iGEM 2025 — Team hwfla-beijing

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"title": "",
"content": '

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **2024, December** | | | | |
|  | First week | Second week | Third week | Fourth week |
| Theory course |  |  |  |  |
| Background  research |  |  |  |  |
| Recruit members |  |  |  |  |

Theory course: Background introduction to the iGEM
challenge

![图片1](https://static.igem.wiki/teams/5748/notebook/1.webp)

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **2025, January** | | | | |
|  | First week | Second week | Third week | Fourth week |
| Theory course |  |  |  |  |
| Topic discussion |  |  |  |  |
| Recruit members |  |  |  |  |
| Material purchase |  |  |  |  |

1. Theory course: Basic research methods of modern molecular biology.
2. Topic discussion: Briefly introduce igem background and scoring criteria, give topic

selection and introduction

3. Theory course: Social Insight and Market Exploration: The art of questionnaire research and interview

![](https://static.igem.wiki/teams/5748/notebook/2.webp)

1. Project discussion: Give a project selection speech and vote for the final project.
2. Theory course: The Art of popular Science Communication: Creating a concise and logical scientific dialogue
3. Theory course: From 0 to 1, the Road to Product Commercialization: Business planning and product Landing
   strategies.

![图片2](https://static.igem.wiki/teams/5748/notebook/3.webp)

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **2025, Feburary** | | | | |
|  | First week | Second week | Third week | Fourth week |
| Project background Review |  |  |  |  |
| Team discussion |  |  |  |  |
| Laboratory safety training |  |  |  |  |
| Experiment conducted |  |  |  |  |
| Recruit members |  |  |  |  |

1. Project background Review
2. Team discussion: team name, Logo, team uniform, etc
3. Laboratory safety training
4. Experiment conducted：

1. LB medium preparation
2. Glycerin inoculator
3. Plasmid extraction
4. The target gene PETase/MHETase was amplified by PCR
5. DNA identification by agarose gel electrophoresis
6. DNA glue recovery
7. Homologous recombinant DNA and vector
8. Convert E.coli DH5α/BL21
9. Plate coating screening
10. The transformant was identified by PCR
11. Select monoclonal culture seed solution
12. Extended culture

![](https://static.igem.wiki/teams/5748/notebook/4.webp)

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **2025, March** | | | | |
|  | First week | Second week | Third week | Fourth week |
| Education scheme |  |  |  |  |
| Basic theory learning |  |  |  |  |

1. Finalization of education scheme（kindergarten, primary school, middle school, high school, public）
2. Create a team account on social media platforms
3. Make and publish a questionnaire about plastic degradation
4. Plan on-campus promotion (post posters, design small games)

![](https://static.igem.wiki/teams/5748/notebook/5.webp)

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **2025, April** | | | | |
|  | First week | Second week | Third week | Fourth week |
| Design business plan |  |  |  |  |
| Expert Interviews |  |  |  |  |
| Discuss project |  |  |  |  |
| BP |  |  |  |  |

1. Design business plan
2. Expert Interviews: Dr. Chen Guangquan, Dr. Li Shiyuan
3. Write a verbatim transcript of the interview
4. Discuss the progress of the current project and the areas that need improvement

![](https://static.igem.wiki/teams/5748/notebook/6.webp)

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **2025,May** | | | | |
|  | First week | Second week | Third week | Fourth week |
| Promotion video |  |  |  |  |
| Questionnaire survey |  |  |  |  |
| Education |  |  |  |  |

1. Write the script for the promotion video
2. Shoot the imitative promotion video of the competition team
3. Sort out the data obtained from the questionnaire survey
4. High school education: Lecture on Plastic-Degrading Enzymes
5. Elect the team leader, the leader of the wet team and the leader of the dry team

![](https://static.igem.wiki/teams/5748/notebook/7.webp)

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **2025,June** | | | | |
|  | First week | Second week | Third week | Fourth week |
| Basic knowledge |  |  |  |  |
| Protein structure |  |  |  |  |
| Wiki website |  |  |  |  |
| Education |  |  |  |  |
| Wet experiment |  |  |  |  |

Learn the basic knowledge that the wet team used in
the subsequent experiments：

1. NCBI gene mining
2. AlphaFold predicts protein structures
3. Molecular docking predicts the binding effect
4. Homologous recombination and plasmid construction of eight variants

Wet team group report:

1. Make a preliminary division of labor for the content writing of the Wiki website
2. Kindergarten education：puppet show -- Enzyme Hero\'s Magic Scissors

![](https://static.igem.wiki/teams/5748/notebook/8.webp)

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **2025,July** | | | | |
|  | First week | Second week | Third week | Fourth week |
| Education |  |  |  |  |
| Wiki design |  |  |  |  |
| Wiki website |  |  |  |  |
| Promotion video |  |  |  |  |

1. Primary school education: Lectures + Handicraft Activities
2. Finalize the design style of the wiki
3. The shooting of the promotion video

![](https://static.igem.wiki/teams/5748/notebook/9.webp)

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| 2025,August | | | | |
|  | First week | Second week | Third week | Fourth week |
| Education |  |  |  |  |
| IHP/BP |  |  |  |  |
| Wiki website |  |  |  |  |
| Promotion video |  |  |  |  |
| Wet experiment |  |  |  |  |

Complete the editing of the promotion video and
upload it to the website

![](https://static.igem.wiki/teams/5748/notebook/10.webp)

**Dry team：**

1. Create pull-up banners and prepare the planning proposal for the 8.27 community education
2. The IHP survey
3. Summarizes Dr. Li Shiyuan\'s presentation content and gains, integrates them and completes the IHP
4. Community education prepares and questionnaires
5. Prepare the interview outline for Professor Huo
6. Wiki writing for primary school education
7. Community education：lectures, promotions, games
8. Interview Outline for Professor Shan
9. Introduction to Education Community wiki Activities
10. Professor Shan\'s IHP reflection
11. Interview with Professor Huo

![](https://static.igem.wiki/teams/5748/notebook/11.webp)

**Wet team：**

1. Review the content of the research topic and the intensive training,
2. The expansion culture of the strain, the determination of OD600, the creation of growth curves
3. The induction of protein expression by IPTG
4. Expand the culture of the strain, OD600, induce protein expression.
5. Ultrasonic disruption of cell walls
6. Learn the principle of nickel column purification and protein purification
7. Divide the tasks of the flowchart
8. SDS-PAGE protein isolation
9. Learn the principle of HPLC
10. Prepare samples of PET degraded by PETase, and take samples for HPLC testing after 48/72 hours
11. Protein purification
12. The remaining proteins from yesterday were identified, stained and decolorized, and Prepare HPLC samples
13. The synergistic effect of PETase and MHETase was identified using PET film
14. Draw flowcharts（BioRender）

![](https://static.igem.wiki/teams/5748/notebook/12.webp)

**Wiki：**

1. Write content for the wiki website
2. Make the first version of the ppt for the project defense
3. Revise the project defense ppt
4. Conduct the first mock project defense

![](https://static.igem.wiki/teams/5748/notebook/13.webp)

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **2025, September** | | | | |
|  | First week | Second week | Third week | Fourth week |
| Write Education |  |  |  |  |
| Write BP |  |  |  |  |
| Wiki website |  |  |  |  |
| Write wet wiki |  |  |  |  |
| **2025, October** | | | | |
|  | First week | Second week | Third week | Fourth week |
| Presentation video |  |  |  |  |
| Presentation |  |  |  |  |

'
}]%}
{% block title %}Notebook{% endblock %}
{% block lead %}

Notebook

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{% extends "layout.html" %}
{% set table\_data = [{
"title": "Overview",
"content": '

Plastic pollution has emerged
as a critical global environmental issue, with polyethylene terephthalate (PET) representing one of the most
persistent and widely used plastics. Current enzymatic degradation methods for PET exhibit limited efficiency,
hindering their application on an industrial scale(Guo etal,2025; V etal, 2020). To address this challenge, we aim to
develop enhanced enzymatic strategies for PET biodegradation and recycling by screening and engineering
high-efficiency plastic-degrading enzymes. Our objective is to improve the catalytic conversion of PET into its
monomers, terephthalic acid (TPA) and ethylene glycol (EG), thereby providing a sustainable and innovative
bioremediation solution(Fig 1).

![图片10](https://static.igem.wiki/teams/5748/engineering/1.webp)

Figure 1. Technical Roadmap

**Design and Optimization of Part
Cycle:**

**Cycle 1:** PETase derived from
Ideonella sakaiensis (positive control), capable of converting PET into MHET.

**Cycle 2:** Screening mutants of
PET-degrading enzyme derived from deep-sea sources, capable of converting PET into MHET.

**Cycle 3:** MHETase derived from
Ideonella sakaiensis, which converts MHET into TPA and EG. The mutant PETase screened in Cycle 2 is used in
combination with the MHETase to degrade PET into recyclable TPA and ethylene glycol (EG).

The resulting engineered enzymes are expected to
demonstrate significantly improved PET degradation performance, with potential applications in environmental
remediation and industrial plastic waste processing.

'
},{
"title": 'Cycle 1: pET28a-IsPETase([BBa\_25BUBMDI](https://registry.igem.org/parts/bba-25bubmdi))',
"titleC": 'Cycle 1: pET28a-IsPETase([BBa\_25BUBMDI](https://registry.igem.org/parts/bba-25bubmdi))',
"content": '

**Design:**

A bacterial strain *Ideonella sakaiensis*
capable of efficiently degrading PET has been discovered. At the same time, a hydrolase secreted by this bacterium
with the ability to degrade PET, named PETase, has also been reported. This enzyme can hydrolyze PET into
bis-2-hydroxyethyl-terephthalate (BHET), mono-2-hydroxyethyl-terephthalate (MHET), and TPA (YOSHIDA etal,2016).The
genes encoding IsPETase fused with 6×His-tag at its C-terminus were optimized and chemically synthesized. The plasmid
map was constructed by snapgene software(Fig. 2), and inserted into the expression vector pET28a by homologous
recombination method, the recombinant plasmids pET28a-IsPETase were further transformed into *E.Coli* DH5α.

![图片7](https://static.igem.wiki/teams/5748/engineering/2.webp)

Fig 2. The plasmid map of pET28a-IsPETase

**Build:**

The pET-28a plasmid vector was linearized for the
construction of the expression plasmid. The plasmid containing the IsPETase gene was constructed using homologous
recombination, followed by confirmation of successful construction via agarose gel electrophoresis. The Fig 3A showed
that the IsPETase gene is between 500bp and 1000bp markers. The results are showed in Fig 3B, the linearized plasmid
pET28a is highlighted 5000bp. The expansion length of gene is consistent with the DNA encoding and indicate the
succeed of amplification. The Fig 3C showed that the recombinant plasmids was transformed into processed
*E.coli* DH5α to copy the plasmids. As shown in Fig 3D, the sequencing results revealed a perfect match to the
target gene sequence without any mutations, demonstrating the successful construction of the plasmid pET28a-IsPETase.

![](https://static.igem.wiki/teams/5748/engineering/3.webp)

Fig. 3.The electrophoresis, plate Assay, and
sequencing of IsPETase. Note: IsPETase：879bp; pET28a：5323bp

Test：

We measured the OD600 at various time points.
As shown in Figure 4A, no significant difference was observed between the growth curves of the control group and the
IsPETase-expressing strain, indicating robust bacterial growth.

We engineered the expression construct to incorporate
a specific gene sequence encoding a hexahistidine (His-tag) affinity tag, enabling ribosomal synthesis of the
recombinant protein with a C-terminal His-tag. The His-tagged protein was subsequently purified using Ni-NTA affinity
chromatography, whereby the immobilized nickel ions selectively bind the His-tag. Non-specifically bound proteins were
removed by washing with buffer, and the target protein was eluted under native conditions using imidazole-containing
elution buffer.

In SDS-PAGE analysis, the crude protein dsPETase
exhibited diffuse banding patterns, indicative of the presence of non-target cellular proteins. Following
purification, the Fig 4B result demonstrated that the molecular weight of the recombinant protein was between 25 kDa
and 35 kDa, which is consistent with its theoretical molecular mass of 33.5 kDa.

![图片70](https://static.igem.wiki/teams/5748/engineering/4.webp)

**Fig 4.The growth curve and SDS-PAGE of
IsPETase**

**Learn:**

In cycle 1, the plasmids are constructed successfully
and transformed in E.coli. The SDS polyacrylamide gel electrophoresis (SDS-PAGE)experiment we conducted yields the
expected proteins’ sizes of IsPETase, which indicates that the proteins IsPETase are successfully expressed in E.coli.
This cycle experiments and proves the positive control, providing essential results for further designs.

