Abstract

This part was designed for the construction of Whole-cell Biocatalysts "BIND-bearPETase." Waseda-Tokyo2024 thoroughly investigated its functionality through wet lab experiments, energetic simulations and mathematical modeling. Additionally, this part holds great value for the iGEM community by addressing the urgent need for better plastic waste management and expanding any enzyme availability.

Agenda(Click to reach)⍝ʕ´•ᴥ•`ʔ⍝

1. Overview
2. Components
3. Cloning & Expression
4. Wet Lab Characterization
• Curli Fiber Formation Assay
• pNPB Hydrolysis Assay
• Storage Activity Assay
• Reusability Assay
• Plastic Pellet Degradation Assay
• PET Bottle Powder Degradation Assay
5. In Silico Energy Simulation
• Affinity Simulation
• Stability Simulation
6. Mathematical Modeling
• Membrane Transport Model
• Curli Fiber-Associated PET Degradation Model
7. Conclusion

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Overview ʕっ•ᴥ•ʔっ♡

This "BIND-bearPETase" offers benefits that address the shortcomings of conventional free PETase shown below.

BearPETase has two meanings. The first comes from the verb “bear,” as BearPETase demonstrates strength in the stability of enzymes and can “bear” burdens. The second meaning relates to the cute animal mascot “Waseda Bear” of our school! ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ

This part encodes the CsgA-bearPETase fusion protein. CsgA is an extracellular fibrous structure-forming factor that constructs Curli Fibers on the surface of the E. coli membrane. By fusing bearPETase to CsgA, we enabled the presentation of bearPETase on the cell membrane surface in a fiber-linked manner.

This enables direct access to substrates without the need for purification, as well as the stabilization of enzyme activity and the reuse of enzymes. This is a technique referred to as the BIND-System ^[1], and whole-cell biocatalysts equipped with PETase are called BIND-PETase ^[2].

The key effort in this part was creating “bearPETase” ,the optimal PETase for the BIND-System. BearPETase, uniquely developed by Waseda-Tokyo 2024, combines mutations from depoPETase (Shi et al., 2023) ^[3] and duraPETase (Cui et al., 2021) ^[4] developed through directed evolution.

We, Waseda-Tokyo2024 thoroughly investigated the characteristics of BIND-bearPETase using a variety of scientific methods; wet lab experiments, mathematical modeling, and energetic simulations.

Furthermore, this part significantly contributes to the iGEM community by expanding enzyme availability. As mentioned above, the BIND-System reduces concerns about purification costs and quality, making them negligible. It also allows for maintaining and reusing proteins with unstable activity. By replacing the bearPETase portion with other BioBricks, any enzyme's use can be simplified.

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Components ʕ – ᴥ – ʔ

I. Optimized RBS for BIND-System (Waseda-Tokyo2024, BBa_K5436005)
This RBS is designed to efficiently drive the BIND-System. In some existing BioBricks, inappropriate RBS strength can either overload E. coli with excessive expression or result in no expression. We've designed an RBS to optimize the amount of CsgA displayed on E. coli’s surface as components of Curli Fibers, which will support future iGEMers using the BIND-System.
II. csgA-taa(Waseda-Tokyo2024, BBa_K5436006)
CsgA-taa is a modified version of BBa_K1583000from iGEM15_TU_Delft, with the stop codon removed, enabling the expression of the desired protein in a fused state after the Curli Fiber formation factor CsgA.
III. BamHI_Linker (Waseda-Tokyo2024, BBa_K5436020)
This uses the BamHI recognition site, which consists of 6 nucleotides, directly as a linker. The BamHI recognition site encodes glycine and serine, which are commonly used amino acids in linker sequences.
IV. bearPETase (Waseda-Tokyo2024, BBa_K5436015)
BearPETase was rationally designed by Waseda-Tokyo 2024 to enhance its enzymatic activity. As shown below, we confirmed that its enzymatic activity surpassed that of existing variants. The existing PETase variants include depoPETase and duraPETase, and combining both was expected to improve enzymatic activity. Based on that consideration, we created 81 combinations, excluding the overlapping mutations Q119Y and Q119R, and generated 3D structures using AlphaFold 2, selecting those with stable structures.
V. 6x HisTag (Waseda-Tokyo2024, BBa_K5436021)
It is useful in protein purification and also beneficial for Western blotting, where anti-His Tag antibodies are used as primary antibodies.

