iGEM 2025 — Team tjusls-china

![Science background](https://static.igem.wiki/teams/5806/static/home/header1-project.webp)

Noteboook

Lv Ziwan

Huang Yixuan

Liu Cheng

Lv Haonan

Wang Cong

Xia Tingting

Yu Yue

Yu Zihan

Zhou Tianchang

ZhuGe xin hao

Zhang Yutian

Liu Chang

Li Qike

Li Menglin

![Science background](https://static.igem.wiki/teams/5806/static/home/header1-project.webp)

Navigation

 [Overview](#part1)
 [Engineering 1 —— Optimization of
Reaction Conditions for
PET05 and 40 New Enzymes](#part2)
[Engineering 2 —— Engineering
Modification of PET05](#part3)

Engineering

# Overview

In our project, we conducted two engineering efforts: we investigated the optimal reaction
conditions and physiological properties of PET05 and 40 new enzymes, and completed the
modification of PET05 for enhancing its enzymatic activity and thermostability.

# Engineering 1 —— Optimization of Reaction Conditions for PET05 and 40 New Enzymes

# 1.1 Optimization of Reaction Conditions for PET05

## ***Cycle 1：***

### Design：

First, we designed the following orthogonal experiment:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| pH | | | NaCl(M) | | | | | | Enzyme Loading (nM) | | | Temperature(℃) | |
| 8 | 9 | 10 | 0 | 1 | 2 | 3 | 4 | 5 | 25 | 50 | 100 | 30 | 50 |
| - | + | - | + | - | - | - | - | - | - | + | - | - | + |
| - | + | - | - | + | - | - | - | - | - | + | - | - | + |
| - | + | - | - | - | + | - | - | - | - | + | - | - | + |
| - | + | - | - | - | - | + | - | - | - | + | - | - | + |
| - | + | - | - | - | - | - | + | - | - | + | - | - | + |
| - | + | - | - | - | - | - | - | + | - | + | - | - | + |
| + | - | - | - | + | - | - | - | - | - | + | - | - | + |
| - | - | + | - | + | - | - | - | - | - | + | - | - | + |
| - | + | - | - | + | - | - | - | - | + | - | - | - | + |
| - | + | - | - | + | - | - | - | - | - | - | + | - | + |
| - | + | - | - | + | - | - | - | - | - | + | - | + | - |

### Build：

The plasmid vector we used was pET-32a(+), and proteins expressed from this vector carry a
TrxA tag at the C-terminus. Based on preliminary experiments, we found that PET05 with the
TrxA tag had no enzymatic activity. Additionally, it is known from literature that PET05 is
highly soluble; therefore, we performed tag removal on PET05. We successfully purified PET05
without the TrxA tag and used NanoDrop to determine the concentration of the purified
protein solution. Using the Expasy - ProtParam website, we calculated that **the
relative
molecular mass of PET05 is 27724.34 Da, the extinction coefficient is 1.431, and the
isoelectric point is 5.52.**

According to the formula:

**Mass (ng) = Enzyme Loading (nM) × Reaction Volume (μL) × Relative Molecular Mass of
the Enzyme (g/mol) × 1000**

we can calculate the mass of PET05 to be added in a 600 μL reaction system.According to the
formula:

**Volume of Enzyme Solution to be Added (μL) = Mass (ng) × Extinction Coefficient /
Concentration of the Enzyme Solution (ng/μL)**

we can calculate the volume of the PET05 enzyme solution to be added.

### Test：

We completed 11 groups of orthogonal experiments with 3 parallel replicates in each group,
and determined the release amounts of the products TPA and MHET after 18 hours of reaction
via ultra performance liquid chromatography (UPLC). The detailed experimental data results
can be found in [here](results#jump1).