'
}, {
"title": 'Cycle 2: pET28a-dsPETaseand mutant([BBa\_25GLIJTL](https://registry.igem.org/parts/bba-25glijtl))',
"titleC": 'Cycle 2: pET28a-dsPETaseand mutant([BBa\_25GLIJTL](https://registry.igem.org/parts/bba-25glijtl))',
"content": '

**Gly8-GIn(BBa\_2542VYML),
Gly8-Ser(BBa\_25N2AZ4A), Gly8-Thr(BBa\_25655FKG), Gly8-Cys(BBa\_25EL7FQM), Gly8-Asn(BBa\_25NSRU73),
Gly8-Tyr(BBa\_2578647W), Arg47-Lys(BBa\_25VTOT4S), and Arg47-His(BBa\_2543N0QT)**

**Design:**

A novel halophilic PET plastic-degrading enzyme with
high activity derived from the hydrothermal vent metagenome (of deep-sea origin) was reported. Meanwhile, a hydrolase
with PET-degrading activity, namely PETase, secreted by this bacterium was also reported. This enzyme can hydrolyze
PET into bis(2-hydroxyethyl) terephthalate (BHET), mono(2-hydroxyethyl) terephthalate (MHET), and terephthalic acid
(TPA) [KANELLI et al,2025].

The genes encoding dsPETase fused with 6×His-tag at
its C-terminus were optimized and chemically synthesized. The plasmid map was constructed by snapgene software(Fig 5),
and inserted into the expression vector pET28a by homologous recombination method, the recombinant plasmids
pET28a-dsPETase were further transformed into *E.Coli* DH5α.

![图片1001](https://static.igem.wiki/teams/5748/engineering/5.webp)

Fig 5. The plasmid map of pET28a-dsPETase

To identify key active sites in dsPETase for
mutagenesis, we constructed a three-dimensional protein structure of dsPETase and performed molecular docking with the
chemical structure of PET(Fig 6). The docking score (binding energy) serves as an indicator to evaluate the affinity
between PET and protein dsPETase. A stronger affinity results in tighter binding and more stable molecular
interactions. A more negative docking score corresponds to a stronger affinity between the molecules.

**![图片3](https://static.igem.wiki/teams/5748/engineering/6.webp)**

Fig 6. Docking results of dsPETase with the PET
molecule; Red represents the PET molecule.

**![图片2](https://static.igem.wiki/teams/5748/engineering/7.webp)**

Fig 7. Schematic diagram of the active site of
dsPETase with the PET molecule.

The optimal binding mode between the PET molecule and
dsPETase is shown in Figs 6 and 7. The molecular docking binding energy was calculated to be −3.249 kcal/mol, with
stabilization primarily achieved through hydrogen bonding (Table 1). Key amino acid residues forming hydrogen bonds
with the PET molecule were glycine at position 8 (Gly8) and arginine at position 47 (Arg47), which enhanced the
stability of the PET molecule within the binding pocket.The molecular docking results validated the feasibility of
selecting Gly8 and Arg47 for mutagenesis, a decision initially made based on expert opinions during the experimental
design phase. However, experimental validation is required to confirm the degradation efficiency of the mutants and
ensure the reliability of the result.

Based on the advice of biology Professor Yao, we
performed site-directed mutagenesis on the active site residues glycine (Gly8) and arginine (Arg47). Specifically,
Gly8 was mutated to amino acids with similar properties—namely, polar neutral R-group residues such as glutamine
(Gln), serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn), and tyrosine (Tyr). This strategy helps
minimize structural perturbations that could lead to loss of protein activity. The arginine at position 47 was mutated
to amino acids with similar properties, specifically basic R-group residues such as lysine (Lys) and histidine (His).
The resulting mutants were designated as Arg47-Lys, Arg47-His, Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and
Gly8-Tyr. As shown in Table 1, the docking scores of these mutants were significantly improved compared to that of
wild-type dsPETase, indicating stronger binding affinity with PET.

**Table 1. Docking scores (binding energy,
kcal/mol) of PET with dsPETase and mutants.**

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Name | dsPETase | Gly8-GIn | Gly8-Ser | Gly8-Thr | Gly8-Cys | Gly8-Asn | Gly8-Tyr | Arg47-Lys | Arg47-His |
| docking score | -3.249 | -5.103 | -4.932 | -5.151 | -4.945 | -5.002 | -5.204 | -5.096 | -5.012 |

The plasmid map was constructed by snapgene
software(Fig 8), and inserted into the expression vector pET28a by homologous recombination method, the recombinant
plasmids were further transformed into *E.Coli* DH5α.

![图片9](https://static.igem.wiki/teams/5748/engineering/8.webp)

Fig 8. The plasmid map of pET28a-IsPETase

**Build:**

![](https://static.igem.wiki/teams/5748/engineering/9.webp)

Fig. 9.The electrophoresis, plate Assay, and
sequencing of dsPETase

The construction of the dsPETase plasmid employed an
identical strategy to that of the IsPETase plasmid, with both based on the method of homologous recombination. As
shown in Fig 9A, the dsPETase gene migrated between 500 and 1000 bp. This is consistent with its actual length of 816
bp, which was determined by sequencing. The linearized pET28a line is around 5000bp and its original length is 5329bp.
We was transformed the pET28a-dsPETase plasmid into *E.coli* in the Fig 9C. In Fig 9D, sequencing of the
plasmid confirmed that the inserted DNA fragment matched the target gene sequence, demonstrating successful ligation
into the vector.

![图片23](https://static.igem.wiki/teams/5748/engineering/10.webp)

**Fig 10. Agarose gel electrophoresis analysis
of the mutant**

We performed site-directed mutagenesis using PCR. As
shown in Fig 10, the DNA fragments migrated between 5000 bp and 7500 bp, with all mutants (Arg47-Lys, Arg47-His,
Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and Gly8-Tyr) exhibiting a consistent length of 6190 bp. The
plasmids were subsequently sent for commercial sequencing(Fig 11), and the results confirmed that the introduced
mutations matched the expected designs.

![图片36](https://static.igem.wiki/teams/5748/engineering/11.webp)

**Fig 11.** The sequencing **of
the mutant**

**Test：**

1. Protein Structure

1.1 Mutant Arg47-Lys and Arg47-His

**![图片34](https://static.igem.wiki/teams/5748/engineering/12.webp)**

Fig 12. Molecular interactions between the protein
and the ligand within the binding pocket. Note: The protein structures of the Arg47-Lys and Arg47-His mutants are
depicted in blue. The PET molecule is shown in orange.

The binding energies between the PET molecule and the
mutant proteins Arg47-Lys and Arg47-His were −5.076 kcal/mol and −5.012 kcal/mol, respectively, indicating strong
molecular interactions. As shown in Fig 12, the PET molecule interacts with the Arg47-Lys mutant primarily through
hydrogen bonds and hydrophobic interactions. Hydrogen bonds were formed with LEU-70 and ALA-71, while hydrophobic
interactions occurred with VAL-43, LEU-70, ALA-71, and ARG-68. In the Arg47-His mutant, the PET molecule was involved
in hydrophobic interactions with the specific residues PHE-44, TYR-118, and THR-117 (Fig 12).

1.2 Mutant (Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser,
Gly8-Thr, and Gly8-Tyr)

![图片30](https://static.igem.wiki/teams/5748/engineering/13.webp)

Fig 13. Molecular interactions between the protein
and ligand within the binding pocket. The mutant protein structure is shown in blue, and the PET molecule is depicted
in orange.

The binding energy between the PET molecule and the
Gly8-Asn mutant protein was calculated to be −5.002 kcal/mol, indicating strong binding affinity. As shown in Fig 13,
the PET molecule interacts with the Gly8-Asn mutant primarily through hydrogen bonding and hydrophobic interactions.
Specifically, a hydrogen bond was formed with TYR-106, while hydrophobic interactions were observed with PHE-44,
THR-117, and TYR-118.

The binding energy for the PET molecule with the
Gly8-Cys mutant was −4.945 kcal/mol, demonstrating favorable binding interactions. Fig 14 shows that the PET molecule
engages in hydrophobic interactions with LEU-103 and LYS-102, as well as electrostatic interactions with LYS-102 and
ARG-99 in the Gly8-Cys mutant.

![图片31](https://static.igem.wiki/teams/5748/engineering/14.webp)

Fig 14. Molecular interactions between the protein
and ligand within the binding pocket. The mutant protein structure is shown in blue, and the PET molecule is depicted
in orange.

The binding energies between the PET molecule and the
Gly8-Gln and Gly8-Ser mutant proteins were calculated to be −5.103 kcal/mol and −4.932 kcal/mol, respectively,
indicating strong binding affinity. As shown in Fig 15, the PET molecule interacts with the Gly8-Gln mutant primarily
through hydrogen bonding and hydrophobic interactions. Specifically, a hydrogen bond was formed with TYR-106, while
hydrophobic interactions were observed with PHE-44, THR-117, and TYR-118. Fig 14 illustrates that the PET molecule
engages in hydrophobic interactions with LEU-103 and LYS-102, as well as electrostatic interactions with LYS-102 and
ARG-99 in the Gly8-Ser mutant.

![图片36](https://static.igem.wiki/teams/5748/engineering/15.webp)

Fig 15. Molecular interactions between the protein
and ligand within the binding pocket. The mutant protein structure is shown in blue, and the PET molecule is depicted
in orange.

The binding energies between the PET molecule and the
Gly8-Thr and Gly8-Tyr mutant proteins were calculated to be −5.151 kcal/mol and −5.204 kcal/mol, respectively,
indicating strong binding interactions. In Fig 15, the PET molecule engages in hydrophobic interactions with PHE-44,
TYR-118, and THR-117 in the Gly8-Thr mutant. Fig 16 illustrates that the PET molecule interacts with the Gly8-Tyr
mutant primarily through hydrogen bonding and hydrophobic interactions. Specifically, a hydrogen bond was formed with
TYR-106, while hydrophobic interactions were observed with PHE-44 and TYR-118.

![dsPETase(1)](https://static.igem.wiki/teams/5748/engineering/16.webp)

**Fig 16.The growth curve of dsPETase and
mutant**

As shown in Figure 17, no significant
difference was observed between the growth curves of the control group and the dsPETase and mutant strain,
indicating robust bacterial growth.

2. Protein Expression

![图片71](https://static.igem.wiki/teams/5748/engineering/17.webp)

**Fig 17. The SDS-PAGE of dsPETase and mutant.
M:marker 1,2:rough protein; 3-5:Wash buffer 6-9:purified protein. DsPETase:31.5kDa**

As shown in Figure 17A, lanes 1–2 exhibited diffuse
bands in the crude protein samples, suggesting the presence of a substantial amount of non-specific proteins. Lanes
3–5, which served as blank controls, showed no detectable target band corresponding to dsPETase. In lanes 6–9, clear
protein bands were observed within the molecular weight range of 25–35 kDa. These samples were purified by
nickel-affinity chromatography, wherein Ni-NTA resin selectively captured His-tagged proteins, wash buffer removed
non-specifically bound impurities, and the target protein was subsequently eluted under appropriate conditions. Figure
17B displays the protein expression profiles of various mutants. Band intensity and thickness reflect relative
expression levels, with the Gly8-Ser mutant showing the darkest and thickest band, indicating its highest expression
among all variants.

3. Determine the content of the product MHET

The chromatographic conditions were optimized using a
Thermo Scientific C18 column (250 × 4.6 mm, 5 μm) with a mobile phase consisting of phosphate buffer (containing 5
mmol/L potassium dihydrogen phosphate and 0.04% phosphoric acid) and methanol in a ratio of 70:30 (v/v). According to
the experimental protocol, standard solutions were serially diluted to appropriate concentrations to establish a
calibration curve. The peak area was plotted on the Y-axis against the concentration of the reference standard on the
X-axis, and regression analysis was performed to obtain the equation for each standard. A good linear relationship was
observed within the concentration range of 1–50 mg/L (Table 2).