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Cloning & Expression ʕ ´•̥̥̥ ᴥ•̥̥̥`ʔ

Designing RBS for BIND-System

The "Optimized RBS for BIND-System (BBa_K5436005)" included in this part was carefully designed by the RBS Calculator from Salis Lab^[5], rather than reusing an existing RBS. Existing RBS used in previous CsgA overexpression experiments did not meet our criteria. The RBS included in the pRha + CsgA (BBa_K1583100) developed by iGEM15_TU_Delft had a transcriptional rate of 40.80, which was insufficient for the expression levels we required. On the other hand, the transcriptional rate of the RBS in Rec-PhoA/CsgA (Addgene #170787)^[6] was approximately 700, and it appeared to meet our requirements. Referring to that order of magnitude, we newly designed an RBS for BIND-PETase (WT) with a transcriptional rate of 800 using the RBS Calculator.

As mentioned later, this optimized RBS was sufficient to induce the expression of BIND-bearPETase.

Molecular Cloning

We used NEBuilder HiFi DNA Assembly ^[7] to obtain plasmids encoding BIND-bearPETase. The DNA fragments encoding bearPETase were prepared with Gene Fragments Synthesis Service (Twist Bioscience).

After culturing and miniprepping, we ran electrophoresis, observing bands near the expected size. Sequence analysis confirmed the correct plasmid sequences.

Western Blotting

Samples induced for the expression of CsgA-bearPETase by IPTG were lysed, and when subjected to Western Blotting using His-Tag as the primary antibody, a clear band was observed around 45 kDa, confirming the overexpression of the target protein. For detailed protocols of the lysis, refer to our wiki, Experiments.

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Wet Lab Characterization ʕ≧ᴥ≦ʔ

A total of 7 wet experiments were conducted to thoroughly investigate the function of BIND-bearPETase. During this process, we compared BIND-bearPETase with its ancestor sequence BIND-PETase (WT) (BBa_K5436130), BIND-duraPETase (BBa_K5436133), and BIND-PETase (ID23) (BBa_K5436123), which is created with a similar design strategy. The results are documented below.

On the Wiki, BIND-bearPETase was evaluated by comparing it with some variants not shown here. The process is detailed in the Engineering Success section of our wiki.

Curli Fiber Formation Assay

The formation of Curli Fibers of BIND-bearPETase was quantitatively measured. Whether Curli Fibers are formed correctly is crucial for the enzyme's stability and reusability.

After centrifuging the BIND-bearPETase suspension, the resulting pellet exhibited a robust structure that did not break apart even after multiple pipetting, as shown in Fig. 8. This suggests that the formation of Curli Fibers due to the overexpression of CsgA-bearPETase led to the development of a biofilm structure in E. coli.

In the Curli Fiber Formation Assay, Congo Red dye is used to stain Curli Fibers, followed by centrifugation to form a pellet. Subsequently, the absorbance of the supernatant is measured to quantify the formation of Curli Fibers. If the Congo Red dye is incorporated into the pellet and the supernatant appears pale, it can be confirmed that Curli Fibers have been properly formed.

The results of Congo Red staining for BIND-bearPETase are shown in Fig. 9. It can be observed that Curli Fibers are formed and stained in a manner dependent on the presence of BIND-bearPETase.

Next, the absorbance of the supernatant was measured and compared between BIND-bearPETase and other variants (Fig. 10).

Although BIND-bearPETase exhibited lower Curli Fiber formation ability compared to BIND-PETase (WT), it had a higher Curli Fiber formation ability than BIND-duraPETase, which is ancient of BIND-bearPETase. Additionally, it was found that BIND-bearPETase and BIND-PETase (ID23) possess a similar level of Curli Fiber formation ability.

