Overview

We consistently adhere to the Design-Build-Test-Learn Cycle methodology to advance our project step by step. This year's project can be broken down into five distinct cycles. In the first cycle, we successfully constructed and tested the PETase synthesis circuit in an E. coli chassis. In the second cycle, in order to improve the PETase efficiency, we use AI tools to generate new PETase mutants and tested in E. coli. In the third cycle, to maintain continued production and functioning of PETase, we designed a signal peptide to allow for efficient extracellular excretion of PETase. In the fourth cycle, we further introduced the PETase into cyanobacteria. Finally, in the fifth cycle, we designed and tailored bioreactors to support the optimized growth and productivity of the engineered cyanobacteria.

Figure 1. Engineering cycle diagram of our project

Cycle 1: PETase from iGEM Distribution Kit

Design

Our goal is to identify enzymes capable of degrading PET at ambient temperatures. Initially, we utilized the PETase available in the iGEM distribution kit and tested their efficiency in PET degradation. Additionally, we took this chance to establish a standardized testing process for PET degradation: measuring the weight loss of E. coli-treated PET particles and a testing method outlined by a previous iGEM team.

Build

From the iGEM distribution kit, we identified five PETase available for use: BBa_K2910000, BBa_K2910003, BBa_J428065, BBa_J428067 and BBa_K3039002. After obtaining these parts, we assembled them into plasmids using the pET28a vector and transformed then into E. coli BL21(DE3).

Figure 2. Snapgene diagrams of the five plasmids constructed

Test

We verified successful plasmid construction through gel electrophoresis.
Figure 3. Gel electrophoresis results
After transforming the plasmid into E. coli, we tested for its efficiency by measuring the weight loss of a 1g PET plastic film following a two-week incubation in our E. coli culture. However, we found out that there were no significant differences prior and post the E. coli treatment.
Figure 4. Graph of initial and final weight.
For additional testing, we adopted the method used by the team iGEM19_Toronto: p-nitrophenyl butryte degradation assay. This assay measures the activity level of PETase through monitoring light absorption at 415 nm. PETase depolymerizes p-nitrophenyl butyrate producing the monomers butyric acid and p-nitrophenolate, which absorb light at a 415nm wavelength. Hence, greater 415nm light absorption correlates with higher enzyme activity. (see protocol)
Figure 5. Diagram of p-nitrophenyl butyrate degradation assay

We obtained positive results when testing with samples extracted from our modified E. coli culture. The relative enzyme efficiency (A415/protein concentration) that we are looking at takes into consideration both the efficiency of the enzyme itself and the PETase synthesis rate of the chassis, since our end goal is to implement the engineered organism in a self-sufficient PET degrading system as a whole.

Figure 6. Result of p-nitrophenyl butyrate degradation assay.

Learn

Our gel results and p-nitrophenyl butryte assay demonstrated that we successfully constructed plasmids that encode PETase in E. coli capable of cleaving the ester bonds in PET polymers. Additionally, we confirmed the p-nitrophenyl butryte assay to be an effective method for measuring PETase activity. However, we were not able to detect any changes in the weight of PET plastic after incubating it with our modified E. coli. We hypothesized that this was due to two factors: (1) the enzymes were not very effective at degrading PET microplastics, and (2) PET plastics have varying degrees of crystallinity depending on their synthesis process and physical aging.

Cycle 2: AI Generated Mutants

Design

Following the completion of Cycle 1, we aimed to further enhance the efficiency and effectiveness of PET degradation by PETase through identifying variants of the PETase enzyme. In a discussion with Professor Qi, we were inspired by his insights on the PETase mutant — LCC-A2, which lead us to explore the potential of improving degradation efficiencies through mutations. Building on to this idea, in addition to reviewing existing literature to identify PETase variants, we also employed AI tools we trained to generate mutants that could possibly result in enzymes that surpass the performance of wild-type PETase.

Build

After conducting research, we identified a PETase variant — BhrPETase. The sequence of BhrPETase was identified by the Shingo group in a metagenomic study on uncultured thermophiles, and was deposited into the NCBI database by the group in 2018 and annotated as a PET hydrolase [1]. Together with the previous PETase obtained from the iGEM distribution kit, we created a series of mutants for both IsPETase and BhrPETase using an AI-assisted protein design tool (see model). These sequences were expressed in E. coli using the pET28a vector.

Figure 7. Table of mutated version of IsPETase/BhrPETase

Test

We successfully verified the construction of plasmids containing the mutated sequences using gel electrophoresis.

Figure 8. Gel electrophoresis Results

The p-nitrophenyl butryte degradation assay, on the other hand, demonstrated that the mutated variants of PETase outperformed wild-type PETase.

Figure 9. Result of of p-nitrophenyl butyrate degradation assay

Learn

From the results we obtained, we determined that certain mutated variants designed by our AI tool are more effective in degrading PET. Yet, we realized a few drawbacks. Firstly, although the p-nitrophenyl butryte degradation assay is a viable method for detecting the hydrolysis of ester bonds, it does not specifically measure the actual degradation of PET. Secondly, lysing the E. coli cells was required when testing for the activity of the enzymes. However, considering that we desire to develop a self-sustaining PET degrading system, breaking down the cells to release the enzyme is an unfeasible method.

Cycle 3: Signal Peptide

Design

With the drawbacks in mind, we consulted Dr. Wang in search for solutions. He suggested the use of signal peptides for the secretion of the enzymes. Following his advice, we inserted an additional sequence into our plasmid design that encoded a signal peptide, which allows the enzyme to exit without causing any disturbance to the cell.