Table 2. The standard curve of MHET

|  |  |  |
| --- | --- | --- |
| **Concentrations**  **(**mg/L) | Peak area | ![](https://static.igem.wiki/teams/5748/engineering/table-2.webp) |
| 1 | 58.41312 |
| 2 | 130.59349 |
| 5 | 357.34796 |
| 10 | 697.24371 |
| 20 | 1393.50171 |
| 50 | 3568.67041 |

We incubated 500 mg of PET plastic substrates with
IsPETase, dsPETase, and their variants at a final concentration of 1 mg/mL, then collected samples after 48 and 72
hours of degradation at room temperature. The peak area of MHET was measured by HPLC, and its concentration in
the experimental samples was calculated based on the standard curve.

As shown in Fig 18A, MHET was detected in the
experimental groups of IsPETase, dsPETase, and their mutants at 48-hour, whereas MHET was undetectable in the CK
control group. Comparative analysis revealed no significant difference in MHET levels between IsPETase and dsPETase
at the 48-hour. In contrast, all tested mutants—namely Arg47-Lys, Arg47-His, Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser,
Gly8-Thr, and Gly8-Tyr—exhibited significantly higher MHET accumulation compared to dsPETase. Notably, the Gly8-Ser
mutant generated markedly elevated MHET levels relative to the other mutants. Furthermore, there was no
statistically significant difference in MHET production between the Arg47-Lys and Arg47-His variants.

In Fig 18B, MHET was detected in the
experimental groups of IsPETase, dsPETase, and their mutants after 72 hours, while no MHET was observed in the CK
control group. The MHET level produced by dsPETase was significantly higher than that of IsPETase at 72h. The MHET
concentration of Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and Gly8-Tyr mutants exhibited markedly higher
than dsPETase. In contrast, no significant difference in MHET levels was observed among Arg47-Lys, Arg47-His, and
dsPETase. These results suggest that Gly8 may represent a critical active site in dsPETase. Notably, the
substitution of Gly8 with Ser significantly enhanced the degradation efficiency of PET.

****![图片90](https://static.igem.wiki/teams/5748/engineering/18.webp)****

**Fig 18. MHET content of IsPETase, dsPETase,
and mutantsArg47-Lys, Arg47-His, Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and Gly8-Tyr) at 48 and 72
hours.**

**Learn:**

In cycle 2, the plasmids for dsPETase and eight
mutants (Arg47-Lys, Arg47-His, Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and Gly8-Tyr) were successfully
constructed and transformed into E. coli. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed that
the expressed proteins corresponded to the expected molecular sizes of dsPETase and all mutants, indicating their
successful expression in E. coli. The experimental data from this cycle not only verified the ability of dsPETase and
the mutants to degrade PET plastic but also enabled us to identify that the Gly8-Ser mutant exhibited higher enzymatic
activity compared to the other mutants. These findings provide critical support for the subsequent dual-enzyme synergy
strategy in cycle 3, making Gly8-Ser an indispensable component for the next phase.

'
}, {
"title": '
Cycle 3：pET28a-MHETase ([BBa\_2582B9BT](https://registry.igem.org/parts/bba-2582b9bt))',
"titleC": 'Cycle 3：pET28a-MHETase ([BBa\_2582B9BT](https://registry.igem.org/parts/bba-2582b9bt))',
"content": '

**Design:**

Mono-2-hydroxyethyl terephthalate (MHET) is an
intermediate with a competitive inhibitory effect in the degradation process of PET, which can be hydrolyzed from
terephthalic acid (TPA) and ethylene glycol (EG) catalyzed by MHET degradation enzymes.

The genes encoding MHETase fused with 6×His-tag at
its C-terminus were optimized and chemically synthesized. The plasmid map was constructed by snapgene software(Fig
19), and inserted into the expression vector pET28a by homologous recombination method, the recombinant plasmids
pET28a- MHETase. were further transformed into *E.Coli* DH5α.

![图片21](https://static.igem.wiki/teams/5748/engineering/19.webp)

Fig 19. The plasmid map of pET28a- MHETase.

**Build:**

![图片18](https://static.igem.wiki/teams/5748/engineering/20.webp)

Fig 20.The electrophoresis, plate Assay, and
sequencing of MHETase.

Note:the length of MHETase is 1818bp. the length of
pET28a is 5324b

The agarose gel electrophoresis result in Fig 20 A
and B showed a band of the MHETase gene at approximately 1500 bp and a linearized plasmid band near the 3000 bp
marker. After ligation, the recombinant plasmids were transformed into E. coli DH5α competent cells for amplification
in the Fig 20C. The subsequent sequencing analysis confirmed the successful insertion of the target gene MHETase into
the vector(Fig 20D).

**Test：**

1. Protein expression

![图片60](https://static.igem.wiki/teams/5748/engineering/21.webp)

Fig 21.The growth curve and SDS-PAGE of
IsPETase. Note:The size of MHETase is 64kDa.

To express the target protein, the plasmid
was transformed into *E. coli* BL21(DE3) cells and induced with 1 mM IPTG, followed by protein extraction and
purification. In Fig 21A, no significant difference was observed between the growth curves of the control group and
the dsPETase and mutant strain, indicating robust bacterial growth. The SDS-PAGE analysis showed that the purified
MHETase protein was approximately 63 kDa, which is consistent with its actual molecular size(Fig 21 B).

2. Scanning electron microscopy (SEM)

![](https://static.igem.wiki/teams/5748/engineering/22.webp)

Fig. 22. Results of scanning electron microscopy
(SEM) of PET. A: control group; B:IsPETase+MHETase; C:DsPETase+MHETase; D:Gly8-Ser+MHETase

Fig 22A shows the PET film from the control group
without enzyme addition, exhibiting a smooth surface without cracks or pores. Fig 22B presents the experimental group
treated with IsPETase and MHETase, where faint fissures observed on the film surface indicate initial enzymatic
activity, though the degradation effect remains suboptimal. In Fig22 C, the sample co-treated with dsPETase and
MHETase demonstrates more distinct fissure structures, suggesting enhanced degradation efficacy compared to the
IsPETase group. Figure 22 D displays the Gly8-Ser variant + MHETase group.
Numerous pronounced fissures can be observed on the film surface, demonstrating the superior degradation performance
of the Gly8-Ser variant.

3. HPLC determination of concentration TPA and EG

The experimental instrument parameters were adjusted
as follows: the column was a Thermo Fisher C18 (250 mm × 4.6 mm, 5 μm), with a column temperature of 30°C, flow rate
of 1 mL/min, and a mobile phase consisting of phosphate buffer solution (containing 5 mmol/L potassium dihydrogen
phosphate and 0.04% phosphoric acid) and methanol at a ratio of 70:30.

According to the experimental protocol, the standard
was diluted to appropriate concentrations to establish a calibration curve. Using the peak area as the ordinate (Y)
and the mass concentration of the reference standard as the abscissa (X), regression analysis was performed to derive
the equation for each standard. A good linear relationship was demonstrated within the concentration range of 1–50
mg/L. The tested concentrations and corresponding calibration curve are presented in Table 3.

Table 3. The standard curve of TPA

|  |  |  |
| --- | --- | --- |
| **Concentrations**  **(**mg/L) | Peak area | ![](https://static.igem.wiki/teams/5748/engineering/table-3.webp) |
| 1 | 85.65078 |
| 2 | 204.93451 |
| 5 | 584.46924 |
| 20 | 2220.17896 |
| 50 | 6006.84082 |

We incubated 1 g PET plastic with 1 mg/mL IsPETase,
dsPETase, and the Gly8-Ser variant, along with MHETase at a final concentration of 1 mg/mL. Under the catalytic action
of PETase and MHETase, PET can be degraded to TPA, which was quantified using HPLC. As shown in Fig 23, the control
group (pET28a vector) showed no peak at 10–12 minutes, whereas distinct peaks were observed for the IsPETase +
MHETase, dsPETase + MHETase, and Gly8-Ser variant + MHETase groups.

![图片100](https://static.igem.wiki/teams/5748/engineering/23.webp)

Fig. 23. Chromatographic peak of TPA

As illustrated in Figure 24, no TPA was detected in
the control (CK) group. The TPA concentrations produced in dsPETase + MHETase and Gly8-Ser + MHETase were
significantly higher than IsPETase + MHETase. Furthermore, the TPA concentrations from the Gly8-Ser+ MHETase
combination was markedly higher than that of dsPETase + MHETase. These results indicate that the degradation
efficiency of dsPETase + MHETase exceeds that of IsPETase + MHETase, and the Gly8-Ser variant in combination with
MHETase demonstrated superior performance compared to all other experimental groups. This evidence confirms that
site-directed mutagenesis enhanced the enzymatic activity of PETase, thereby significantly improving the overall
degradation efficiency.

![图片57](https://static.igem.wiki/teams/5748/engineering/24.webp)

Fig. 24. The concentration of TPA of CK, IsPETase +
MHETase, dsPETase + MHETase, and Gly8-Ser+ MHETase

EG (Ethylene Glycol) was qualitatively and
quantitatively analyzed in complex mixtures using Gas Chromatography-Mass Spectrometry (GC-MS). The instrument
parameters were set as follows:

Gas Chromatograph: Agilent GC 8860 equipped with CTC
PAL3 autosampler

Mass Spectrometer: Agilent 5977B MSD

Column: Agilent DB-WAX (30 m × 0.25 μm)

Carrier Gas: High-purity helium (≥99.99999%)

Chromatographic conditions:

Detector temperature: 240°C

Injector temperature: 220°C

Flow rate: 1.0 mL/min

Temperature gradient: Initial temperature 80°C (hold
5 min), then ramp at 5°C/min to 240°C (hold 1 min).

![图片54](https://static.igem.wiki/teams/5748/engineering/25.webp)

Fig. 25. The chromatographic peak, ratio, mass
scharge ratio of EG.

As shown in Fig 25, the detection results of ethylene
glycol (EG) in the samples were obtained using high-performance liquid chromatography (HPLC) and mass spectrometry
(MS). The control group exhibited no detectable peaks, indicating the absence of EG in the sample. Distinct
chromatographic peaks were observed in the experimental groups containing IsPETase + MHETase, dsPETase + MHETase, and
the Gly8-Ser + MHETase.The concentration of EG in the samples was calculated based on the chromatographic peak areas
using the standard curve equation y = 6.306x + 363.8. As shown in Figure 26 A, the calibration curve exhibited an
excellent fit with a coefficient of determination (R²) greater than 0.99.

In Fig 26B, the EG concentration obtained with
dsPETase + MHETase was significantly higher than that with IsPETase + MHETase. Notably, the Gly8-Ser variant + MHETase
combination yielded a substantially greater EG concentration compared to both the dsPETase and IsPETase. These results
indicate that the Gly8-to-Ser mutation at position 8 in dsPETase enhances enzymatic activity, significantly improving
the conversion efficiency of EG.

![图片81](https://static.igem.wiki/teams/5748/engineering/26.webp)

Fig 26. The concentration of EG of CK, IsPETase + MHETase, dsPETase + MHETase, and Gly8-Ser+MHETase

**Learn:**

Through this study, we demonstrate the feasibility of
employing a dual-enzyme system—comprising dsPETase and MHETase—for synergistic degradation of polyethylene
terephthalate (PET), enabling a closed-loop recycling process. Our work primarily focused on enhancing the
depolymerization efficiency via structure-guided engineering of dsPETase. Using molecular docking, we predicted key
residues within the enzyme’s active site and conducted site-directed mutagenesis at positions Arg47 and Gly8,
substituting them with amino acids of similar properties to minimize functional disruption.

Experimental results revealed that the Gly8-Ser
mutation significantly improved PET degradation efficiency, whereas the Arg47 variant showed no notable effect.
Further optimization of catalytic activity may be achieved through additional rational mutagenesis at predicted active
sites. Moreover, the combined action of the Gly8-Ser and MHETase synergistically catalyzed the complete decomposition
of PET into its monomers, terephthalic acid (TPA) and ethylene glycol (EG), which can be reused as raw materials for
plastic synthesis.

In summary, this study proposes a novel enzymatic
strategy for sustainable plastic degradation and recycling. These findings provide a theoretical foundation for the
development of biocatalytic recycling processes and highlight potential applications in the bioremediation of plastic
pollution.

'
},{
"title": "References",
"content": '

Guo, Z., Li, Y., Wang, M., & Ma, D. (2025).
Catalytic upcycling of PET: From waste to chemicals and degradable polymers. Accounts of Chemical Research.

KANELLI M, VASILAKOS S, NIKOLAIVITS E, LADAS S,
CHRISTAKOPOULOS P, TOPAKAS E. Surface modification of poly(ethylene terephthalate) (PET) fibers by a cutinase from
Fusarium oxysporum[J]. Process Biochemistry, 2015, 50(11): 1885-1892. DOI:10.1016/j.procbio.2015.08.013

V T ,M C T ,A G , et al.An engineered PET
depolymerase to break down and recycle plastic bottles.[J].Nature,2020,580(7802):216-219.