Based on these results, it can be concluded that bearPETase is more suited for the BIND-System in terms of Curli Fiber formation ability among the many improved PETases.

pNPB Hydrolysis Assay

The activity of BIND-bearPETase was investigated in an easy way(Fig. 11). Para-nitrophenyl butyrate (pNPB) produces yellow para-nitrophenol (pNP) upon hydrolysis, and we measured this product. However, the magnitude of hydrolytic activity against pNPB does not necessarily correspond to the activity against PET polymers.
Therefore, it is important to note that the pNPB Hydrolysis Assay only provides a simplified assessment of activity. (As will be discussed in the PET Bottle Powder Degradation Assay section, BIND-bearPETase demonstrated the highest practical degradation of PET among these variants.)

It was confirmed that the activities of BIND-bearPETase and BIND-PETase (ID23) increased compared to their ancestors, BIND-PETase (WT) and BIND-duraPETase. BIND-bearPETase and BIND-PETase (ID23) designed by Waseda-Tokyo demonstrated superior performance, suggesting they possess more advantageous features for the practical application of PETase.

Storage Activity Assay & Reusability Assay

Here, we document the experimental results that verify the strengths of BIND-bearPETase regarding the stability and reusability of the enzyme in the social implementation of PETase.

Storage Activity Assay

Since various BIND-PETases are whole-cell biocatalysts utilizing live E. coli, proper storage conditions allow for protein expression and bacterial growth, which can maintain or enhance their activity.
The activities of BIND-bearPETase were evaluated on days 0, 5, and 11 after expression using the pNPB Hydrolysis Assay (Fig. 13). Additionally, we assessed the increase in activity when the storage temperature was changed to either 4°C or room temperature.

During storage, both BIND-bearPETase and BIND-PETase (ID23) exhibited a greater increase in activity over time compared to BIND-PETase (WT) and BIND-duraPETase.This is believed to be due to the nature of the whole-cell biocatalyst, which may have allowed cell proliferation and protein expression during the storage period.

However, we cannot conclude our analysis for further characterization of the part is necessary. One point to consider is that while some samples showed increased activity after storage, others did not.

We hypothesized that there are various factors that inhibit BIND-PETase activity in a temperature-dependent manner. The following are potential factors.

• Denaturation of PETase
• Degradation of PETase
• Detachment of Curli Fiber from E. coli body
• Detachment of PETase from Curli Fiber

Assuming that the rates of these factors inhibiting BIND-PETase activity are temperature-dependent, this becomes a reasonable hypothesis. If we assume the relationship shown in Fig. 14 exists, the results of Fig. 13 are consistent.

Based on this hypothesis, BIND-bearPETase is considered to have a Storage Activity that is advantageous for industrial applications. In BIND-PETase (WT), the rate of activity-inhibiting factors exceeded the growth rate of E. coli and the rate of PETase expression. However, in the improved BIND-bearPETase, the structural stability of PETase was enhanced, making it less susceptible to the effects of the activity-inhibiting factors, which likely led to the observed increase in activity.

The same trend can likely be observed in the Reusability Assay, which will be discussed later.

Reusability Assay

BIND-bearPETase could be reused three times after a single reaction, with the presence of activity confirmed through the pNPB Hydrolysis Assay. The activity after reuse was also observed for BIND-PETase (WT) and other variants (Fig. 15).

It was observed that the activity of BIND-PETase except for WT increased after reuse. Although this may be due to the contamination of the reaction product, pNP, during the collecting stage of BIND-PETases, we attempted to conduct washing operations as thoroughly as possible to achieve the most accurate measurements. Additionally, the promotion of PETase enzyme folding due to the initial reaction may also contribute to the observed increase in activity.

BIND-duraPETase, BIND-PETase (ID23), and BIND-bearPETase exhibited an increase in activity during reuse. While the exact reasons for the activity increase upon reuse could not be identified, it was confirmed that at least BIND-bearPETase does not significantly lose activity even after reuse, indicating its advantage for practical applications.

