iGEM 2024 — Team jiangnan-china

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Wet lab

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Team

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![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/notebook.png)
![](https://static.igem.wiki/teams/5157/wikimages/project/description/bgicon1.png)
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![](https://static.igem.wiki/teams/5157/wikimages/project/description/bgcha1.png)
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March-April
![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/three-gang.png)

## First weekly team meeting:

1. Members gave brief self-introductions and were devided into five groups: wet lab, dry lab, human
practice, design, and network.

2. PI and instructor gave an speech about the specifics of our laboratory.

## Laboratory safety training:

1. Instructor trained us about experimental safety, including appointment of instruments, norms
for the use of laboratory reagents.

2. Test on laboratory and test on experimental skills: members of wet lab need to pass the test
about lab safety norms and acknowledge basic experimental skills before they are allowed access
to the laboratory.

## Advisor speech:

1. They introduced the basic rules of the iGEM competition, and gave us advice on project
selection and project organization.

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/1.png)

Fig. 1   Safety
training

## Social and documentary research:

1. We conducted research on social hotspots in the hope of discovering pressing and researchable
issues around us, and we searched and read the literature on these issues.

2. We screened the project topics suggested by our members and solicited advice from PI. Finally,
we determined that our the goal of project is to solve the problem of how to achieve efficient
degradation of PET plastics by enzymatic degradation.

## Learning laboratory operations:

1. Learn about preparation of culture medium (LB, TB, ZYM) and other reagents.

**2.** Learn about culturing *Escherichia coli* strain.

## Coordination with dry lab:

1. Assist dry lab in finding and organizing protein PDB files.

## Reading literature about PET degradation and PETase:

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/2.png)

Fig. 2   Media
preparation

May![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/three-gang.png)

Week 1 2024.5.6-2024.5.12

## Screening of chassis cells:

We constructed recombinant plasmids and introduced them into different host cells to detect
fusion protein expression.

Week 2-3 2024.5.13-2024.5.26

## Constructing recombinant plasmids:

pET-21b-40-1-C-G4S-PETase,    pET-21b-40-2-C-G4S-PETase,   pET-21b-40-3-C-G4S-PETase,

pET-21b-40-4-C-G4S-PETase,
pET-21b-50-1-C-G4S-PETase,
   pET-21b-50-3-C-G4S-PETase,
pET-21b-50-4-C-G4S-PETase,    pET-21b-50-5-C-G4S-PETase.

## Shaking flask-level culture:

1. We picked single colonies and incubated in LB media with 0.03 mg/mL ampicillin for 10 hours.

2. We sent samples to sequence.

## Shaking flask-level fermentation:

1. We inoculated the bacterial fluid and culture for 12 hours, then vaccinated 5% bacterial
suspension into 50 mL TB medium incubated for 48 hours.

2. We isolated fusion protein and determined enzyme activities.

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/3.png)

Fig. 3   Shaking
flask-level fermentation

(a). Amplification culture. (b). Precipitate of bacteria
after fermentation.

June![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/three-gang.png)

Week 4-5 2024.5.27-2024.6.15

## Constructing recombinant plasmids:

pET-21b-60-1-C-G4S-PETase,    pET-21b-60-2-C-G4S-PETase,   pET-21b-60-3-C-G4S-PETase,

pET-21b-60-4-C-G4S-PETase,
pET-21b-70-1-C-G4S-PETase,
   pET-21b-70-2-C-G4S-PETase,
pET-21b-70-3-C-G4S-PETase,    pET-21b-70-4-C-G4S-PETase.

## Shaking flask-level culturing and sequencing

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/4.png)

Fig. 4   The PCR
identification result of pET-21b and fragment

## Shaking flask-level fermentation.

## Connecting pepetides to the -NH2 terminus of PETase:

1. After pre-experiment, we found that enzyme activities were significantly different when the
PET-binding peptides were connected to the -NH2 terminus or the -COOH terminus of PETase. We tried to
construct recombinant plasmids by assembling these 16 peptides at the -NH2 and
-COOH terminus of PETase.

2. Sequencing and fermentating on the shaking flask-level when sequencing correct.

## Replacing linker:

1. We replaced linkers of fusion protein with SLE, AG, 10A, EAAAK, Tr. Then, we constructed
recombinant plasmids.

2. Plasmids were sequencing and several were re-constructed.

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/5.png)

Fig. 5
Construction of fusion protein

Week 6 2024.6.16-2024.6.22

## Shaking flask-level fermentation:

1. Recombinant plasmids of correct sequence were fermentated for 48 hours:

pET-21b-40-1-N-SLE-PETase,    pET-21b-50-1-C-SLE-PETase,   pET-21b-50-1-N-SLE-PETase,

pET-21b-50-4-N-SLE-PETase,
pET-21b-50-5-C-SLE-PETase,
   pET-21b-70-1-C-SLE-PETase.