### Learn：

We found that PET05 only **exhibits enzymatic activity in buffer solutions with a NaCl
concentration of 4-5 M.**

## ***Cycle 2：***

### Design：

Next, we increased the overall NaCl concentration of the reaction system and designed the
following second round of orthogonal experiment:

|  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| pH | | | NaCl(M) | | | Enzyme Loading (nM) | | | | Temperature | | |
| 8 | 9 | 10 | 4 | 4.5 | 5 | 45 | 50 | 55 | 60 | 50 | 500 | 1000 |
| + | - | - | - | - | + | - | + | - | - | - | + | - |
| - | + | - | - | - | + | - | + | - | - | - | + | - |
| - | - | + | - | - | + | - | + | - | - | - | + | - |
| - | + | - | + | - | - | - | + | - | - | - | + | - |
| - | + | - | - | + | - | - | + | - | - | - | + | - |
| - | + | - | - | - | + | + | - | - | - | - | + | - |
| - | + | - | - | - | + | - | - | + | - | - | + | - |
| - | + | - | - | - | + | - | - | - | + | - | + | - |
| - | + | - | - | - | + | - | + | - | - | + | - | - |
| - | + | - | - | - | + | - | + | - | - | - | - | + |

### Build：

Same as the first round.

### Test：

We completed 10 groups of orthogonal experiments with 3 parallel replicates in each group.
The treatment method is the same as the first round. The detailed experimental data results
can be found in [here](results#jump2).

### Learn：

After two rounds of orthogonal experiments, we concluded that the optimal reaction
conditions for PET05 are as follows:**the optimal pH is 9, the optimal NaCl
concentration is
5 M, the optimal reaction temperature is 55 °C, and saturation of enzyme amount is
achieved
when the Enzyme Loading is 50 nM.**

# 1.2 Testing and Optimization of Reaction Conditions for 40 New Enzymes

### Design：

To ensure that a comparison of properties can be made between our 40 new enzymes and PET05,
we first designed an orthogonal experiment identical to the first round for PET05:

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| pH | | | NaCl(M) | | | | | | Enzyme Loading (nM) | | | Temperature | |
| 8 | 9 | 10 | 0 | 1 | 2 | 3 | 4 | 5 | 25 | 50 | 100 | 30 | 50 |
| - | + | - | + | - | - | - | - | - | - | + | - | - | + |
| - | + | - | - | + | - | - | - | - | - | + | - | - | + |
| - | + | - | - | - | + | - | - | - | - | + | - | - | + |
| - | + | - | - | - | - | + | - | - | - | + | - | - | + |
| - | + | - | - | - | - | - | + | - | - | + | - | - | + |
| - | + | - | - | - | - | - | - | + | - | + | - | - | + |
| + | - | - | - | + | - | - | - | - | - | + | - | - | + |
| - | - | + | - | + | - | - | - | - | - | + | - | - | + |
| - | + | - | - | + | - | - | - | - | + | - | - | - | + |
| - | + | - | - | + | - | - | - | - | - | - | + | - | + |
| - | + | - | - | + | - | - | - | - | - | + | - | + | - |

### Build：

We successfully purified the 40 new enzymes and constructed the reaction system following
the same process as that used for PET05.

### Test：

We completed 11 groups of orthogonal experiments with 3 parallel replicates in each group.
The treatment method is the same as that for PET05's rounds. The detailed experimental data
results can be found in [here](notebook#jump3).

### Learn：

Among all the enzymes, we identified four of them that exhibit PET-degrading enzyme
activity:

### GOMC ID Numbers and Optimal Conditions

| Part Name | Name/Short Description | Part Type | ID Number in GOMC | Description | | |
| --- | --- | --- | --- | --- | --- | --- |
| Optimum pH | Optimal NaCl Concentration/M | Optimum Temperature/℃ |
| BBa\_25ERRSY9 | B6 | Coding | >SRR8062318\_GL0674422 | 8 | 4 | 50 |
| BBa\_25NLL6X5 | B7 | Coding | >SRR7479623\_GL0026367 | 8 | 4 | 50 |
| BBa\_25A3MGA6 | B8 | Coding | >SRR100298\_GL0002528 |  | 3 |  |
| BBa\_25P0HHP3 | B14 | Coding | >SRR6207735\_GL6219842 | 8 | 3 | 50 |

\* Due to its relatively low melting temperature (Tm), the enzymatic activity of B8
decreases sharply with temperature changes. Consequently, its optimal NaCl
concentration
and optimum temperature were not determined in the experiment.

In Engineering 1, we found that among all the marine-derived PET-degrading enzymes we
studied, **PET05 exhibits the best properties**. Both its enzymatic activity
and thermostability
are higher than those of the newly identified enzymes. **Thus, we decided to select
PET05 as
the target for engineering modification**.

