respectively.^[7]

Protein Strip Test

The same purified His-tagged proteins of the 4 gene combinations before SDS-PAGE were added dropwise to protein test strips, to confirm the presence of His-tagged proteins. After the four samples were added to protein test strips, all of the test strips experienced a change in colour, which indicated that the modified PETase enzymes have been expressed by the E. coli and were purified as His-tagged proteins successfully.

SDS-PAGE

To separate the proteins by the molecular weight, we have performed SDS-PAGE after preparing the bacteria culture medium. His-tag purification is performed prior to running the gel electrophoresis to obtain the protein of the E. coli. During the first engineering cycle, no bands were seen on the gel, and thus no meaningful results were obtained. The protein ladder is the Thermo Scientific PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa.

Results (Cycle 2):

From the results, it can be seen that none of the bands of protein samples besides HotPETase can be visible even after staining, with the bands from HotPETase being unclear and faint. The molecular weight of the HotPETase proteins are similar to those provided by the ladder protein.

Analysis:

During the engineering cycles of the SDS-PAGE, the extracted samples were not thoroughly heated and thus resulted in incomplete denaturation, which resulted in the proteins retaining their tertiary or secondary structures. Additionally, we hypothesized that the concentration of PETase in the TE buffer for DNA solubilization and stabilization was too low, likely resulting in the faint bands shown for the HotPETase, while the rest were not visible at all. Most importantly, however, was the absence of hexafluoroisopropanol (HFIP), which is essential in CsgA amyloid depolymerization. Due to the high aggregation propensity and colloidal instability of the CsgA gene^[3], the insoluble CsgA fibrils may have been retained in the wells, and the aggregates may have interfered with the staining of the bands^[4], which can potentially decrease the visibility of said bands.

Scanning Electron Microscopy (SEM)

To further investigate the action of the modified PETase on PET, we have prepared the digested samples of PET to be observed under a scanning electron microscope under the assistance of HKU. The purpose of the test is to determine the effect on digestion of the PETase with the BaCBM2 gene towards the surface of the PET.

Results

Control

HotPETase-(G4S)2-BaCBM2

CsgA-(G4S)2-Hotpetase-(G4S)2-BaCBM2

Analysis

As the frames display, the surface of the PET has been digested on a thorough and large-scale basis. Compared to the control sample, there are minimal to no areas that appear untouched by the action of the 2 modified PETase. In addition, the digestion of the enzyme has penetrated quite deeply into the surface, forming numerous cracks and interconnected grooves across the area, which showcases the effective digestion of the modified enzyme.

Under the SEM, HotPETase forms pit-like structures on the surface of PET^[5] instead of the trenches shown on the surface of the samples. Considering that enzymes inherently target less amorphous regions of the surface of crystalline structures,^[6] along with the fact that the PET plastic samples obtained have a high likelihood of being scratched or damaged, it is plausible that the when the modified PETase attaches to the PET surface, the BaCBM2 gene resulted in a linear, directional degradation of plastic due to its improved adhesion of the PETase on the scratched surface, proving the digestion is not due to randomness.

Plasmid Detection Test

The plasmid detection test was carried out to prove the introduction of the modified plasmid into the E. coli for the 4 gene combinations. After obtaining the purified sample through plasmid miniprep (refer to Experiments section), 2 trials were carried out using gel electrophoresis to measure the length of the linear plasmid vector. 2 enzymes, BamHI and HindIII, were used to perform a restriction digest on the plasmid.

1st Trial

Components	Volume
Deionized water	30μL
FastDigest Green Buffer	4μL
DNA	4μL
Fast Digest Enzyme (BamHI / HindIII)	2μL
Total Volume	40μL

2nd Trial

Components	Volume
Deionized water	24μL
FastDigest Green Buffer	4μL
DNA	8μL
Fast Digest Enzyme (BamHI / HindIII)	4μL
Total Volume	40μL

Results:

1st Trial (BamHI enzyme)

1st Trial (HindIII enzyme)

2nd Trial (BamHI enzyme)

2nd Trial (HindIII enzyme)

Analysis:

The base pairs for each PETase range between 6000 and 7000. The 2 ladder bands that move the slowest represent 6000 base pairs and 10000 base pairs.

As shown in the results, both trials successfully showed a singular, clear band between the 6000 base pairs and 10000 base pairs ladder band. Not only does this prove the introduction of plasmid into the E. coli, but it also proved the sample for each well were not contaminated and have the same number of base pairs.

However, the bands for BC could not be seen in both trials. This abnormality can be attributed to the fact that the concentration of pellets for BC was significantly lower than the rest during the resuspension process of the pellets.

In addition, the brightness of the bands during the 1st trial was quite low, in which we concluded that it was due to low concentration of DNA. Therefore, the ratio of components during sample preparation were altered to double the concentration of DNA, which led to greater brightness of the bands during the 2nd trial, confirming our hypothesis.

References

[1] Bell, Elizabeth & Smithson, Ross & Kilbride, Siobhan & Foster, Jake & Hardy, Florence & Ramachandran, Saranarayanan & Tedstone, Aleksander & Haigh, Sarah & Garforth, Arthur & Day, Philip & Levy, Colin & Shaver, Michael & Green, Anthony. (2022). Directed Evolution of an Efficient and Thermostable PET Depolymerase. 10.21203/rs.3.rs-1350765/v1.

[2] Pirillo, V., Pollegioni, L., & Molla, G. (2021). Analytical methods for the investigation of enzyme‐catalyzed degradation of polyethylene terephthalate. FEBS Journal, 288(16), 4730–4745. https://doi.org/10.1111/febs.15850

[3] Bu F, Dee DR, Liu B.2024.Structural insight into Escherichia coli CsgA amyloid fibril assembly. mBio15:e00419-24. https://doi.org/10.1128/mbio.00419-24

[4] Kurien BT, Scofield RH. Common artifacts and mistakes made in electrophoresis. Methods Mol Biol. 2012;869:633-640. doi:10.1007/978-1-61779-821-4_58

[5] Bell, E.L., Smithson, R., Kilbride, S. et al. Directed evolution of an efficient and thermostable PET depolymerase. Nat Catal 5, 673–681 (2022). https://doi.org/10.1038/s41929-022-00821-3

[6] Ermis, H. A mini-review on the role of PETase in polyethylene terephthalate degradation. Rev Environ Sci Biotechnol 24, 545–555 (2025). https://doi.org/10.1007/s11157-025-09737-3

[7] Ajmal MR. Protein Misfolding and Aggregation in Proteinopathies: Causes, Mechanism and Cellular Response. Diseases. 2023 Feb 9;11(1):30. doi: 10.3390/diseases11010030. PMID: 36810544; PMCID: PMC9944956.