Tournier, V., et al. (2023). Enzymatic recycling of
PET plastic: From discovery to industrial implementation. Nature Reviews Chemistry, 7(12),

YOSHIDA S, HIRAGA K, TAKEHANA T, TANIGUCHI I, YAMAJI
H, MAEDA Y, TOYOHARA K, MIYAMOTO K, KIMURA Y, ODA K. A bacterium that degrades and assimilates poly(ethylene
terephthalate). *Science*, 2016, 351(6278): 1196-1199. [DOI:10.1126/science.aad6359](http://dx.doi.org/10.1126/science.aad6359%22%20%5Co%20%22%E7%82%B9%E5%87%BB%E6%B5%8F%E8%A7%88%E5%8E%9F%E6%96%87%22%20%5Ct%20%22https%3A//actamicro.ijournals.cn/html/actamicrocn/2023/1/_blank)

'
}]%}
{% block title %}Engineering{% endblock %}
{% block lead %}

Engineering

{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Plasmid
Construction",
"content": '

![图片3-](https://static.igem.wiki/teams/5748/experiment/1.webp)

',
"children": [{
"title": "1. Preparation of LB Medium",
"content": '

Target：Make LB medium

Material：

|  |  |
| --- | --- |
| Material | Dosage/Usage |
| Tryptone | 2.5g |
| Yeast Extract | 1.25g |
| NaCl | 2.5g |
| ddH2O | 250mL |
| Agar (for solid) | 3.75g (1.5% of gross) |
| Sealing film | 1 |
| high-pressure steam sterilization pot | 121 Celsius,20min(1) |
| Scale | 1 |
| Pan paper | 4-5 sheets |
| Flask | 1 |
| Graduated cylinder | 1 |

Procedure：

1. Place the weighing paper on the scale and weigh the net weight.
2. Weigh out 2.5 grams of tryptone on a scale and add it to the flask.
3. Place the weighing paper on the scale and weigh the net weight.
4. Weigh out 1.25 grams of yeast extract on a scale and add it to the flask.
5. Place the weighing paper on the scale and weigh the net weight.
6. Weigh out 2.5 grams of NaCl on a scale and add it to the flask.
7. Measure out 250mL of ddH2O with a graduated cylinder and add it to the flask.
8. Write the name of the medium on the flask and the sealing film, then seal the mouth of the flask with the sealing
   film.
9. Shake the flask gently to even out the medium.
10. Put it into the high-pressure steam sterilization pot, sterilize for 121 degree Celsius, 20 minutes.

'
},{
"title": "2. Bacterial Culture",
"content": '

Target：Culture bactreia

Material：

|  |  |
| --- | --- |
| Material | Dosage/Usage |
| LB medium | 3mL |
| cell culture tube | 8 |
| Kan+ | 3μL(0.003mL)(Kan+：LB = 1：1000) |
| bacteria solution(PETase[Is,ds],MHET, PET28a) | 5μL(0.005mL) |
| Pipette | 1(2-20μL) |
| Injection syringe | 1(5mL) |
| Aseptic console | 1 |
| Ethyl alcohol | ——(75%) |
| Sealing film | A big piece |
| constant temperature shaker | 1 |

Procedure：

1. The hands were disinfected with ethyl alcohol, and the hands were put into the aseptic console to do the
   experiment.
2. Lable the cell culture tube (name of the bacteria solution, operator’s name, date).
3. Use a injection syringe to add 3mL LB medium to the cell culture tube.
4. Use a pipette to add 3μL Kan+ to the cell culture tube.
5. Use a pipette to add 5μL IsPETase to the cell culture tube.
6. Gently shake the tube and wrap the sealing film around the side of the lid.
7. Make another tube with the same solution.
8. Repeat the process with dsPETase.
9. Repeat the process with MHET.
10. Repeat the process with PET28a.
11. Bundle four in groups and put them in a constant temperature shaker at 37 degrees Celsius for 12-16 h.

'
},{
"title": "3. Plasmid Extraction",
"content": '

Target：To extract the target plasmid pET28a

Material：

1. Buffer Sp1：to give a suitable environment
2. Buffer Sp2：Lysis of cell membrane
3. Buffer Sp3：stop the reaction
4. adsorption column
5. collecting pipe
6. centrifuge tube
7. Adsorption film：uptake plasmid
8. Wash solution：flushing impurity
9. Elution buffer：flushing target plasmid
10. Nanodrop：test DNA concentration

Procedure：

1. Check that RNase A has been added to Buffer SP1.
2. Check whether anhydrous ethanol has been added to the Wash Solution.
3. Check whether Buffer SP2 and SP3 are precipitated.
4. Adsorption column balance: Add 500μL Buffer S to the adsorption column (the adsorption column is put into the
   collection tube), centrifuge for 1min at 12,000 Xg, dump the waste liquid in the collection tube, and put the
   adsorption column back into the three collection tubes.
5. Take 1.5-5mL of overnight cultured bacterial solution, centrifuge 8,000Xg for 2 minutes to collect bacterial
   bodies, and discard the medium.
6. Add 250μL Buffer SP1 to the precipitate and completely suspend the bacteria.
7. Add 250 μL Buffer SP2 and immediately gently reverse the centrifugal tube for 10 times and mix well. Let stand at
   room temperature for 2-4 minutes.
8. Add 350 μL Buffer SP3 and immediately gently reverse the centrifuge tube 5-10 times to mix.
9. 12,000Xg centrifuge for 5-10 min. Transfer the supernatant into the adsorption column, centrifuge 8,000Xg for 30
   seconds, and drain the liquid in the collection tube.
10. (Optional) Add 500 μL Buffer DW1, centrifuge 9,000Xg for 30 seconds, and drain the liquid in the collection tube.
11. Add 500 μL Wash Solution, centrifuge 9,000Xg for 30 seconds, and drain the liquid in the collection tube.
12. Repeat Step 9 once .
13. The empty adsorption column was centrifuged at 9,000Xg for 1 minute.
14. Put the adsorption column into a clean 1.5mL centrifuge tube, add 50-100 μL Elution Buffer in the center of the
    adsorption film, stand at room temperature for 1 minute, and centrifuge for 1 minute. Preserve the DNA bath in the
    tube.

'
},{
"title": "4. PCR(polymerase chain reaction)",
"content": '

(In vitro amplification: A section or loop of DNA
that is duplicated to increase its number)

Target：To amplify the target gene
(IsPETase,dsPETase,MHET, PET28a) by PCR

Principle:

1. Denaturation (high temperature) : Splitting DNA
into single strands (breaking hydrogen bonds)

2. Annealing: Bind primer

3. Extension: Insert the base with DNA polymerase
(starting at the 3 \'- end of the primer)

4. Cycle: n= number of cycles, 2^n DNA

Material:

PCR
tube,Template(DNA),Primer(dsPETase-F,dsPETase-R)Mix(DNA polymerase,DNTP,MgCl2,buffer)--attenuation for 2x

ddH2O , PCR amplifierMVortexor, Centrifugal machine

Procedure：

PCR system：

|  |  |
| --- | --- |
| Ingredient | Volume(μL) |
| Mix | 25 |
| DdH2O | 19 |
| DsPETase(Template) | 2 |
| DsPETase-F | 2 |
| DsPETase-R | 2 |

PCR procedure：

|  |  |  |  |
| --- | --- | --- | --- |
| Step | Temperature(degree Celsius) | Time | Remark |
| predegeneration | 98 | 10min |  |
| degeneration | 98 | 30s |  |
| .annealing | 60 | 30s |  |
| .Extension | 72 | Determined by length | V：30s,1000bp  dsPETase: 816bp |
| Extension | 72 | 10min | At the end, to check for gaps |
| Preservation | 4 | ∞ |  |

Note: 30 cycles of 2,3,4

'
},{
"title": "5. Agarose Gel Electrophoresis",
"content": '

Target: To use agarose gel electrophoresis to analyze
the molecular weight of nucleic acids

Material：

|  |  |
| --- | --- |
| Material | Dosage |
| Agarose | 1g |
| TAE buffer loom |  |
| Nucleic acid dye | 5μL |
| PCR product | 50μL |
| Uv photoscope |  |
| Marker |  |

Procedure：

1. Gel: agarose 1g+TAE buffer loom, heat, cool, add nucleic acid dye 5μL, add into the mold (against the negative
   pole po4^2-) to form glue block, add marker
2. Sample addition: 50μL PCR product
3. Running glue: 180v, 20min
4. Photoscope: UV photoscope (Gel imaging system)

'
},{
"title": "6. Gel extraction",
"content": '

Target：To recover the DNA from the target gel.

Material：

|  |  |
| --- | --- |
| Material | Dosage/usage |
| Gel of the target fragment | 1 |
| Buffer B2 |  |
| adsorption column |  |
| collecting pipe |  |
| centrifuge tube |  |
| Adsorption film |  |
| Wash solution |  |
| Elution buffer |  |
| Vortexor  Centrifugal machine |  |
| Water bath | 50 degree Celsius |

Procedure：

1. Preparation：

Check whether anhydrous ethanol has been added to the
Wash Solution.

Check Buffer B2 for precipitation.

Set the water bath to 50°C.

2. Cut the glue block containing the target fragment from the agarose gel and weigh it.
3. Add 3-6 times the weight of the glue block Buffer B2, 50°C water bath for 5-10 minutes sol.
4. (Optional) For fragments <500bp, add 1/3 Buffer B2 volume of isopropyl alcohol.
5. Transfer the sol solution into the adsorption column and centrifuge for 30 seconds at 8,000Xg.Drain the liquid
   from the collection pipe.
6. Add 500μL Wash Solution, centrifuge 9,000Xg for 30 seconds, and drain the liquid in the collection tube.
7. Repeat Step 6.
8. Centrifuge the empty adsorption column at 9,000Xg for 1 min.
9. Put the adsorption column into a clean 1.5mL centrifuge tube, add 15-40μL Elution Buffer in the center of the
   adsorption film, leave at room temperature for 1 minute, and centrifuge for 1 minute.Preserve the DNA solution in
   the tube.

'
},{
"title": "7. Homologous recombination",
"content": '

Target：To allow DNA molecules containing homologous
sequences to recombine between or within them

Material:

|  |  |
| --- | --- |
| Material | Dosage |
| IsPETase | 1.3µL |
| PET28a-1 | 1µL |
| Homologous recombinase | 5.7µL |
| 5xbuffer | 2µL |
| centrifuge tube | 1 |
| pipettor | 1 |

Procedure:

1. Sequentially add 2µL of 5xbuffer, 5.7µL of homologous recombinase, 1µL of PETase28a-1, and 1.3µL of IsPETase.
2. Vortex for one minute and centrifuge for 30s.
3. Put the centrifuge tube in 37℃ for 15 minutes.

|  |  |
| --- | --- |
| Material | Dosage |
| MHET | 4.2µL |
| PET28a-2 | 1µL |
| Homologous recombinase | 2.8µL |
| 5xbuffer | 2µL |
| centrifuge tube | 1 |
| pipettor | 1 |

Procedure：

1. Sequentially add 2µL of 5xbuffer, 2.8µL of homologous recombinase, 1µL of PETase28a-2, and 4.2µL of MHET.
2. Vortex for one minute and centrifuge for 30s.
3. Put the centrifuge tube in 37℃ for 15 minutes.

|  |  |
| --- | --- |
| Material | Dosage |
| dsPETase | 1µL |
| PET28a-1 | 1µL |
| Homologous recombinase | 6µL |
| 5xbuffer | 2µL |
| centrifuge tube | 1 |
| pipettor | 1 |

Procedure:

1. Sequentially add 2µL of 5xbuffer, 6µL of homologous recombinase, 1µL of PETase28a-1, and 1µL of dsPETase into a
   centrifuge tube.
2. Vortex for one minute and centrifuge for 30s.
3. Put the centrifuge tube in 37℃ for 15 minutes.

Notes：

1. MHET connects PET28a-2; IsPETase, dsPETase connect PET28a-1
2. Formula: 0.02\* Length of target gene fragment (ng)/actual DNA concentration (ng/μL) = actual added volume (μL)
3. Why choose PET28a carrier?