# Engineering 2 —— Engineering Modification of PET05

# 2.1 Mutation of the Enzyme Active Site Pocket Based on Structural Homology and Molecular Docking Results

## ***Cycle 1：Single-point Mutation***

### Design：

To directly enhance the catalytic activity of 05PET enzyme, we successively utilized
structural homology and molecular docking to locate its pocket, selected hot spot amino
acids and conducted mutation and wet experiments for verification.

We first used AutoDock Vina to perform molecular docking of 05PET enzyme with trimeric PET
long-chain molecules. By comparing with the structure of LCC, we excluded some non-active
pocket areas. Based on this, we delineated a more reliable docking region and re-performed
molecular docking. After identifying the sequence sites corresponding to the active pocket
space, we took 05PET enzyme as the basis and conducted mutations based on the sequences of
other PET enzymes, determining the following single-point mutation schemes:

`G55A T56A T56Y T56F T57A T58G T62S T62A Y63I Y63L Y63A H123A H123W S124A Q125M Q125A Y148A Y148W I150A I150T I150L I150S G151A G152A D174A I176A I186V L199A L199I S200N S200A S200D S200R G201A G201N A202G S203A S203T H204A F205A F205L F205S E206A E206C E206V V208N V208A G209A G209I G209T G209S S200A S200N S200G S200P A211N A211D G212A G212T G212K G212S D213A D213I D213N D213T D234A D242A D246A D248A D248E`

### Build：

We designed forward and reverse primers for each mutant, and performed PCR using 32a-PET05
(i.e., the wild-type PET05 gene constructed in the pET-32a(+) plasmid vector) as the PCR
template. The PCR products were subjected to agarose gel electrophoresis and gel extraction.
The gel-extracted products were then subjected to homologous recombination. The homologous
recombination products were used for slow transformation of E. coli DH5α. After
transformation, the Ampicillin-resistant (Amp-resistant) plate medium was incubated at 37°C
for 12 hours, and then sent to a sequencing company for sequencing. The sequencing and
assembly results were checked. The detailed construction procedure can be found in notebook
[here](results#jump4).

Plasmids with correct sequencing results were returned to the laboratory by the company. We
transformed the plasmids into E. coli BL21(DE3), followed by bacterial culture and protein
purification. We determined the concentration (mg/mL) of the purified protein solution and
the A260/280 ratio.

### Test：

We constructed the same reaction system for both PET05 mutants and PET05 as follows:

Reaction buffer (600 μL): 50 mM Glycine, 5 M NaCl, pH = 9

Reaction time: 18 hours

Reaction temperature: 55 ℃

Rotation speed: 300 rpm

Reactant: flaky lcPET (with a uniform diameter)

According to the formula:

**Mass (ng) = Enzyme Loading (nM) × Reaction Volume (μL) × Relative Molecular Mass
of the Enzyme (g/mol) × 1000**

we can calculate the mass of the enzyme solution to be added in a 600 μL reaction
system.

According to the formula:

**Volume of the Enzyme Solution to be Added (μL) = Mass (ng) × Extinction
Coefficient / Concentration of the Enzyme Solution (ng/μL)**

we can calculate the volume of the enzyme solution to be added.

We completed experiments for several mutants and PET05, with 3 parallel replicates in each
group. The release amounts of the products TPA and MHET after 18 hours of reaction were
determined via ultra performance liquid chromatography (UPLC). The detailed experimental
data results can be found in [here](results#jump5).

### Learn：

After testing, we identified 10 mutants with higher enzymatic activity than the wild-type:

`I150A I150T I150L I150S I176A L199A S200A S200N S203T F205A`

We will combine these mutations for the next round of engineering cycle.