2. Recombinant plasmids of wrong sequence were re-constructed and sequencing.

## Enzyme activity measurement:

1. We measured enzyme activities of fusion protein by crude enzyme solution.

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/6.png)

Fig. 6   SDS-PAGE
samples

Week 7 2024.6.23-2024.6.29

## Protein purification:

1. Expressible fusion proteins were purified by Ni-NTA affinity chromatography column.

2. Enzyme solution was freeze-dried after dialysis for 24 hours.

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/7.png)

Fig. 7   Protein
purification instrument

July![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/three-gang.png)

Week 8 2024.6.30-2024.7.6

## PET degradation

1. Enzyme solution of G4S-liner fusion protein (protein concentration 4 μM) was added into the
glass tube containing 25 mg PET amorphous particles. The degradation was reacted in 60℃ for 96
hours.

2. Samples were taken every 24 hours.

## Optimization of fermentation conditions

1. We took TB, LB, and ZYM were selected as culture media to investigate the optimal medium
type. IPTG concentrations of 0.025 mmol/L, 0.050 mmol/L, 0.075 mmol/L, and 0.100 mmol/L
were selected to investigate the optimal IPTG concentration. 16℃ and 25℃ were fermentation
temperature to investigate the optimal fermentation temperature. 24 h and 48 h were fermentation
time to investigate the optimal fermentation time.

2. Then, we measured enzyme activities of fusion protein by crude enzyme solution.

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/8.png)

Fig. 8   PET
degradation samples

Week 9-10 2024.7.7-2024.7.20

## EPA and MHET detection by HPLC:

1. We took PET degradation samples (24h, 48h, 72h, 96h) of week 8 to test the degradation
product by HPLC.

## PET degradation:

1. Expressible enzyme solution of SLE-linker fusion protein reacted with PET for 96 hours.

2. Samples were taken every 24 hours.

Week 11 2024.7.21-2024.7.27

## EPA and MHET detection by HPLC:

1. We took PET degradation samples (24h, 48h, 72h, 96h) of week 10 to test the degradation
product by HPLC.

2. We organized and analyzed data.

August![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/three-gang.png)

Week 12-13 2024.7.28-2024.8.10

## Single spot mutation

We selected three short peptides (50-4-N-G4S, 50-5-C-G4S, 60-2-N-SLE) with better effects
and performed mutant construction based on the mutation sites screened by dry lab group.

1. Mutants construction:

| WildType | Mutation Sites |
| --- | --- |
| 50-4-N-G4S | R4D, S14A, V19I, L23K, Y33F, S61A |
| 50-5-C-G4S | I283V, R287V, C293V, R301V, A325S |
| 60-2-N-SLE | L7F, A13S, S26D, N29D, R32K, L44I, Q55L, L60I |

2. We designed primers and constructed recombinant plasmids.

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/9.png)

Fig. 9
Construction of single spot mutants

## Shaking flask-level culturing and sequencing :

1. Plasmids were sequencing and several were re-constructed.

2. Recombinant plasmids of correct sequence were fermentated for 48 hours.

## Enzyme activity measurement:

1. We measured enzyme activities of fusion protein by crude enzyme solution.

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/10.png)

Fig. 10
Fermentation samples

Week 14 2024.8.11-2024.8.17

## Protein purification depending on results of enzyme activities.

## PET degradation:

1. Expressible enzyme solution of mutants fusion protein reacted with PET for 96 hours.

2. Samples were taken every 24 hours.

## Costruction of eGFP-fusion protein:

1. We selected pET-20b as vector, design primers to assembly eGFP and peptides (50-4-N-G4S, 50-5-C-G4S,
60-2-N-SLE) with better effects.

2. We sequenced those recombinant plasmids and re-constructed wrong plasmids.

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/11.png)

Fig. 11   PET
degradation of single spot mutants

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/12.png)

Fig. 12
Construction of eGFP-fusion protein

(a). The PCR samples. (b). The PCR identification result of
fusion protein.

Week 15 2024.8.18-2024.8.24

## EPA and MHET detection by HPLC:

1. We took PET degradation samples (24h, 48h, 72h, 96h) of mutants to test the degradation
product by HPLC.

## Shaking flask-level fermentation:

1. Recombinant plasmids of eGFP-fusion protein were fermentated for 48 hours.

Week 16 2024.8.25-2024.8.31

## Characterization of eGFP-fusion Protein

1. eGFP-fusion Protein were reacted with PET thin films at 37℃ for 24 hours.

2. Remaining fluorescence adsorption intensity of PET films are observed under a fluorescence
microscope.

September![](https://static.igem.wiki/teams/5157/wikimages/wetlab/notebook/three-gang.png)

**We organized and analyzing data.**

**We wrote documents and graphs.**

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# Project

[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
[Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)
[Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
[Safety](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27safety%27%29%20%7D%7D)

# Model

[Introduction & Related work](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
[Methology](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27methology%27%29%20%7D%7D)
[Experiment & Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiment_results%27%29%20%7D%7D)

# Wet lab

[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)
[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
[Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)

# Human Practices

[Integrated Human Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
[Education & Communication](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27education%27%29%20%7D%7D)

# Team

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© 2024 - Content on this site is licensed under a
[Creative Commons Attribution 4.0 International License](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at
[gitlab.igem.org/2024/jiangnan-China](https://gitlab.igem.org/2024/jiangnan-china).