Plastic Pellet Degradation Assay

Furthermore, Waseda-Tokyo 2024 evaluated the practical degradation activity of BIND-bearPETase with the aim of utilizing this part outside the lab.

In this assay, composite plastic pellets (PETPEPP) used in actual recycling plants and single-material pellets (PET(N)) were utilized as substrates. After adding BIND-bearPETase suspension to the reaction system at pH 7.0 and pH 9.0 and allowing it to act for five days, mass reduction was confirmed in both types of pellets. The negative control did not show any weight loss (data not shown). For comparison, BIND-PETase (ID23) was also included.

It was demonstrated that BIND-bearPETase and BIND-PETase(ID23) are capable of degrading the pellets. However, due to the pellets' heterogeneity, quantitative experiments are needed for accurate activity comparisons between variants.

The pellets were provided by the recycling company esa Inc., and we would like to take this opportunity to express our gratitude. For more detail, refer to our wiki, IHP.

PET Bottle Powder Degradation Assay

Next, we conducted HPLC analysis using PET bottle powder to perform a more quantitative comparison of BIND-bearPETase activity with other variants. In the previously mentioned pellet degradation experiments, the heterogeneity of the pellets made it difficult to accurately compare enzyme activities. Therefore, quantitative validation was crucial.

It was confirmed that BIND-bearPETase possesses the highest practical activity against PET powder compared to other variants.
PETase decomposes the PET polymer, resulting in the formation of TPA, MHET, and BHET (Fig. 17).

Waseda-Tokyo 2024 quantified the products TPA, MHET, and BHET, generated by BIND-bearPETase, using High-Performance Liquid Chromatography (HPLC).
PET bottles, commonly used in everyday life, were ground with sandpaper, and BIND-bearPETase was applied.

In addition to pH 7.0, the reaction was also carried out at pH 9.0, as many PETases are reported to have optimal conditions at pH 8.5 or higher ^[9]. The results were measured 1 day and 3 days after the reaction.

In this way, it was confirmed that the products TPA, MHET, and BHET were generated by BIND-bearPETase. Additionally, it was suggested that the optimal pH for BIND-bearPETase is also pH 9.0.

Furthermore, we quantitatively compared the amounts of these degradation products (Fig. 20). Contrary to the pNPB hydrolysis assay mentioned earlier, BIND-bearPETase degraded PET bottle powder more effectively than BIND-PETase (ID23). BIND-bearPETase exhibited 10 times the activity of its ancestor BIND-duraPETase and 1.5 times that of its sibling BIND-PETase (ID23). These findings suggest that bearPETase, developed by Waseda-Tokyo, is well-suited for the BIND-System and demonstrates high practical activity.

Summary of Wet Lab Characterization

We showed that this part works as expected through a series of distinctive experiments. This evidence suggests that..

• Demonstrates usefulness to the community with potential for societal applications.
• Exhibits stability and reusability of the enzyme when tested in a device.
• Effectively works with substrates based on industrial plants and commonly available materials.

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In Silico Energy Simulation ᕦʕ •ᴥ•ʔᕤ

Since thermodynamic validations, such as free energy and docking energy, cannot be performed through wet experiments, we used the following tools to evaluate its affinity for PET and stability more effectively:

• AutoDock Vina ^[10]
• PyRosetta ^[11]
• FoldX ^[12]

We can assess binding affinity from the energy values provided by AutoDock Vina, where lower energy indicates higher binding affinity. Higher binding affinity suggests greater activity in actual wet-lab experiments. PyRosetta outputs score values that allow us to evaluate the structural stability of bearPETase. FoldX also provides energy values that help assess bearPETase's stability.

Affinity Simulation

Method

In Affinity Simulation, we performed molecular docking using AutoDock Vina, preparing the PDBQT files of PET dimer along with those of PETase (WT), duraPETase, PETase (ID23), and bearPETase. The reason we conducted the validation on PETase instead of BIND-PETase is that we focused on the PETase domain, which is directly related to enzymatic activity. Using the energy values output by AutoDock Vina, we evaluated the binding affinity of bearPETase to the PET dimer.