--!! Can be used for E.coli, the usual carrier

--Strong promoter -- T7 promoter, efficient
expression of protein

4. E.coli ：

-- DH5α : high copy number, short cycle, generally
used for plasmid construction

-- BL21 (DE3) : Short cycle, good protein expression
effect (high protein content)

'
},{
"title":"8. Transform",
"content": '

Target: Transform using the heat shock method.

Material:

|  |  |
| --- | --- |
| Material | Dosage |
| competent cell(CaCl₂) | 10µL |
| Homologous recombination product | 10µL |
| fluid Luria-Bertani broth | 900µL |
| centrifugal machine | 1 |
| centrifuge tube | 1 |
| pipettor | 1 |
| ice | No limit |

Procedure：Heat shock method

1. The 10μL homologous recombinant product was put into 10μL receptive cells
2. Leave on ice for 30 minutes
3. Water bath 42 degrees Celsius, 45s
4. 3 minutes on ice
5. Add 900μL LB medium, 37 ° C, 1h (resuscitation)
6. Centrifuge 5000xg, 5min, discard part of the supernatant
7. Apply the remaining E.coliDN5α/BL21 (DE3) to a petri dish with antibiotic Kan+ (kill bacteria)
8. Invert the petri dish and put it in the incubator at 37 degrees Celsius, 12-16h

'
},{
"title": "9. Monoclonal colony verification",
"content": '

![图片36](https://static.igem.wiki/teams/5748/experiment/2.webp)

Target：Verify that the transform is successful

Material：

|  |  |
| --- | --- |
| Material | Dosage |
| PCR tube | Some(20μL) |
| 2x Mix | 10μL |
| F | 1μL |
| R | 1μL |
| Template | A colony(0μL) |
| ddH2O | 8μL |
| Agarose | 1g |
| TAE buffer loom |  |
| Nucleic acid dye | 5μL |
| PCR product | 10μL |
| Uv photoscope |  |
| Marker |  |
| PCR amplifier |  |
| Vortexor |  |
| Centrifugal machine |  |
| Pipette |  |

Procedure:

-- PCR system：

|  |  |
| --- | --- |
| Ingredient | Volume(μL) |
| Mix | 10 |
| DdH2O | 8 |
| Template(colony) | 0(Use pipette to pick into PCR tube) |
| DsPETase-F | 1 |
| DsPETase-R | 1 |

-- PCR procedure：

|  |  |  |  |
| --- | --- | --- | --- |
| Step | Temperature(degree Celsius) | Time | Remark |
| 1.predegeneration | 98 | 10min |  |
| 2.degeneration | 98 | 30s |  |
| 3.annealing | 60 | 30s |  |
| 4.Extension | 72 | 1 min 30s | V：30s,1000bp  1700bp |
| 1. Extension | 72 | 10min | At the end, to check for gaps |
| 1. Preservation | 4 | ∞ |  |

Note: 30 cycles of 2,3,4

-- Agarose Gel Electrophoresis(check whether the
expansion is successful)

1. Gel: agarose 1g+TAE buffer loom, heat, cool, add nucleic acid dye 5μL, add into the mold (against the negative
   pole po4^2-) to form glue block, add marker
2. Sample addition: 10μL PCR product
3. Running glue: 180v, 20min
4. Photoscope: UV photoscope (Gel imaging system

'
}]
}, {
"title": "Mutant
construction",
"content": '

![图片-1](https://static.igem.wiki/teams/5748/experiment/3.webp)',
"children": [{
"title": "1. Plasmid extraction",
"content": '

Target：To extract the target plasmid dspET28a

Material：

1. Buffer Sp1：to give a suitable environment
2. Buffer Sp2：Lysis of cell membrane
3. Buffer Sp3：stop the reaction
4. adsorption column
5. collecting pipe
6. centrifuge tube
7. Adsorption film：uptake plasmid
8. Wash solution：flushing impurity
9. Elution buffer：flushing target plasmid
10. Nanodrop：test DNA concentration

Procedure：

1. Check that RNase A has been added to Buffer SP1.
2. Check whether anhydrous ethanol has been added to the Wash Solution.
3. Check whether Buffer SP2 and SP3 are precipitated.
4. Adsorption column balance: Add 500μL Buffer S to the adsorption column (the adsorption column is put into the
   collection tube), centrifuge for 1min at 12,000 Xg, dump the waste liquid in the collection tube, and put the
   adsorption column back into the three collection tubes.
5. Take 1.5-5mL of overnight cultured bacterial solution, centrifuge 8,000Xg for 2 minutes to collect bacterial
   bodies, and discard the medium.
6. Add 250μL Buffer SP1 to the precipitate and completely suspend the bacteria.
7. Add 250 μL Buffer SP2 and immediately gently reverse the centrifugal tube for 10 times and mix well. Let stand at
   room temperature for 2-4 minutes.
8. Add 350 μL Buffer SP3 and immediately gently reverse the centrifuge tube 5-10 times to mix.
9. 12,000Xg centrifuge for 5-10 min. Transfer the supernatant into the adsorption column, centrifuge 8,000Xg for 30
   seconds, and drain the liquid in the collection tube.
10. (Optional) Add 500 μL Buffer DW1, centrifuge 9,000Xg for 30 seconds, and drain the liquid in the collection tube.
11. Add 500 μL Wash Solution, centrifuge 9,000Xg for 30 seconds, and drain the liquid in the collection tube.
12. Repeat Step 9 once .
13. The empty adsorption column was centrifuged at 9,000Xg for 1 minute.
14. Put the adsorption column into a clean 1.5mL centrifuge tube, add 50-100 μL Elution Buffer in the center of the
    adsorption film, stand at room temperature for 1 minute, and centrifuge for 1 minute. Preserve the DNA bath in the
    tube.

'
},{
"title": "2. PCR(polymerase chain reaction) ",
"content": '

(In vitro amplification: A section or loop of DNA
that is duplicated to increase its number)

Target：To amplify the target gene (Gly8-GIn,
Gly8-Ser, Gly8-Thr, Gly8-Cys, Gly8-Asn, Gly8-Tyr, Arg47-Lys, and Arg47-His) by PCR

Principle:

1. Denaturation (high temperature) : Splitting DNA
into single strands (breaking hydrogen bonds)

2. Annealing: Bind primer

3. Extension: Insert the base with DNA polymerase
(starting at the 3 \'- end of the primer)

4. Cycle: n= number of cycles, 2^n DNA

Material:

PCR tube,Template(DNA),Primer,Mix(DNA
polymerase,DNTP,MgCl2,buffer)--attenuation for 2x

ddH2O , PCR amplifierMVortexor, Centrifugal machine

Procedure：

PCR system：

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Ingredient | Volume(μL) | Ingredient | 25 | Ingredient | 25 | Ingredient | 25 |
| Mix | 25 | Mix | 19 | Mix | 19 | Mix | 19 |
| DdH2O | 19 | DdH2O | 2 | DdH2O | 2 | DdH2O | 2 |
| DsPETase | 2 | DsPETase | 2 | DsPETase | 2 | DsPETase | 2 |
| Gly8-Cys-F | 2 | Gly8-GIn-F | 2 | Gly8-Ser-F | 2 | Gly8-Thr-F | 2 |
| Gly8-Cys-R | 2 | Gly8-GIn-R | 25 | Gly8-Ser-R | 25 | Gly8-Thr-R | 25 |
| Mix | 25 | Mix | 25 | Mix | 25 | Mix | 25 |
| DdH2O | 19 | DdH2O | 19 | DdH2O | 19 | DdH2O | 19 |
| DsPETase | 2 | DsPETase | 2 | DsPETase | 2 | DsPETase | 2 |
| Gly8-Asn-F | 2 | Gly8-Tyr-F | 2 | Arg47-Lys-F | 2 | Arg47-His-F | 2 |
| Gly8-Asn-R | 2 | Gly8-Tyr-R | 2 | Arg47-Lys-R | 2 | Arg47-His-R | 2 |

PCR procedure：

|  |  |  |  |
| --- | --- | --- | --- |
| Step | Temperature(degree Celsius) | Time | Remark |
| predegeneration | 98 | 10min |  |
| degeneration | 98 | 30s |  |
| .annealing | 60 | 30s |  |
| .Extension | 72 | 3min |  |
| Extension | 72 | 10min | At the end, to check for gaps |
| Preservation | 4 | ∞ |  |

Note: 30 cycles of 2,3,4

'
},{
"title": "3. Agarose Gel Electrophoresis",
"content": '

Target: To use agarose gel electrophoresis to analyze
the molecular weight of nucleic acids

Material：

|  |  |
| --- | --- |
| Material | Dosage |
| Agarose | 1g |
| TAE buffer loom |  |
| Nucleic acid dye | 5μL |
| PCR product | 50μL |
| Uv photoscope |  |
| Marker |  |

Procedure：

1. Gel: agarose 1g+TAE buffer loom, heat, cool, add nucleic acid dye 5μL, add into the mold (against the negative
   pole po4^2-) to form glue block, add marker
2. Sample addition: 50μL PCR product
3. Running glue: 180v, 20min
4. Photoscope: UV photoscope (Gel imaging system)

'
},{
"title": "4. Gel extraction",
"content": '

Target：To recover the DNA from the target gel.

Material：

|  |  |
| --- | --- |
| Material | Dosage/usage |
| Gel of the target fragment | 1 |
| Buffer B2 | / |
| adsorption column | / |
| collecting pipe | / |
| centrifuge tube | / |
| Adsorption film | / |
| Wash solution |  |
| Elution buffer | / |
| Vortexor  Centrifugal machine | / |
| Water bath | 50 degree Celsius |

Procedure：

1. Preparation：

Check whether anhydrous ethanol has been added to the
Wash Solution.

Check Buffer B2 for precipitation.

Set the water bath to 50°C.

2. Cut the glue block containing the target fragment from the agarose gel and weigh it.
3. Add 3-6 times the weight of the glue block Buffer B2, 50°C water bath for 5-10 minutes sol.
4. (Optional) For fragments <500bp, add 1/3 Buffer B2 volume of isopropyl alcohol.
5. Transfer the sol solution into the adsorption column and centrifuge for 30 seconds at 8,000Xg.Drain the liquid
   from the collection pipe.
6. Add 500μL Wash Solution, centrifuge 9,000Xg for 30 seconds, and drain the liquid in the collection tube.
7. Repeat Step 6.
8. Centrifuge the empty adsorption column at 9,000Xg for 1 min.
9. Put the adsorption column into a clean 1.5mL centrifuge tube, add 15-40μL Elution Buffer in the center of the
   adsorption film, leave at room temperature for 1 minute, and centrifuge for 1 minute.Preserve the DNA solution in
   the tube.

'
},{
"title": "5. Homologous recombination",
"content": '

Target：To allow DNA molecules containing homologous
sequences to recombine between or within them

Material:

|  |  |
| --- | --- |
| Material | Dosage |
| Gly8-GIn, Gly8-Ser, Gly8-Thr, Gly8-Cys, Gly8-Asn, Gly8-Tyr, Arg47-Lys, and Arg47-His | 1.3µL |
| PET28a-1 | 1µL |
| Homologous recombinase | 5.7µL |
| 5xbuffer | 2µL |
| centrifuge tube | 1 |
| pipettor | 1 |

Procedure:

1. Sequentially add 2µL of 5xbuffer, 5.7µL of homologous recombinase, 1µL of PETase28a-1, and 1.3µL of IsPETase.
2. Vortex for one minute and centrifuge for 30s.
3. Put the centrifuge tube in 37℃ for 15 minutes.

'
},{
"title": "6. Transform",
"content": '

Target: Transform using the heat shock method.

