▼ ENGINEERING SUCCESS ▼

Cycle 1

Design

Our objective was to develop a modified PETase which improves degradation efficiency, and lays the foundation for scalable bioremediation. Therefore, our focus initially began by identifying key bottlenecks in the efficiency of PET degradation, mainly low enzyme-substrate affinity, poor enzyme stability, and an inefficient deployment in environmental settings.^[1] To address these, we designed a fusion protein system composed of HotPETase (BBa_25FWBRO7), CsgA (BBa_25HOIJ11) and BaCBM2 (BBa_25322080) genes. The plasmid consists of 3 core components and a linker: CsgA gene, producing a protein that forms curli fibers on the bacterial surface and serves as a scaffold for enzyme display^[2]; HotPETase gene, producing an enzyme that can steadily maintain its ability to break down PET plastic under elevated temperatures^[3]; BaCBM2 gene, a cellulose-binding module from Bacillus anthracis that increases PET binding affinity^[4]; and a flexible peptide linker composed of repeating (G4S)2 units (BBa_25ZERWUB, BBa_25WIHZ1B), which connect the genes together to preserve proper folding and enzymatic activity. When expressed, the HotPETase is expected to be displayed onto the curli fibers, coating the surface of E. coli with active HotPETase and enabling direct interaction with plastic substrates for enhanced degradation.

We also designed constructs expressing individual modules consisting of HotPETase alone, CsgA HotPETase, and HotPETase with BaCBM2 to know more about the contributions of each gene to the efficiency and performance of the final gene combination and to also identify possible conflicts between the genes.

Build

During the Build phase, we synthesized our gene constructs. Then, the plasmids were transformed into E. coli DH5α for cloning and BL21(DE3) for protein expression. Protein expression was induced with IPTG. After His-tag purification, SDS-PAGE was used to confirm protein size.

Test

In the Test phase, we conducted a series of assays to evaluate the performance of our engineered system. SDS-PAGE analysis showed no detectable bands at the expected molecular weight for our fusion constructs, suggesting a potential failure in protein expression, instability of the fusion protein, or rapid proteolytic degradation.^[5] Thermostability tests were conducted on the modified PETase across temperatures 25 ˚C, 60 ˚C, and 70 ˚C, however, the PET plastics demonstrated trivial degradation over time, as there was a negligible decrease in weight after each measurement. The absorbance of the solutions was also measured, but displayed abnormally high readings in absorbance initially before decreasing. Absorbance of the PBS solutions was measured from 220nm to 300nm to detect terephthalic acid (TPA) release.^[6] However, we observed abnormally high initial readings that decreased over time, suggesting potential interference from media components or cell debris.

Learn

From the Learn phase, we gained several insights that informed our next round of design improvements. While our initial tests were inconclusive, our design rationale suggested that surface display and the BaCBM2 gene should, in theory, enhance efficiency and binding. The critical failures we needed to address first were the lack of protein expression in SDS-PAGE and the unreliable microbial growth in YESCA medium, which prevented us from testing our core hypotheses. Our initial design assumed that the (G4S)2 linker would provide optimal flexibility.^[7] However, the lack of detected protein prevented us from validating this. The anomalous absorbance data led us to hypothesize that the in-house prepared YESCA medium was a key point of failure, potentially inhibiting consistent microbial growth. Also, the gel electrophoresis in SDS-PAGE demonstrated no bands formed from the His-tagged proteins, leading to the second cycle of testing.

Cycle 2

Design

Since our Build phase in Cycle 1 indicated that the transformation was successful as multiple bacterial colonies were formed, we proceeded with the original gene constructs. This allowed us to focus on troubleshooting the experimental and protein purification protocols instead of the DNA design.

YESCA Medium Refinement

The components of the medium were formed using raw materials, including dried yeast, peptone and sodium hydroxide. As such, the chance of the self-prepared YESCA medium containing hidden preservatives or being contaminated when stored is considerably high. Therefore, we suspected that the YESCA medium had been improperly prepared. To address this, we reformulated the medium with casamino acid and yeast extract and the stock was stored in a sealed container whenever not in use, minimizing the risk of contamination and unsuitable pH.

Gel Electrophoresis Enhancement

In parallel, our initial gel electrophoresis results for plasmid detection were inconclusive, with faint or missing bands. We hypothesized that low DNA concentration was the limiting factor. To tackle the issue, we doubled the concentration of extracted plasmid samples from 1:10 to 1:5 and optimized the loading volume. Protein testing was performed to ensure that His-tagged proteins were present in the sample before carrying out gel electrophoresis.

Build

The original gene constructs were reused.