Results

The energy values output by AutoDock Vina from the molecular docking performed on each PETase are shown in Table 1. The graph is presented in Fig. 21. Note that in Fig. 21, the values are plotted with the negative values facing upwards.

It was confirmed that bearPETase has higher binding affinity than PETase(WT) and duraPETase. This suggests that it is likely to exhibit higher activity in wet experiments as well. In fact, as shown in Fig. 20 of PET Powder Degradation Assay section, BIND-bearPETase demonstrates greater activity compared to BIND-duraPETase, consistent with the simulation results. However, the energy value for bearPETase is the same as that of PETase (ID23), which contradicts the results shown in Fig. 20. This discrepancy requires further discussion. The process by which PETase breaks down PET molecules is as follows ^[13].

1. PET molecules dock onto PETase.
2. PETase breaks down the PET molecules.
3. The PET molecules are released from PETase.

However, molecular docking tools like AutoDock Vina only simulate the first stage, where PET molecules dock onto PETase. Therefore, it is likely that the differences in results observed in Fig. 20 and Fig. 21 are influenced by stages 2 and 3.

Nonetheless, we can at least conclude that bearPETase is expected to exhibit higher activity than PETase (WT) and duraPETase.

Finally, we visualized the docking interaction between bearPETase and the PET molecule using PyMOL ^[14] (Fig. 22, Fig. 23).

In Fig. 22 and Fig. 23, the red dots indicate the binding site of BIND-bearPETase. The PET molecule is successfully bound to this binding site, visually confirming the high binding affinity of BIND-bearPETase.

From these results, it is demonstrated from a computational simulation perspective that BIND-bearPETase has higher binding affinity than BIND-PETase (WT) and its ancestor, BIND-duraPETase.

Stability Simulation

Method

To evaluate the whole structural stability of the fusion protein BIND-bearPETase, we conducted validation using PyRosetta and FoldX. For this, we input the PDB files of BIND-PETase (WT), BIND-duraPETase, BIND-PETase (ID23), and BIND-bearPETase. By comparing the score values output by PyRosetta and the Gibbs free energy output by FoldX, we assessed the structural stability of BIND-bearPETase in relation to the others.

Results

First, the score values output by PyRosetta are shown below (Fig. 24, Table 2). Note that in Fig. 24, the negative values are plotted upwards. Additionally, the values represent the output after each BIND-PETase structure was optimized to minimize energy before being input into PyRosetta.

Our analysis from Table 2 and Fig. 24 shows that the PyRosetta score for BIND-bearPETase is lower than that of BIND-PETase(WT) and BIND-duraPETase. This indicates that the structure of BIND-bearPETase is more stable compared to BIND-PETase(WT) and BIND-duraPETase. However, the PyRosetta score for BIND-bearPETase is higher than that of BIND-PETase(ID23), suggesting that BIND-bearPETase is less stable than BIND-PETase(ID23).

Next, the Gibbs free energy values output by FoldX for each BIND-PETase variant are shown in Table 3. The corresponding graph is presented in Fig. 25. Note that, unlike Fig. 21 and Fig. 24, the values in Fig. 25 increase as we move up the graph.

From Table 3 and Fig. 25, we observe that the Gibbs free energy value output by FoldX for BIND-bearPETase is the lowest. This indicates that BIND-bearPETase has the most stable structure among the variants

The results of our validation using PyRosetta and FoldX indicate that the BIND-PETase variant with the most stable structure differed between the two methods. Based on these simulation results alone, it is not possible to conclusively determine which structure is more stable, BIND-PETase(ID23) or BIND-bearPETase. To resolve this, additional validation using other sophisticated computational techniques, such as Molecular Dynamics (MD), would be necessary.

Nevertheless, based on the PyRosetta score and the Gibbs free energy values from FoldX, BIND-bearPETase has been shown to have a more stable structure compared to BIND-PETase(WT) and its ancestral variant, BIND-duraPETase.