Material:

|  |  |
| --- | --- |
| Material | Dosage |
| competent cell(CaCl₂) | 10µL |
| Homologous recombination product | 10µL |
| fluid Luria-Bertani broth | 900µL |
| centrifugal machine | 1 |
| centrifuge tube | 1 |
| pipettor | 1 |
| ice | No limit |

Procedure：Heat shock method

1. The 10μL homologous recombinant product was put into 10μL receptive cells
2. Leave on ice for 30 minutes
3. Water bath 42 degrees Celsius, 45s
4. 3 minutes on ice
5. Add 900μL LB medium, 37 ° C, 1h (resuscitation)
6. Centrifuge 5000xg, 5min, discard part of the supernatant
7. Apply the remaining E.coliDN5α/BL21 (DE3) to a petri dish with antibiotic Kan+ (kill bacteria)
8. Invert the petri dish and put it in the incubator at 37 degrees Celsius, 12-16h

'
}]
},{
"title": "Protein expression and
identification",
"content": '',
"children": [{
"title": "1. Growth curve",
"content": '

Goal: The goal of this process is to monitor the
colony density of the bacteria

Materials:

|  |
| --- |
| Name |
| LB base |
| bacteria sample |
| constant temperature shaker |
| UV-spectrophotometer |

Process

1. Modulate the culture solution(15ml LB-base, 1ml bacteria sample)
2. Culture the solution in the shaker, wait for 30minutes
3. After time is over, extract 1ml solution and replace the solution in the shaker
4. Move the shaker into the UV-spectrophotometer, record the first three data from the output
5. Repeat the process for a time interval of 1hour, 2 hours, 4 hours, 6 hours, 10 hours,16h,24h until the
   colony density(CD600 (600 stands for wavelength)) reaches a value between 0.6 to 0.8

'
},{
"title": "2. Protein introduction",
"content": '

**![图片4](https://static.igem.wiki/teams/5748/experiment/4.webp)**

Goal: Using the IPTG to trigger bacteria to produce
enzyme as protein

Material:

|  |  |
| --- | --- |
| Name | Amount |
| Culture solution from previous experiment |  |
| IPTG solution | 2ml per each solution |
| Constant temperature shaker |  |
| LB base | 5 liter |

Process:

1. When OD600 are about 0.6, add 1mm of IPTG, the ratio is 1:1000
2. Shake equally when IPTG is added, put them in constant temperature shaker for 12 hours, 16 degree Celsius.

'
},{
"title": "3. Break the cell membrane",
"content": '

Goal: Break the cell membrane and extract the protein

The goal of this process is to break the
cell membrane and extract the protein.

Materials:

|  |
| --- |
| Name |
| Ice |
| Bacterial sample |
| Ultrosonic equipment |
| Lysis buffer |
| Centrifuge |

 Process:

1. Centrifuge the bacterial sample solution to precipitate the solid.(7000W power, 30 minutes)
2. Remove the supernatant, add 20mL of Lysis buffer, and stir up the precipitate to blend with the buffer.
3. Put the solution on ice and operate with an ultrasonic instrument. (300W power, on for 3 seconds, off for 3
   seconds, soak for 30 minutes)

'
},{
"title": "4. Protein purification by nickel column",
"content": '

Goal: Collect the target protein by taking the
advantage of the binding and elution mechanism of nickel column.

Material

|  |
| --- |
| Name |
| Washing solution |
| Ice |
| timer |
| nickel column |
| Bacterial sample |
| 10 small test tube |
| Centrifuge |

Process:

1. Place the solution that has undergone ultrasonic disruption of the cell membrane into a centrifuge for
   centrifugation.
2. Load the nickel column and place it on ice for preparation.
3. After the protein solution has been centrifuged, take the supernatant and pour it into a clean test tube. Place
   the precipitate on ice for later use.
4. Pour a certain amount of the supernatant into the container with the nickel column for rinsing, so that the nickel
   column is dissolved in the supernatant. Then, rotate and refrigerate it at 4 degrees Celsius for two hours.
5. After refrigeration, remove the refrigerated supernatant and first filter it using the nickel column loading tube.
6. After filtration, place the loading tube with the nickel column on top of the small test tube and add 500
   microliters of washing solution to it. Wait for it to filter, repeat this step five times.
7. After elution is completed, seal the loading tube and add 500 microliters of elution solution to it. Shake until
   the solution becomes cloudy and then let it stand for two minutes.
8. After two minutes of standing, place the loading tube on top of the small test tube and wait for the liquid to
   filter.
9. Repeat steps 7 and 8 five times to obtain five samples.

'
},{
"title": "5. SDS-PAGE",
"content": '

Goal: Adding protein sample to gel and observe the
protein pillar(success if protein pillar on the gel is obvious)

Preparation:Take the protein samples out from ice and
pick number 1,2,7,8,9. Transfer 200ul of them to new tube. Then add 20ul of blue coloring to them and put them in
centrifuge, to make sure the solution are stable. Then put them in heater for 10 minutes.

Materials:

|  |  |
| --- | --- |
| Name | Amount |
| Lower layer buffer solution(2X) | 4ml |
| Lower layer adhesive solution | 4ml |
| Coagulant accelerator | Upper:20ul, lower:80ul |
| Upper layer buffer solution(2X) | 1ml |
| Upper layer adhesive solution(2X) | 1ml |
| Ethanol |  |
| Staining solution | 20ul per each protein sample |
| Electrophoresis model |  |
| Protein sample from previous experiment | 30ul when transfer them to the gel |

1. Organize the gel model first, assemble the two layers of glass together, remain 1.5mm space between them.
2. Put all devices in the model together, then verify whether it’s sealed by adding purifies water between the glass
   carefully, them observe it and check whether there is water in bottom.
3. If it’s sealed, pour the water out then make the lower gel.
4. Add 4ml of lower layer glue buffer solution and adhesive solution to the flask by using pipette, then add 80ul
   coagulant accelerator, mix them together.
5. When it’s mixed, transfer the solution to the model using pipette, take 1ml a time and slowly drip them in the
   gap, make sure to drip equally in both side of the glass.
6. Left it still about 15 minutes, and see if it becomes solid, then add some ethanol on the top of gel to make the
   surface of the gel flat. After that, make the upper layer.
7. Add 1ml of upper layer glue buffer solution with pink color and adhesive solution to another flask, then add 20ul
   coagulant accelerator to it and mix them together. Using pipette to transmit the mixture to the model, the process
   is similar to lower layer one. Then add a comb like devices to make spaces for protein substance to intake.
8. Left it still for 15 minutes until it become solid.
9. Take the protein sample from previous experiment out of the heater with blue coloring and use pipette to transfer
   these substance to the gel, drip them vertically and make sure it do not spill to other gap.
10. Wait for about 2 hours until the marking are appeared in the gel, them analyze the results.

'
}]}, {
"title": "Functional Test",
"content": '

****![图片2-](https://static.igem.wiki/teams/5748/experiment/5.webp)****

',
"children": [{
"title": "1. Determination of MHET by HPLC",
"content": '

Goal:Using HPLC method to test the degrading rate of
MHET

Materials:

|  |  |
| --- | --- |
| Name | Amounts |
| plastic particles | 0.5g |
| 8 protein of each mutant | 100ul |
| MHETase | 100ul |

Sample process:

1. Write the name of protein name on new microtude.
2. Weight 0.5g PETase particles and transfer them to new microtube
3. Add 200ul protein samples(such as IsPETase,dsPETase, His, Gln, Asn) to that microtude and put them on scroll
   machine for about 10 seconds, until the mixture are will mixed.

**MHET HPLC process:**

**a. Experimental Instrument Parameters**

Detector: DAD

Column: Thermo Scientific C18, 250 mm \* 4.6 mm i.d.,
5 μm particle size

Column Temperature: 30 °C

Flow Rate: 1 mL/min

Wavelength: 242 nm

Injection Volume: 20 μL

Mobile Phase: Phosphate buffer solution (containing 5
mmol/L potassium dihydrogen phosphate, 0.04% phosphoric acid) : Methanol = 70 : 30 (v/v)

**b. Standard Solution Preparation**

An accurate amount of ethylene phthalate (PET)
standard was weighed into a 5 mL brown volumetric flask. An appropriate amount of 0.1 mol/L NaOH solution was added,
and the mixture was sonicated to dissolve the standard. The solution was then diluted to the mark with ultrapure
water, mixed thoroughly to prepare a 1 mg/mL single-standard stock solution, and stored at -20 °C.

The standard working curve was prepared using
ultrapure water for dilution.

**c. Sample Extraction Process**

The sample was homogenized, filtered through a 0.45
μm membrane filter, and then subjected to HPLC analysis.

'
},{
"title": "2. Determination of TPA and EG by HPLC",
"content": '

Goal: Calculating the speed of plastic degrading and
determain the most efficient one.

Material:

|  |  |
| --- | --- |
| Name | Amount |
| plastic particle | 5 g total |
| MHETase | 400ul |
| IsPETase | 400ul |
| DsPETase | 400ul |
| Ser | 400ul |
| ddH2O | 800ul |
| HPLC |  |

Sample Process:

1. Weigh 1g of plastic particles in each test tube and write the tag, write the name that is going to add.
2. Add the according enzyme to the test tube about 400ul for each, then add 400ul of MHETase to each test tube about
   400ul.
3. Left it in the refrigerator for 48h and 72h, add additional enzyme if it’s not enough.
4. Take some sample to test the degrading rate after 72 hours.

**TPA and EG HPLC process:**

1. **Experimental Instrument Parameters**

Detector: DAD

Column: Thermo Scientific C18, 250 × 4.6 mm, 5 μm

Column Temperature: 30 °C

Flow Rate: 1 mL/min

Wavelength: 242 nm

Injection Volume: 20 μL

Mobile Phase: Phosphate buffer solution (containing 5
mmol/L potassium dihydrogen phosphate and 0.04% phosphoric acid) : Methanol = 70 : 30

**2. Standard Solution Preparation**

Accurately weigh an appropriate amount of
terephthalic acid standard into a 5 mL brown volumetric flask. Add a suitable volume of 0.1 mol/L NaOH solution, and
dissolve by sonication. Dilute to the mark with ultrapure water, mix well to prepare a 1 mg/mL single-standard stock
solution, and store at -20 °C.

Prepare the standard working curve using ultrapure
water.

**3. Sample Treatment**

Mix the sample homogenously, filter through a 0.45 μm
membrane filter, and then analyze by HPLC.

'
},{
"title": "3. Scanning transmission electron microscope",
"content": '

Goal: Determain the rate to degrading PET by using
PETase and MHETase

Materials:

|  |  |
| --- | --- |
| Name | Amount |
| Plastic film | 5 |
| MHETase | 100ul |
| DsPETase, IsPETase, Ser, ddH2O | 100ul |
| Scanning transmission electron microscopoe |  |

Process

1. Cut 5 plastic film, the size is 1cm time 1cm.
2. Put them on cultivating base, make sure to make it flat.
3. Add 100ml of MHETase and 100ml of Ser/isPETase/dsPETase/Ser on the plastic, make sure not to make the plastic film
   curve and drip these enzyme equally.

'
}]
}]%}
{% block title %}Experiments{% endblock %}
{% block lead %}

Experiments

{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Summary",
"content": '

Plastic pollution has emerged as a critical global
environmental issue, with polyethylene terephthalate (PET) representing one of the most persistent and widely used
plastics. Current enzymatic degradation methods for PET exhibit limited efficiency, hindering their application on an
industrial scale(Guo etal,2025; V etal, 2020). To address this challenge, we aim to develop enhanced enzymatic
strategies for PET biodegradation and recycling by screening and engineering high-efficiency plastic-degrading
enzymes. Our objective is to improve the catalytic conversion of PET into its monomers, terephthalic acid (TPA) and
ethylene glycol (EG), thereby providing a sustainable and innovative bioremediation solution(Fig 1). The resulting
engineered enzymes are expected to demonstrate significantly improved PET degradation performance, with potential
applications in environmental remediation and industrial plastic waste processing.

![1758001182746](https://static.igem.wiki/teams/5748/results/1.webp)

Fig 1. Degradation process of PET plastic and key
enzymes [Yoshida etal,2016]

'
},{
"title": "Results of
work",
"content": '',
"children": [{
"title": "Section 1: PETase (IsPETase,
dsPETase, Mutants)",
"content": '

1. ****Plasmid Construction****
   1. ****pET28A-IsPETase****

   ![](https://static.igem.wiki/teams/5748/results/2.webp)

   Fig. 2.The electrophoresis, plate Assay, and
   sequencing of IsPETase. Note: IsPETase：879bp; pET28a：5323bp

   The pET-28a plasmid vector was linearized for the
   construction of the expression plasmid. The plasmid containing the IsPETase gene was constructed using homologous
   recombination, followed by confirmation of successful construction via agarose gel electrophoresis. The Fig 2A showed
   that the IsPETase gene is between 500bp and 1000bp markers. The results are showed in Fig 2B, the linearized plasmid
   pET28a is highlighted 5000bp. The expansion length of gene is consistent with the DNA encoding and indicate the
   succeed of amplification. The Fig 2C showed that the recombinant plasmids was transformed into processed
   *E.coli* DH5α to copy the plasmids. As shown in Fig 2D, the sequencing results revealed a perfect match to the
   target gene sequence without any mutations, demonstrating the successful construction of the plasmid pET28a-IsPETase.