Test

Scanning electron microscopy (SEM) revealed the formation of cracks and grooves on the PET surface, providing physical evidence consistent with degradation. However, the SDS-PAGE only confirmed the display of HotPETase on the cell surface. While this confirmed the successful expression of the fusion protein and the presence of the HotPETase domain, the same couldn’t be said for the other 3 proteins. Therefore, we tested the extracted samples using protein test strips, with all 4 His-tagged enzymes confirmed to be present. In addition, the second electrophoresis run to detect the presence of plasmids in E. coli, produced a singular, clear, and well-defined band for all plasmids besides CsgA-(G4S)2-HotPETase-(G4S)2-BaCBM2, which confirmed the successful transformation of E. coli and validated most of our constructs’ integrity. PET plastic assays demonstrated measurable degradation over time, with a decrease in mass observed over the experimental period. Larger and heavier pieces of PET plastic were used to reduce the percentage uncertainty of the assays. The revised YESCA formulation led to significantly higher absorbance values, confirming that medium quality was a decisive factor in surface display efficiency. The PBS solutions were centrifuged and the absorbances, from 220nm to 300nm^[6] , of supernatant were measured to ensure the increase in absorbance was due to the breakdown of PET into TPA and other smaller molecules instead of bacterial growth.

Learn

After the second cycle, much of our previous hypotheses, such as the subpar quality of the original YESCA medium and the concentration of plasmid were confirmed. The SEM also proved the action of the modified PETase towards PET. However, the SDS-PAGE for both cycles yielded unsatisfactory results. Therefore, we propose two key improvements for gel electrophoresis in SDS-PAGE. First, we should concentrate purified His-tagged protein samples by using a smaller elution volume during extraction to ensure a high enough concentration for clear visualization. Second, we should optimize the denaturation protocol for our thermostable fusion protein by testing higher heating temperatures such as over 90°C for longer durations, and the addition of denaturants to ensure complete unfolding. Considering that the HotPETase is thermostable even at 60 degrees celsius, the denaturing process is a crucial step in gel electrophoresis for SDS-PAGE and must be thoroughly designed to prevent poor separation.

To explain the lack of results for CsgA-(G4S)2-HotPETase-(G4S)2-BaCBM2, we offer 2 hypotheses: Firstly, the concentration of pellets for the CsgA-(G4S)2-HotPETase-(G4S)2-BaCBM2 was lower than that of the other fusion constructs. Secondly, the larger size of the fusion construct may lead to slower folding kinetics. We will go into further detail when analysing the results (direct to Results)

Future Directions

To resolve the persistent SDS-PAGE issues, a future cycle will design and test an optimized protein analysis workflow, including sample concentration and sensitive detection methods like Silver Staining. Looking ahead, our Future Directions include testing our enzymes in real world conditions to investigate the ability to effectively degrade PET plastics and integrating our engineered E. coli into bioreactors or environmental platforms. These steps will help us move closer to deploying a sustainable, biologically-driven solution to plastic pollution.

Transformation Success

The four plasmids, each containing a different gene, were successfully transformed into E. coli. This was confirmed by the growth of multiple bacterial colonies on kanamycin-containing agar plates, indicating that the cells had acquired the antibiotic resistance gene from the plasmid.

Figure 1. E. coli colonies after transformation with the plasmid containing HotPETase (BBa_25OAK6TB). The "B" plate shows the BL21(DE3) strain, and the "D" plate shows the DH5α strain.

Figure 2. E. coli colonies after transformation with the plasmid containing CsgA-HotPETase (BBa_252IGL39). The "B" plate shows the BL21(DE3) strain, and the "D" plate shows the DH5α strain.

Figure 3. E. coli colonies after transformation with the plasmid containing HotPETase-BaCBM2 (BBa_25IHX9R7). The "B" plate shows the BL21(DE3) strain, and the "D" plate shows the DH5α strain.

Figure 4. E. coli colonies after transformation with the plasmid containing CsgA-HotPETase-BaCBM2 1576ng (BBa_251QH76Y). The "B" plate shows the BL21(DE3) strain, and the "D" plate shows the DH5α strain.

References

[1] Satta, A., Zampieri, G., Loprete, G. et al. Metabolic and enzymatic engineering strategies for polyethylene terephthalate degradation and valorization. Rev Environ Sci Biotechnol 23, 351–383 (2024). https://doi.org/10.1007/s11157-024-09688-1

[2] Wang, C., Long, R., Lin, X., Liu, W., Zhu, L., & Jiang, L. (2024). Development and characterization of a bacterial enzyme cascade reaction system for efficient and stable PET degradation. Journal of Hazardous Materials, 472, 134480. https://doi.org/10.1016/j.jhazmat.2024.134480

[3] 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

[4] Wang, T., Yang, W., Gong, Y., Zhang, Y., Fan, X., Wang, G., Lu, Z., Liu, F., Liu, X., & Zhu, Y. (2024b). Molecular engineering of PETase for efficient PET biodegradation. Ecotoxicology and Environmental Safety, 280, 116540. https://doi.org/10.1016/j.ecoenv.2024.116540

[5] Mtoz Biolabs https://www.mtoz-biolabs.com/why-are-protein-bands-not-visible-after-destaining-in-sds-page.html

[6] 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

[7] Joshua S. Klein, Siduo Jiang, Rachel P. Galimidi, Jennifer R. Keeffe, Pamela J. Bjorkman, Design and characterization of structured protein linkers with differing flexibilities, Protein Engineering, Design and Selection, Volume 27, Issue 10, October 2014, Pages 325–330, https://doi.org/10.1093/protein/gzu043