As demonstrated through wet experiments, protein structural stability is a significant factor for BIND-bearPETase's storage activity and reusability. Thus, the validation using PyRosetta and FoldX supports the superior structural stability of BIND-bearPETase over the previously developed BIND-PETase variants.

Summary of In Silico Energy Simulation

In this chapter, we demonstrated the following:

• Using AutoDock Vina, we showed the binding affinity of bearPETase, suggesting the potential for high activity in wet experiments.
• Through PyRosetta and FoldX, we confirmed that the structure of BIND-bearPETase is stable.

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Mathematical Model for Functional Analysis ʕ·͡ᴥ፝֟·ʔ

Since we found it difficult to study the mechanism of PET degradation by BIND-bearPETase through wet lab experiments alone, the modeling approach was indispensable. Based on the following results, we gained a better understanding of the PET degradation mechanism by BIND-bearPETase. The details are explained below.

The validation of this composite part using mathematical modeling is divided into two stages. The 1st stage, the "Membrane Transport Model," quantifies the process of BIND-bearPETase being transported outside the membrane. The 2nd stage, the "Curli Fiber-Associated PET Degradation Model," quantifies the efficiency of bearPETase, fused with Curli fibers, in acting on and degrading PET molecules.

Membrane Transport Model

Here, we used mathematical formulas to simulate the transport of BIND-bearPETase, and estimate the expression level of Curli Fibers formed by the surface display.

The Membrane Transport Model provides important insights into the extracellular transport of BIND-bearPETase, which cannot be understood through wet-lab experiments. While the formation of Curli fibers can be quantified, the wet experiment ”Curli Fiber Formation Assay” with Congo Red Dye does not account for BIND-bearPETase that was transported outside but did not form Curli fibers, or that were formed but detached from E. coli. A model that takes all of these factors into consideration could be useful for a more detailed analysis of experimental results.

From this model, the concentration of extracellular CsgA(BIND-bearPETase) and B were found to be gradually increasing. The specific formulas and results are shown in our wiki(Model).

Curli Fiber-Associated PET Degradation Model

We demonstrated that this part works as expected by modeling the mechanism by which BIND-bearPETase on the E. coli membrane degrades PET polymers. The specific assumptions and formulas are shown in our wiki (Model).

It is clearly shown that the length of the PET R(t) and r_j(t) will decrease and converge to zero, which does not contradict our prediction. Thus, the model for PET degradation surely represents the degradation of PET by BIND-bearPETase on Curli Fiber, which means this mathematical model has been proven to be quantitative and valid.

Summary of Mathematical Model for Functional Analysis

Our modeling data indicates that it is useful to the community by providing insights into the efficiency of this degradation process. We characterized this part by predicting the time-dependent changes in PET degradation.

Conclusion ʕ •ɷ•ʔฅ

We, Waseda-Tokyo 2024 team has developed a novel enzyme system called "BIND-bearPETase," which makes the use of PETase more accessible and efficient. This technology can also be applied to other enzymes, suggesting that the BIND-System can reduce enzyme purification costs and improve convenience.

In Wet Experiments, it was confirmed that BIND-bearPETase has higher hydrolytic activity compared to other BIND-PETase variants. Additionally, experiments verified that BIND-bearPETase does not require purification, can be stored for approximately two weeks, and can be reused up to 3 times. Furthermore, it was demonstrated that BIND-bearPETase can be applied to PET from everyday PET bottles, showcasing the practical potential of this part.

In Dry Experiments, energy simulations were used to verify the stability of PETase-PET docking and the structural stability, which could not be confirmed in Wet Experiments. For deeper characterization of part functions, a mathematical model allowed for the examination of outer membrane transport and Curli Fiber-Associated PET Degradation.

The detailed documentation of BIND-bearPETase will serve as a crucial guide for future iGEMers who wish to use or modify and apply this system.

ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ ʕ•ᴥ•ʔ

References