   2. ****pET28A-dsPETase****

   ![](https://static.igem.wiki/teams/5748/results/3.webp)

   Fig. 3.The electrophoresis, plate Assay, and
   sequencing of dsPETase

   The construction of the dsPETase plasmid employed an
   identical strategy to that of the IsPETase plasmid, with both based on the method of homologous recombination. As
   shown in Fig 3A, the dsPETase gene migrated between 500 and 1000 bp. This is consistent with its actual length of 816
   bp, which was determined by sequencing. The linearized pET28a line is around 5000bp and its original length is 5329bp.
   We was transformed the pET28a-dsPETase plasmid into *E.coli* in the Fig 3C. In Fig 3D, sequencing of the
   plasmid confirmed that the inserted DNA fragment matched the target gene sequence, demonstrating successful ligation
   into the vector.

   To identify key active sites in dsPETase for
   mutagenesis, we constructed a three-dimensional protein structure of dsPETase and performed molecular docking with the
   chemical structure of PET(Fig 4). The docking score (binding energy) serves as an indicator to evaluate the affinity
   between PET and protein dsPETase. A stronger affinity results in tighter binding and more stable molecular
   interactions. A more negative docking score corresponds to a stronger affinity between the molecules.

   **![图片3](https://static.igem.wiki/teams/5748/results/4.webp)**

   Fig 4. Docking results of dsPETase with the PET
   molecule; Red represents the PET molecule.

   **![图片2](https://static.igem.wiki/teams/5748/results/5.webp)**

   Fig 5. Schematic diagram of the active site of
   dsPETase with the PET molecule.

   The optimal binding mode between the PET molecule and
   dsPETase is shown in Figs 4 and 5. The molecular docking binding energy was calculated to be −3.249 kcal/mol, with
   stabilization primarily achieved through hydrogen bonding (Table 1). Key amino acid residues forming hydrogen bonds
   with the PET molecule were glycine at position 8 (Gly8) and arginine at position 47 (Arg47), which enhanced the
   stability of the PET molecule within the binding pocket.The molecular docking results validated the feasibility of
   selecting Gly8 and Arg47 for mutagenesis, a decision initially made based on expert opinions during the experimental
   design phase. However, experimental validation is required to confirm the degradation efficiency of the mutants and
   ensure the reliability of the result.

   Based on the advice of biology Professor Yao, we
   performed site-directed mutagenesis on the active site residues glycine (Gly8) and arginine (Arg47). Specifically,
   Gly8 was mutated to amino acids with similar properties—namely, polar neutral R-group residues such as glutamine
   (Gln), serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn), and tyrosine (Tyr). This strategy helps
   minimize structural perturbations that could lead to loss of protein activity. The arginine at position 47 was mutated
   to amino acids with similar properties, specifically basic R-group residues such as lysine (Lys) and histidine (His).
   The resulting mutants were designated as Arg47-Lys, Arg47-His, Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and
   Gly8-Tyr. As shown in Table 1, the docking scores of these mutants were significantly improved compared to that of
   wild-type dsPETase, indicating stronger binding affinity with PET.

   Table 1. Docking scores (binding energy,
   kcal/mol) of PET with dsPETase and mutants.

   |  |  |  |  |  |  |  |  |  |  |
   | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
   | Name | dsPETase | Gly8-GIn | Gly8-Ser | Gly8-Thr | Gly8-Cys | Gly8-Asn | Gly8-Tyr | Arg47-Lys | Arg47-His |
   | docking score | -3.249 | -5.103 | -4.932 | -5.151 | -4.945 | -5.002 | -5.204 | -5.096 | -5.012 |

   3. ****Mutant****

   **1.3.1 Mutant Arg47-Lys and Arg47-His**

   **![图片34](https://static.igem.wiki/teams/5748/results/6.webp)**

   Fig 6. Molecular interactions between the protein and
   the ligand within the binding pocket. Note: The protein structures of the Arg47-Lys and Arg47-His mutants are depicted
   in blue. The PET molecule is shown in orange.

   The binding energies between the PET molecule and the
   mutant proteins Arg47-Lys and Arg47-His were −5.076 kcal/mol and −5.012 kcal/mol, respectively, indicating strong
   molecular interactions. As shown in Fig 6, the PET molecule interacts with the Arg47-Lys mutant primarily through
   hydrogen bonds and hydrophobic interactions. Hydrogen bonds were formed with LEU-70 and ALA-71, while hydrophobic
   interactions occurred with VAL-43, LEU-70, ALA-71, and ARG-68. In the Arg47-His mutant, the PET molecule was involved
   in hydrophobic interactions with the specific residues PHE-44, TYR-118, and THR-117 (Fig 6).

   **1.3.2 Mutant (Gly8-Gln, Gly8-Asn, Gly8-Cys,
   Gly8-Ser, Gly8-Thr, and Gly8-Tyr)**

   ![图片30](https://static.igem.wiki/teams/5748/results/7.webp)

   Fig 7. Molecular interactions between the protein and
   ligand within the binding pocket. The mutant protein structure is shown in blue, and the PET molecule is depicted in
   orange.

   The binding energy between the PET molecule and the
   Gly8-Asn mutant protein was calculated to be −5.002 kcal/mol, indicating strong binding affinity. As shown in Figure
   7, the PET molecule interacts with the Gly8-Asn mutant primarily through hydrogen bonding and hydrophobic
   interactions. Specifically, a hydrogen bond was formed with TYR-106, while hydrophobic interactions were observed with
   PHE-44, THR-117, and TYR-118.

   The binding energy for the PET molecule with the
   Gly8-Cys mutant was −4.945 kcal/mol, demonstrating favorable binding interactions. Figure 7 shows that the PET
   molecule engages in hydrophobic interactions with LEU-103 and LYS-102, as well as electrostatic interactions with
   LYS-102 and ARG-99 in the Gly8-Cys mutant.

   ![图片31](https://static.igem.wiki/teams/5748/results/8.webp)

   Fig 8. Molecular interactions between the protein and
   ligand within the binding pocket. The mutant protein structure is shown in blue, and the PET molecule is depicted in
   orange.

   The binding energies between the PET molecule and the
   Gly8-Gln and Gly8-Ser mutant proteins were calculated to be −5.103 kcal/mol and −4.932 kcal/mol, respectively,
   indicating strong binding affinity. As shown in Fig 8, the PET molecule interacts with the Gly8-Gln mutant primarily
   through hydrogen bonding and hydrophobic interactions. Specifically, a hydrogen bond was formed with TYR-106, while
   hydrophobic interactions were observed with PHE-44, THR-117, and TYR-118. Fig 8 illustrates that the PET molecule
   engages in hydrophobic interactions with LEU-103 and LYS-102, as well as electrostatic interactions with LYS-102 and
   ARG-99 in the Gly8-Ser mutant.

   ![图片36](https://static.igem.wiki/teams/5748/results/9.webp)

   Fig 9. Molecular interactions between the protein and
   ligand within the binding pocket. The mutant protein structure is shown in blue, and the PET molecule is depicted in
   orange.

   The binding energies between the PET molecule and the
   Gly8-Thr and Gly8-Tyr mutant proteins were calculated to be −5.151 kcal/mol and −5.204 kcal/mol, respectively,
   indicating strong binding interactions. In Fig 9, the PET molecule engages in hydrophobic interactions with PHE-44,
   TYR-118, and THR-117 in the Gly8-Thr mutant. Fig 9 illustrates that the PET molecule interacts with the Gly8-Tyr
   mutant primarily through hydrogen bonding and hydrophobic interactions. Specifically, a hydrogen bond was formed with
   TYR-106, while hydrophobic interactions were observed with PHE-44 and TYR-118.

   ![图片23](https://static.igem.wiki/teams/5748/results/10.webp)

   Fig 10. Agarose gel electrophoresis analysis
   of the mutant

   We performed site-directed mutagenesis using PCR. As
   shown in Fig 10, the DNA fragments migrated between 5000 bp and 7500 bp, with all mutants (Arg47-Lys, Arg47-His,
   Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and Gly8-Tyr) exhibiting a consistent length of 6190 bp. The
   plasmids were subsequently sent for commercial sequencing(Fig 11), and the results confirmed that the introduced
   mutations matched the expected designs.

   ![图片36](https://static.igem.wiki/teams/5748/results/11.webp)

   Fig 11. The sequencing of
   the mutant
2. ****Protein Expression****

![图片70](https://static.igem.wiki/teams/5748/results/12.webp)

Fig 12.The growth curve and SDS-PAGE of
IsPETase

We measured the OD600 at various time points.
As shown in Figure 12A, no significant difference was observed between the growth curves of the control group and
the IsPETase-expressing strain, indicating robust bacterial growth.

We engineered the expression construct to incorporate
a specific gene sequence encoding a hexahistidine (His-tag) affinity tag, enabling ribosomal synthesis of the
recombinant protein with a C-terminal His-tag. The His-tagged protein was subsequently purified using Ni-NTA affinity
chromatography, whereby the immobilized nickel ions selectively bind the His-tag. Non-specifically bound proteins were
removed by washing with buffer, and the target protein was eluted under native conditions using imidazole-containing
elution buffer.

In SDS-PAGE analysis, the crude protein dsPETase
exhibited diffuse banding patterns, indicative of the presence of non-target cellular proteins. Following
purification, the Fig 12B result demonstrated that the molecular weight of the recombinant protein was between 25 kDa
and 35 kDa, which is consistent with its theoretical molecular mass of 33.5 kDa.

![dsPETase(1)](https://static.igem.wiki/teams/5748/results/13.webp)

Fig 13.The growth curve of dsPETase and
mutant

As shown in Figure 13, no significant
difference was observed between the growth curves of the control group and the dsPETase and mutant strain,
indicating robust bacterial growth.

![图片71](https://static.igem.wiki/teams/5748/results/14.webp)

Fig 14. The SDS-PAGE of dsPETase and mutant.
M:marker 1,2:rough protein; 3-5:Wash buffer 6-9:purified protein. DsPETase:31.5kDa

As shown in Figure 14A, lanes 1–2 exhibited diffuse
bands in the crude protein samples, suggesting the presence of a substantial amount of non-specific proteins. Lanes
3–5, which served as blank controls, showed no detectable target band corresponding to dsPETase. In lanes 6–9, clear
protein bands were observed within the molecular weight range of 25–35 kDa. These samples were purified by
nickel-affinity chromatography, wherein Ni-NTA resin selectively captured His-tagged proteins, wash buffer removed
non-specifically bound impurities, and the target protein was subsequently eluted under appropriate conditions. Figure
14B displays the protein expression profiles of various mutants. Band intensity and thickness reflect relative
expression levels, with the Gly8-Ser mutant showing the darkest and thickest band, indicating its highest expression
among all variants.

3. ****Function test****

The chromatographic conditions were optimized using a
Thermo Scientific C18 column (250 × 4.6 mm, 5 μm) with a mobile phase consisting of phosphate buffer (containing 5
mmol/L potassium dihydrogen phosphate and 0.04% phosphoric acid) and methanol in a ratio of 70:30 (v/v). According to
the experimental protocol, standard solutions were serially diluted to appropriate concentrations to establish a
calibration curve. The peak area was plotted on the Y-axis against the concentration of the reference standard on the
X-axis, and regression analysis was performed to obtain the equation for each standard. A good linear relationship was
observed within the concentration range of 1–50 mg/L (Table 2).

Table 2. The standard curve of MHET

|  |  |  |
| --- | --- | --- |
| **Concentrations**  **(**mg/L) | Peak area | ![](https://static.igem.wiki/teams/5748/results/tabel-2.webp) |
| 1 | 58.41312 |
| 2 | 130.59349 |
| 5 | 357.34796 |
| 10 | 697.24371 |
| 20 | 1393.50171 |
| 50 | 3568.67041 |

We incubated 500 mg of PET plastic substrates with
IsPETase, dsPETase, and their variants at a final concentration of 1 mg/mL, then collected samples after 48 and 72
hours of degradation at room temperature. The peak area of MHET was measured by HPLC, and its concentration in
the experimental samples was calculated based on the standard curve.