Build

ChatGPT provided us with several preliminary signal peptide sequences.

Figure 10. Answer from ChatGPT

After reading more papers and doing more research, we decided to use pelB as our signal peptide; when fused to the front end of a protein, this signal peptide allows the protein to be transported across the two membranes of E. coli. Plasmids were reconstructed with this sequence inserted before the enzyme sequence.

Figure 11. Diagram of new plasmid including the signal peptide sequences

Test

The effectiveness of the signal peptides was measured using the p-nitrophenyl butryte degradation assay. We extracted cultures from both E. coli containing the newly designed plasmid and E. coli containing the original plasmid. After centrifuging the samples, we retained the supernatant for the assay. Theoretically, enzymes with the additional signal peptide will be able to leave the cell and be present in the supernatant, thus result in better enzyme activity. Final results indicated that signal peptides allowed for the secretion of the enzymes.

Figure 12. Result of of p-nitrophenyl butyrate degradation assay

Subsequently, to verify the enzymes secreted were in fact the PETase enzymes, we performed SDS-PAGE.

Figure 13. SDS-PAGE results

In hopes of detecting PETase activity through a more intuitive approach, we utilized a scanning electron microscope (SEM) to directly observe the degradation of PET. Different plastic samples were placed in the culture medium of the engineered E. Coli. After two weeks, the samples were observed under an SEM for any alterations in the surface of the plastic by technicians at Shenzhen University. The results demonstrated that plastic with low level of crystallinity were degraded under the exposure to PETase synthesized by our engineered E. coli. Further, the fact that plastic with high crystallinity did not show any significant changes addresses our hypothesis in Cycle 1: PET degradation is affected by the crystallinity of the plastic, which varies depending on its manufacturing process.

Figure 14. Results from SEM

Learn

Upon analyzing our results, we determined that the signal peptide pelB successfully enables the secretion of the actual PETase synthesized within then engineered E. coli cells. Using a SEM we were also able to detect the degradation of low crystallinity plastics by the secreted enzymes. Still, the results are not sufficient; the alteration of the plastic surfaces might also be due to physical damages and not chemical degradation. Additionally, we realized another problem concerning the use of E. coli as our chassis. To ensure the E. coli cells are still living, we fed them with growth medium. However, this contradicts with our vision of a fully self-sustaining PET degradation system.

Cycle 4: E. coli to Cyanobacteria

Design

Following the progress in cycle 3 and considering the problems identified, we consulted more experts and decided to use a new chassis — cyanobacteria Synechococcus elongatus PCC 7942. Hence, we reconstructed our plasmids using a transfer vector that transport the target gene into cyanobacteria to be incorporated into its genome.

Build

The plasmids were reconstructed using a transfer vector, with the signal peptide and PETase sequence inserted between promoter PpsbA2 and terminator Bba_B0015.

Figure 15. Snapgene diagram of plasmid for cyanobacteria

Test

The reconstructed plasmids were transformed into cyanobacteria and coated on BG11 plates containing the antibiotic spectacular ionomycin. Single colonies appeared after two weeks of incubation.

Figure 16. Cyanobacteria culture plate

The single colonies were cultured and samples were extracted for the p-nitrophenyl butryte degradation assay. Results demonstrated that the engineered cyanobacteria can synthesize active PETases.

Figure 17. Result of of p-nitrophenyl butyrate degradation assay

To address the limitations existing in our previous methods for measuring the effectiveness of PETase in degrading PET, we performed high performance liquid chromatography to detect the amount of PET monomers — tetrephtalic acid (TPA) — present after treatment of PET with engineered E. coli and cyanobacteria. Samples were prepared and sent to technicians at Shenzhen Univeristy, whom performed HPLC. The results, were positive for both.

Figure 18. HPCL result

Learn

We concluded that cyanobacteria is a suitable chassis for PETase synthesis. We also obtained positive results from the alternative PET degradation testing method, HPLC, directly indicating that the PETase synthesized are capable of breaking down the polymer PET into its monomers. Yet, our end goal is to achieve complete degradation of PET into harmless, naturally occurring compounds. Moreover, our current engineered organisms lack a clear application scenario.

Cycle 5.1: Co-culture System

Design

Upon doing more research, we learned that further degradation of terephthalic acid (TPA) into carbon dioxide and water can be achieved by the bacteria Rhodococcus jostii. This led us to consider co-culturing cyanobacteria with Rhodococcus to attain further degradation of PET.

Build

To culture the two different organism together, their respective growth mediums, R2A for rhodobacteria and BG-11 for cyanobacteria, were mixed together in different ratios: 1:1, 1:2, and 1:3 (R2A: BG-11).

Test

During the two week incubation, pictures were taken weekly to observe the growth of the rhodobacteria and cyanobacteria. Results demonstrate the the organisms performed the best at 1:3 R2A to BG-11 ratio.

Figure 19. Cyanobacteria and Rhodococcus co-culture results

Learn

We determined that cyanobacteria can be co-cultured with Rhodococcus jostii.

Cycle 5.2: Bioreactors

Design

After successfully designing a PET degrading biological system, we aim to give it a real life application through implementing our co-culture system in bioreactors. Involving discussions with our design group, and after interviews with experts Dr. Fu and Dr. Zhou, we created several bioreactor design.

Figure 20. Bioreactor designs

Build

Out of the multiple designs, a few were constructed.

Figure 21. Hardware

Test

Out of the ones constructed, some demonstrated to be viable, such as the life-based version.

Figure 22. Life-based hardware

Learn

Although some of our bioreactor designs are feasible, there still exists some limitations. Further improvement still needs to be made in the future. (see hardware for more details)