# Cycle 2：Double-Point Combinatorial Mutation

### Design：

Based on the 70 sequences in section 2.3, we selected 10 mutants with enzymatic activity
higher than that of the wild-type PET05. These 10 mutants were combined in pairs to form 38
types of double-point combinatorial mutants. We then designed the PCR templates and primers
required for constructing these mutants:

### Mutants, Templates, and Corresponding Primers

| Name | Template | Mutant primer (F) | Mutant primer (R) | Mutant |
| --- | --- | --- | --- | --- |
| D1 | 32a-PET05-I150A | I176A-F | I176A-R | I150A-I176A |
| D2 | 32a-PET05-I150A | L199A-F | L199A-R | I150A-L199A |
| D3 | 32a-PET05-I150A | S200A-F | S200A-R | I150A-S200A |
| D4 | 32a-PET05-I150A | S200N-F | S200N-R | I150A-S200N |
| D5 | 32a-PET05-I150A | S203T-F | S203T-R | I150A-S203T |
| D6 | 32a-PET05-I150A | F205A-F | F205A-R | I150A-F205A |
| D7 | 32a-PET05-I150T | I176A-F | I176A-R | I150T-I176A |
| D8 | 32a-PET05-I150T | L199A-F | L199A-R | I150T-L199A |
| D9 | 32a-PET05-I150T | S200A-F | S200A-R | I150T-S200A |
| D10 | 32a-PET05-I150T | S200N-F | S200N-R | I150T-S200N |
| D11 | 32a-PET05-I150T | S203T-F | S203T-R | I150T-S203T |
| D12 | 32a-PET05-I150T | F205A-F | F205A-R | I150T-F205A |
| D13 | 32a-PET05-I150L | I176A-F | I176A-R | I150L-I176A |
| D14 | 32a-PET05-I150L | L199A-F | L199A-R | I150L-L199A |
| D15 | 32a-PET05-I150L | S200A-F | S200A-R | I150L-S200A |
| D16 | 32a-PET05-I150L | S200N-F | S200N-R | I150L-S200N |
| D17 | 32a-PET05-I150L | S203T-F | S203T-R | I150L-S203T |
| D18 | 32a-PET05-I150L | F205A-F | F205A-R | I150L-F205A |
| D19 | 32a-PET05-I150S | I176A-F | I176A-R | I150S-I176A |
| D20 | 32a-PET05-I150S | L199A-F | L199A-R | I150S-L199A |
| D21 | 32a-PET05-I150S | S200A-F | S200A-R | I150S-S200A |
| D22 | 32a-PET05-I150S | S200N-F | S200N-R | I150S-S200N |
| D23 | 32a-PET05-I150S | S203T-F | S203T-R | I150S-S203T |
| D24 | 32a-PET05-I150S | F205A-F | F205A-R | I150S-F205A |
| D25 | 32a-PET05-I176A | L199A-F | L199A-R | I176A-L199A |
| D26 | 32a-PET05 | L199A-S200A-F | L199A-S200A-R | L199A-S200A |
| D27 | 32a-PET05 | L199A-S200N-F | L199A-S200N-R | L199A-S200N |
| D28 | 32a-PET05 | L199A-S203T-F | L199A-S203T-R | L199A-S203T |
| D29 | 32a-PET05 | L199A-F205A-F | L199A-F205A-R | L199A-F205A |
| D30 | 32a-PET05-I176A | S200A-F | S200A-R | I176A-S200A |
| D31 | 32a-PET05 | S200A-S203T-F | S200A-S203T-R | S200A-S203T |
| D32 | 32a-PET05 | S200A-F205A-F | S200A-F205A-R | S200A-F205A |
| D33 | 32a-PET05-I176A | S200N-F | S200N-R | I176A-S200N |
| D34 | 32a-PET05 | S200N-S203T-F | S200N-S203T-R | S200N-S203T |
| D35 | 32a-PET05 | S200N-F205A-F | S200N-F205A-R | S200N-F205A |
| D36 | 32a-PET05-I176A | S203T-F | S203T-R | I176A-S203T |
| D37 | 32a-PET05 | S203T-F205A-F | S203T-F205A-R | S203T-F205A |
| D38 | 32a-PET05-I176A | F205A-F | F205A-R | I176A-F205A |

### Build：

The method is the same as Cycle 1.

### Test：

The method is the same as Cycle 1.

### Learn：

We have completed the construction and testing of a total of 16 double mutants:The mutants
D2, D3, D4, D5, D6, D9, D11, D14, D16, D17, D21, D25, D30, D33, D36, and D38 exhibit
significantly higher enzymatic activity than the wild-type. Among them, the D5 mutant
achieves a product release amount of 1.28 μM within 18 hours, representing a 62% increase
compared to wild-type PET05.