As shown in Fig 15A, MHET was detected in the
experimental groups of IsPETase, dsPETase, and their mutants at 48-hour, whereas MHET was undetectable in the CK
control group. Comparative analysis revealed no significant difference in MHET levels between IsPETase and dsPETase
at the 48-hour. In contrast, all tested mutants—namely Arg47-Lys, Arg47-His, Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser,
Gly8-Thr, and Gly8-Tyr—exhibited significantly higher MHET accumulation compared to dsPETase. Notably, the Gly8-Ser
mutant generated markedly elevated MHET levels relative to the other mutants. Furthermore, there was no
statistically significant difference in MHET production between the Arg47-Lys and Arg47-His variants.

In Fig 15B, MHET was detected in the
experimental groups of IsPETase, dsPETase, and their mutants after 72 hours, while no MHET was observed in the CK
control group. The MHET level produced by dsPETase was significantly higher than that of IsPETase at 72h. The MHET
concentration of Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and Gly8-Tyr mutants exhibited markedly higher
than dsPETase. In contrast, no significant difference in MHET levels was observed among Arg47-Lys, Arg47-His, and
dsPETase. These results suggest that Gly8 may represent a critical active site in dsPETase. Notably, the
substitution of Gly8 with Ser significantly enhanced the degradation efficiency of PET.

****![图片90](https://static.igem.wiki/teams/5748/results/15.webp)****

Fig 15. MHET content of IsPETase, dsPETase,
and mutantsArg47-Lys, Arg47-His, Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and Gly8-Tyr) at 48 and 72
hours.

'
},{
"title": "Section 2：MHETase",
"content": '

1. ****Plasmid Construction****

![](https://static.igem.wiki/teams/5748/results/16.webp)

Fig 16.The electrophoresis, plate Assay, and
sequencing of MHETase.

Note:the length of MHETase is 1818bp. the length of
pET28a is 5324bp

The agarose gel electrophoresis result in Fig 16A and
B showed a band of the MHETase gene at approximately 1500 bp and a linearized plasmid band near the 3000 bp marker.
After ligation, the recombinant plasmids were transformed into E. coli DH5α competent cells for amplification in the
Fig16 C. The subsequent sequencing analysis confirmed the successful insertion of the target gene MHETase into the
vector(Fig 16 D).

2. ****Protein Expression****

![图片60](https://static.igem.wiki/teams/5748/results/17.webp)

Fig 17.The growth curve and SDS-PAGE of
IsPETase. Note:The size of MHETase is 64kDa.

To express the target protein, the plasmid
was transformed into *E. coli* BL21(DE3) cells and induced with 1 mM IPTG, followed by protein extraction and
purification. In Figure 17A, no significant difference was observed between the growth curves of the control group
and the dsPETase and mutant strain, indicating robust bacterial growth. The SDS-PAGE analysis showed that the
purified MHETase protein was approximately 63 kDa, which is consistent with its actual molecular size.

3. ****Function test****

****3.1 S**canning electron
microscopy (SEM)**

![](https://static.igem.wiki/teams/5748/results/18.webp)

Fig. 18. Results of scanning electron microscopy
(SEM) of PET. A: control group; B:IsPETase+MHETase; C:DsPETase+MHETase; D:Gly8-Ser+MHETase

Fig 18A shows the PET film from the control group
without enzyme addition, exhibiting a smooth surface without cracks or pores. Fig1 8 B presents the experimental group
treated with IsPETase and MHETase, where faint fissures observed on the film surface indicate initial enzymatic
activity, though the degradation effect remains suboptimal. In Fig18 C, the sample co-treated with dsPETase and
MHETase demonstrates more distinct fissure structures, suggesting enhanced degradation efficacy compared to the
IsPETase group. Figure D displays the Gly8-Ser variant + MHETase group.
Numerous pronounced fissures can be observed on the film surface, demonstrating the superior degradation performance
of the Gly8-Ser variant.

**3.2 HPLC determination of concentration TPA
and EG**

The experimental instrument parameters were adjusted
as follows: the column was a Thermo Fisher C18 (250 mm × 4.6 mm, 5 μm), with a column temperature of 30°C, flow rate
of 1 mL/min, and a mobile phase consisting of phosphate buffer solution (containing 5 mmol/L potassium dihydrogen
phosphate and 0.04% phosphoric acid) and methanol at a ratio of 70:30.

According to the experimental protocol, the standard
was diluted to appropriate concentrations to establish a calibration curve. Using the peak area as the ordinate (Y)
and the mass concentration of the reference standard as the abscissa (X), regression analysis was performed to derive
the equation for each standard. A good linear relationship was demonstrated within the concentration range of 1–50
mg/L. The tested concentrations and corresponding calibration curve are presented in Table 3.

Table 3. The standard curve of TPA

|  |  |  |
| --- | --- | --- |
| **Concentrations**  **(**mg/L) | Peak area | ![https://static.igem.wiki/teams/5748/results/tabel-3.webp](https://static.igem.wiki/teams/5748/results/table-3.webp) |
| 1 | 85.65078 |
| 2 | 204.93451 |
| 5 | 584.46924 |
| 20 | 2220.17896 |
| 50 | 6006.84082 |

We incubated 1 g PET plastic with 1 mg/mL IsPETase,
dsPETase, and the Gly8-Ser variant, along with MHETase at a final concentration of 1 mg/mL. Under the catalytic action
of PETase and MHETase, PET can be degraded to TPA, which was quantified using HPLC. As shown in Fig 19, the control
group (pET28a vector) showed no peak at 10–12 minutes, whereas distinct peaks were observed for the IsPETase +
MHETase, dsPETase + MHETase, and Gly8-Ser variant + MHETase groups.

![图片100](https://static.igem.wiki/teams/5748/results/19.webp)

Fig. 19. Chromatographic peak of TPA

As illustrated in Figure 20, no TPA was detected in
the control (CK) group. The TPA concentrations produced in dsPETase + MHETase and Gly8-Ser + MHETase were
significantly higher than IsPETase + MHETase. Furthermore, the TPA concentrations from the Gly8-Ser+ MHETase
combination was markedly higher than that of dsPETase + MHETase. These results indicate that the degradation
efficiency of dsPETase + MHETase exceeds that of IsPETase + MHETase, and the Gly8-Ser variant in combination with
MHETase demonstrated superior performance compared to all other experimental groups. This evidence confirms that
site-directed mutagenesis enhanced the enzymatic activity of PETase, thereby significantly improving the overall
degradation efficiency.

![图片57](https://static.igem.wiki/teams/5748/results/20.webp)

Fig. 20. The concentration of TPA of CK, IsPETase +
MHETase, dsPETase + MHETase, and Gly8-Ser+ MHETase

EG (Ethylene Glycol) was qualitatively and
quantitatively analyzed in complex mixtures using Gas Chromatography-Mass Spectrometry (GC-MS). The instrument
parameters were set as follows:

Gas Chromatograph: Agilent GC 8860 equipped with CTC
PAL3 autosampler

Mass Spectrometer: Agilent 5977B MSD

Column: Agilent DB-WAX (30 m × 0.25 μm)

Carrier Gas: High-purity helium (≥99.99999%)

Chromatographic conditions:

Detector temperature: 240°C

Injector temperature: 220°C

Flow rate: 1.0 mL/min

Temperature gradient: Initial temperature 80°C (hold
5 min), then ramp at 5°C/min to 240°C (hold 1 min).

![图片54](https://static.igem.wiki/teams/5748/results/21.webp)

Fig. 21. The chromatographic peak, ratio, mass
scharge ratio of EG.

As shown in Fig 21, the detection results of ethylene
glycol (EG) in the samples were obtained using high-performance liquid chromatography (HPLC) and mass spectrometry
(MS). The control group exhibited no detectable peaks, indicating the absence of EG in the sample. Distinct
chromatographic peaks were observed in the experimental groups containing IsPETase + MHETase, dsPETase + MHETase, and
the Gly8-Ser + MHETase.The concentration of EG in the samples was calculated based on the chromatographic peak areas
using the standard curve equation y = 6.306x + 363.8. As shown in Figure 22A, the calibration curve exhibited an
excellent fit with a coefficient of determination (R²) greater than 0.99.

In Fig 22B, the EG concentration obtained with
dsPETase + MHETase was significantly higher than that with IsPETase + MHETase. Notably, the Gly8-Ser variant + MHETase
combination yielded a substantially greater EG concentration compared to both the dsPETase and IsPETase. These results
indicate that the Gly8-to-Ser mutation at position 8 in dsPETase enhances enzymatic activity, significantly improving
the conversion efficiency of EG.

![图片81](https://static.igem.wiki/teams/5748/results/22.webp)

Fig 22. The concentration of EG of CK, IsPETase + MHETase, dsPETase + MHETase, and Gly8-Ser+MHETase

'
}]
}, {
"title": "Analysis and Discussion",
"content": '

Based on the experimental results, the plasmids for
IsPETase, dsPETase, MHETase, and their mutants were successfully constructed, and the target proteins were effectively
expressed. As predicted in Figure 5, dsPETase possesses 13 potential active sites. According to the data presented in
Figure 15, the Gly8-Gln, Gly8-Asn, Gly8-Cys, Gly8-Ser, Gly8-Thr, and Gly8-Tyr mutants exhibited significantly enhanced
enzymatic activity. This suggests that Glycine at position 8 may be a critical active site, while other unmutated
sites could also potentially contribute to catalytic function, representing a promising direction for future
investigation. Additionally, site-directed mutagenesis of dsPETase may alter its optimal pH and temperature profiles.
Subsequent studies should explore the effects of varying pH and temperature on PETase activity to identify the optimal
reaction conditions (Arnal et al., 2023; Guangyi et al., 2024).

In addition, the synergistic action of dsPETase and
MHETase enables the depolymerization of PET into terephthalic acid (TPA) and ethylene glycol (EG), achieving effective
recycling and utilization of plastic waste. As shown in Results 19 and 21, the TPA and EG yields in the
Gly8-Ser+MHETase group were significantly higher than those in other groups, demonstrating the feasibility of the
dual-enzyme synergistic system (McGeehan et al., 2020). Future work may explore the use of a single microbial strain
for co-production of both enzymes, for instance, by employing a pETDuet co-expression vector to achieve dual-gene
expression.

Our research holds significant implications for
humanity by reducing petroleum resource consumption, decreasing carbon emissions, and enabling the recycling of PET,
which effectively mitigates the greenhouse effect. For instance, the production energy consumption of recycled PET is
only 30% of that of virgin PET (La et al., 2025). Furthermore, it helps control white pollution by reducing the
accumulation of microplastics in soil and water bodies, thereby protecting ecosystems. Ultimately, we aim to achieve a
"plastic → monomers → new plastic" zero-waste circular model that promotes sustainable development. In summary, PET
recycling provides critical support for sustainable development through synergistic environmental, economic, and
social benefits, while advanced technologies such as enzymatic degradation are accelerating the industrialization of
this process (Arnal et al., 2023).

'
},{
"title": "References",
"content": '

An engineered PET-depolymerase to break down and
recycle plastic bottles. Nature, 580(7802), 216-219.

Arnal, G., et al. (2023). The diversity of PET
degrading enzymes: A systematic review of sequence, structure, and function. Protein Science, 34(10), e70282.
https://doi.org/10.1002/pro.70282

Guo, Z., Li, Y., Wang, M., & Ma, D. (2025).
Catalytic upcycling of PET: From waste to chemicals and degradable polymers. Accounts of Chemical Research.

Guangyi Fan, Global marine microbial diversity and
its potential in bioprospecting, Nature (2024). DOI: 10.1038/s41586-024-07891-2.
www.nature.com/articles/s41586-024-07891-2

La Mantia, F. P., Titone, V., Botta, L., &
Glerean, P. (2025). Mechanical recycling processes of PET in bottle-to-bottle circular manufacturing. Journal of
Applied Polymer Science.

McGeehan, J. E., et al. (2020). Characterization and
engineering of a two-enzyme system for plastics depolymerization. Proceedings of the National Academy of Sciences of
the United States of America, 117(41), 25476–25485. https://doi.org/10.1073/pnas.2006753117

V T ,M C T ,A G , et al.An engineered PET
depolymerase to break down and recycle plastic bottles.[J].Nature,2020,580(7802):216-219.

Tournier, V., et al. (2023). Enzymatic recycling of
PET plastic: From discovery to industrial implementation. Nature Reviews Chemistry, 7(12), 875–890.
https://doi.org/10.1038/s41570-023-00527-5

Yoshida, S., et al. (2016). A bacterium that degrades
and assimilates poly(ethylene terephthalate). Science, 351(6278), 1196–1199. https://doi.org/10.1126/science.aad6359

'
}]%}
{% block title %}Results{% endblock %}
{% block lead %}

Results

{% endblock %}