# 2.2 Enzyme Thermostability Engineering Based on Deep Learning and Physicochemical Algorithms

## ***Cycle 1 :Single-point mutation***

### Design：

Overall process: ThermoMPNN->AlphaFold->FoldX

Therefore, we first performed structure prediction on the 05PET enzyme using AlphaFold, and then
ran ThermoMPNN with the predicted structure of PET05 as input. After obtaining a 20×257 heatmap
and its corresponding CSV file, we screened out mutations with a model-predicted ddG (delta
delta Gibbs free energy) greater than 0, and used AlphaFold to conduct structure prediction for
these single-point mutations. Subsequently, FoldX, a software capable of rapidly calculating
protein stability, was employed to sequentially perform "Repair" operations and predict the
folding free energy of both PET05 and all its mutants. Through this process, ten single-point
mutations with a ddG of less than -6 kcal/mol were identified.

To prevent the mutants from affecting the catalysis of the enzyme's active pocket, we first
determined the active pocket position of LCC (a reference enzyme) and then identified the
catalytic pocket of PET05 by leveraging homology analysis. Molecular docking within the
catalytic pocket of the 05PET enzyme was carried out using AutoDock Vina, and the single-point
mutation sites identified in the previous step were further verified based on the set of amino
acid residues involved in the interaction with the substrate. Finally, we screened out 9
single-point mutations:

| T142L | V208L | D213R | D234K | Q146F | D234S | D242R | D248M | D248K |

Disulfide bonds represent a potential structural feature capable of enhancing enzyme stability.
To further improve the stability of the PET05 enzyme, we employed Disulfide by Design 2.0 to
engineer disulfide bonds into PET05. Disulfide by Design 2.0 is an algorithm that utilizes
protein structures to design disulfide bonds. We executed the algorithm using the structure of
PET05 as input. The program output possible disulfide bond mutations along with associated
metrics. By applying the threshold criteria recommended in the literature, we selected two
triple-disulfide bond variants and utilized AlphaFold for structural prediction to validate the
feasibility of forming three disulfide bonds. The simultaneous presence of all three disulfide
bonds was observed in one structural prediction outcome, confirming the viability of the
disulfide bond design. The three disulfide bonds are as follows:

| A58C-T62C | Q159C-R189C | A121C-A143C |

### Build：

The method is the same as Cycle 1 in Section 2.1

### Test：

The method is the same as Cycle 1 in Section 2.1

### Learn：

After testing, we excluded the Q146F and T142L mutants, whose activity was significantly reduced.
We then determined the Tm values of the other mutants to rule out any impact on thermostability.

## ***Cycle 2 :Composite mutant***

### Design：

After ruling out potential impacts on the active pocket, we combined the single-point mutations,
predicted their structures following the same protocol as described in section 2.1, performed
repairs, and calculated the folding free energies. Finally, we integrated both the single-point
mutations and the three disulfide bonds into the sequences simultaneously, generating multiple
sequence variants for subsequent wet laboratory experiments.

### Combination Mutants and Their Designations

| Name | Mutant |
| --- | --- |
| C1 | A58C\_T62C\_Q159C\_R189C\_ |
| C2 | q159w\_v208l\_ |
| C3 | v208l\_d213r\_ |
| C4 | d213r\_d234k\_ |
| C5 | d234k\_d242r\_ |
| C6 | d242r\_d248k\_ |
| C7 | q159w\_v208l\_d213r\_d234k\_d242r\_d248k\_A58C\_T62C\_Q159C\_R189C\_ |

### Build：

The method is the same as Cycle 1 in Section 2.1

### Test：

The method is the same as Cycle 1 in Section 2.1

### Learn：


![Science background](https://static.igem.wiki/teams/5806/static/home/header1-project.webp)

Navigation

[Standard Curve](#part1)
[PET05](#part2)
 [Mining of PET Degrading Enzymes from Marine Sources](#part3)
 [The Engineering Modification of PET05](#part4)

Result

# Standard Curve

To quantify the enzymatic activity of PET-degrading enzymes in our experiment, we fabricated
standard curves of TPA and MHET concentrations versus absorbance. The original data is shown in
the following table.

![](https://static.igem.wiki/teams/5806/result/result-1.avif)

The standard curve drawn is as follows.

![](https://static.igem.wiki/teams/5806/result/result-standard-curve-2.webp)

To facilitate subsequent calculations, we swapped the X-axis and Y-axis and obtained a linear
function of the integral of the product concentration with respect to the absorbance value.

![](https://static.igem.wiki/teams/5806/result/result-standard-curve-3.webp)

# PET05

### The Optimal Reaction Conditions of PET05

We tested the optimal enzymatic activity conditions for the degradation of low-crystalline PET by
PET05 through two sets of orthogonal experiments. The data from the first orthogonal test are as
follows.

![](https://static.igem.wiki/teams/5806/result/result-pet05-11.avif)

![](https://static.igem.wiki/teams/5806/result/result-pet05-2.webp)

We found that PET05 only had enzymatic activity at a near-saturated NaCl concentration of 4-5
meters. So we conducted the second orthogonal experiment, and the data are as follows.

![](https://static.igem.wiki/teams/5806/result/result-pet05-33.avif)

![](https://static.igem.wiki/teams/5806/result/result-pet05-4.webp)

We concluded that the optimal **pH value for the PET05 reaction is 9, the optimal sodium
chloride
concentration is 5M, the optimal reaction temperature is 55℃, and the Enzyme Loading is
50nM**,
which can achieve enzyme saturation.

### Long-term Reaction of PET05

Under the optimal conditions of enzyme activity, we conducted a long-term reaction using PET05
for up to 312 hours. Sampling was conducted at different times of the reaction, PET films were
taken under a stereomicroscope, and the product concentration was determined using a UPLC. The
state of PET sheets at different reaction durations is as shown in the figure below.

![](https://static.igem.wiki/teams/5806/result/result-pet05-5.webp)

The product concentrations at different reaction durations are as shown in the table below.

### Enzyme activity（TPA+MHET released/µM）

| hours | 1 | 2 | 3 | mean |
| --- | --- | --- | --- | --- |
| 0.5 | 0.004991837 | 0.00267749 | 0.003266831 | 0.003645386 |
| 1 | 0.005815201 | 0.017494123 | 0.01765414 | 0.013654488 |
| 2 | 0.05125206 | 0.057176795 | 0.018797215 | 0.04240869 |
| 3 | 0.028209588 | 0.10294138 | 0.119168082 | 0.083439684 |
| 6 | 0 | 0.081811266 | 0.594488362 | 0.22543321 |
| 12 | 0.569991996 | 0.803092286 | 0.652898886 | 0.675327723 |
| 18 | 0.826369577 | 0.80382015 | 0.705473866 | 0.778554531 |
| 24 | 1.031834406 | 1.153923982 | 0.445321476 | 0.877026621 |
| 48 | 1.503339831 | 0.916822999 | 0.847155996 | 1.089106275 |
| 72 | 2.208111014 | 1.52159562 | 2.36479914 | 2.031501924 |
| 96 | 4.828925256 | 1.961029356 |  | 3.394977306 |
| 120 |  |  |  |  |
| 144 | 6.06728164 | 5.431115065 | 6.331766881 | 5.943387862 |
| 168 | 6.411591421 | 5.539353767 | 6.495993598 | 6.148979595 |
| 192 | 6.581324815 |  |  | 6.581324815 |
| 216 |  |  |  |  |
| 240 | 6.657914471 | 6.495560289 |  | 6.57673738 |
| 264 |  |  |  |  |
| 288 | 6.784582732 | 6.343612036 |  | 6.564097384 |

![](https://static.igem.wiki/teams/5806/result/result-pet05-6.webp)

From the data and line chart, it can be seen that the maximum reaction duration PET05 can
achieve under the optimal conditions for enzyme activity is approximately 200 hours. This is
another highly representative characteristic of PET05, in addition to its high salt tolerance.

# Mining of PET Degrading Enzymes from Marine Sources

We purified and subjected 40 protein sequences, which were mined and screened from in silico
experiments, to enzyme activity assays. These sequences were cloned into the pET-32a(+) plasmid,
as shown in the figure.

![](https://static.igem.wiki/teams/5806/result/result-marine-11.webp)

Ultimately, we identified four of these protein sequences that exhibit PET-degrading enzyme
activity. Their orthogonal experiment results are shown in the figure below.

![](https://static.igem.wiki/teams/5806/result/result-marine-22.webp)

![](https://static.igem.wiki/teams/5806/result/result-marine-3.webp)

B6

![](https://static.igem.wiki/teams/5806/result/result-marine-4.webp)

B7

![](https://static.igem.wiki/teams/5806/result/result-marine-5.webp)

B8

![](https://static.igem.wiki/teams/5806/result/result-marine-6.webp)

B14

Below are the AlphaFold-predicted 3D structures of these four enzymes.

![B6](https://static.igem.wiki/teams/5806/static/home/61.webp)

B6

![B7](https://static.igem.wiki/teams/5806/static/home/71.webp)

B7

![B8](https://static.igem.wiki/teams/5806/static/home/81.webp)

B8

![B14](https://static.igem.wiki/teams/5806/static/home/141.webp)

B14

Upon validation through wet-lab experiments, three of the four obtained PET hydrolases were
found to belong to Cluster 42\_29. An evolutionary tree was constructed using these sequences
together with the nine known PET hydrolases, as shown in the figure below. Cluster 42\_29 can be
considered a potential family of PET hydrolases, though this requires further wet-lab
experimental validation.

![](https://static.igem.wiki/teams/5806/result/result-marine-77.webp)

# The Engineering Modification of PET05

### Enzyme Active Site Pocket Mutagenesis Based on Structural Homology and Molecular Docking Results

Based on the mutation sites designed from in silico experiments, we first designed a pair of
primers for each mutant, and performed PCR amplification using pET-32a(+)-PET05 as the template.
The figure shows the results of agarose gel electrophoresis, where 10 μL was taken from the 50
μL PCR system for characterization.

![](https://static.igem.wiki/teams/5806/result/result-modification-1.webp)

![](https://static.igem.wiki/teams/5806/result/result-modification-1.webp)

After PCR, the target fragments were subjected to self-homologous recombination, and sequencing
was performed to verify whether the mutant construction was successful. We cultured and purified
70 successfully constructed mutant proteins. The protein expression is shown in the following
SDS-PAGE gel electrophoresis image.

![](https://static.igem.wiki/teams/5806/result/result-modification-2.webp)

They were reacted with low-crystallinity PET under the same conditions as the wild-type PET05,
and the enzyme activity was detected by UPLC. The results are shown in the following figure
(mutants with higher enzyme activity than the wild-type are marked with orange fluorescence in
the figure).

![](https://static.igem.wiki/teams/5806/result/result-normalization-3.avif)

We have completed the construction and testing of 16 double combinatorial mutants, with the
results shown in the following figure. Among them, the top-performing one in characterization is
D5 (PET05-I150A-S203T), whose enzyme activity has increased by 62% compared with the wild type.
Although the enzyme activity—both before and after modification—is relatively low and lacks
practical application value, the increase in the enzyme activity of the modified protein proves
that our modification strategy is effective.

![](https://static.igem.wiki/teams/5806/result/result-normalization-1.avif)

### Enzyme Thermostability Engineering Based on Deep Learning and Physicochemical Algorithms

The construction and testing procedures for nine single-point mutations and two pairs of
disulfide bond mutants were consistent with those described in Section 4.1. The enzymatic
activity data are provided below.

![](https://static.igem.wiki/teams/5806/result/result-normalization-2.avif)

We determined the Tm values of several proteins using a real-time quantitative PCR instrument.
With the exception of protein B8, the Tm values of most proteins were higher than that of the
wild-type PET05.This indicates that after our modification, the thermal stability of PET05 has
achieved a significant improvement.

![](https://static.igem.wiki/teams/5806/result/tem-1.avif)

A3 D234S Tm:74.5℃

![](https://static.igem.wiki/teams/5806/result/tem-2.avif)

A4 D234K Tm:70℃

![](https://static.igem.wiki/teams/5806/result/tem-3.avif)

B3 V208L Tm:51.5℃

![](https://static.igem.wiki/teams/5806/result/tem-4.avif)

F2 D248M Tm：76℃

![](https://static.igem.wiki/teams/5806/result/tem-5.avif)

B3 V208L Tm:51.5℃

{% endblock %}