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Wet lab

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[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
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![](https://static.igem.wiki/teams/5157/wikimages/project/engineering/engineering.png)
![](https://static.igem.wiki/teams/5157/wikimages/project/description/bgicon1.png)
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![](https://static.igem.wiki/teams/5157/wikimages/project/description/yinfu10.png)

Overview

Cycle1: PET-binding Peptides Mining
Based on Deep Learning Models

Cycle2: Construction and Expression of
PETase-PET-binding
Peptides Fusion Protein

Cycle3: Optimization of Fermentation Conditions

Cycle4: Identification of PET-binding Peptides

Cycle5: Verification of the Binding Capability of
PET-binding
Peptides with PET Microplastics

Cycle6: Replacement of the Linker

Cycle7: Replacing the Linker with SLE

Cycle8: Modification of PET-binding Peptides

Overview

Engineering is a critical process in synthetic biology research. An engineering cycle consists
of four stages: design, build, test and learn. In our project, we continuously executed eight
cycles. The accumulation of engineering results eventually led us to achieve the final goal of this
project.

Our project aims to enhance the binding and degradation capabilities of PETase toward PET
microplastics by the fusion of PET-binding peptides.

![](https://static.igem.wiki/teams/5157/wikimages/project/engineering/1.png)

Cycle1:
PET-binding
Peptides Mining
Based on
 Deep
Learning Models

# Design

In the case of insufficient binding ability of PETase towards PET, improving the binding
efficiency becomes a key goal. Given the complexity, inefficiency, and time-consuming nature of
traditional experimental screening processes, we introduced deep learning models to accelerate
PET-binding peptide screening.

# Build

We constructed two models: LSTM and GCN. The LSTM model was used for the
preliminary screening of peptides, focusing on capturing long-range dependencies within the
sequence. Next, the GCN model was applied to analyze the three-dimensional structural
information of the peptides and to build node features, thereby improving the accuracy of
screening and the reliability of predicting biological binding capabilities.

# Test

We trained and tested the LSTM and GCN models, evaluating their ability to screen peptides,
respectively. Preliminary experiments were conducted to validate the effectiveness of the models
in predicting the binding affinity between peptides and PET plastic. Based on the experimental
results, we continuously adjusted the model parameters to improve the recognition rate of target
peptides.

# Learn

Based on the evaluation of model performance, the screening accuracy of the LSTM model
gradually increased to 93.4%, while the accuracy of the GCN model reached 90.4%. The GCN
model successfully identified
[16 target peptides](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D#part2)
with potential high PET-binding affinity. Based on
the obtained peptide data, we further optimized the screening accuracy and enhanced the
performance of the deep learning models through dataset expansion and model structure
optimization. And we conducted experimental validation using the predicted peptides.

Cycle2:
Construction and Expression of PETase-PET-binding
Peptides Fusion Protein

# Design

Since the attachment of short peptides to the amino and carboxyl termini can lead to
differences in their position and functional characteristics within the peptide chain or protein, as
well as potentially affecting the overall properties of the protein, we first selected PET-binding
peptides 50-1, 50-3, 50-4 and 50-5 obtained from deep learning screening, as examples, to fuse
with the N-terminus and C-terminus of PETase, respectively. We constructed fusion proteins for
validation and characterization.

# Build

We fused the aforementioned short peptides to the N-terminus or C-terminus of PETase using
the linker G4S, and their encoding genes were linked to pET-21b to construct recombinant
plasmids, respectively. The correctly verified recombinant plasmids were then transformed into
 *Escherichia coli* BL21 (DE3) for shake-flask fermentation to obtain the fusion proteins.

# Test

We used *p*NPB as the substrate to test the catalytic activity of the fusion protein under
conditions of 60°C and pH 8.0.

![](https://static.igem.wiki/teams/5157/wikimages/project/engineering/2.png)

Fig. 1   Enzymatic activity of fusion
proteins with peptides 50-1 to 50-5

(a). Enzymatic activity of C-terminal
fusion proteins with
peptides 50-1 to
50-5. (b). Enzymatic activity of N-terminal fusion proteins with peptides 50-1 to 50-5.

# Learn

We compared the enzymatic activity of fusion proteins with the same peptide linked to the
N-terminus and C-terminus of PETase and found that there are differences depending on the
position of the peptide attachment. Therefore, attaching the peptides to different ends of PETase
does
indeed affect its degradation efficiency. However, we still face the problem of low fusion protein
expression.

Cycle3:
Optimization of Fermentation Conditions

# Design

Since the mined peptides are derived from non-*E. coli* hosts and possess strong
hydrophobicity, they may have a significant negative effect on the expression of the fusion protein.
We took 50-4-N-G4S-PETase
([BBa\_K5157051](https://parts.igem.org/Part%3ABBa_K5157051)) as an example to optimize the
types of culture medium, inducer
concentration, fermentation temperature, and fermentation time.

# Build

We conducted optimization of fermentation conditions at the shake-flask level: we selected
TB, LB, and ZYM as culture media to explore the most suitable medium type; we tested IPTG
final concentrations of 0.025 mmol/L, 0.050 mmol/L, 0.075 mmol/L, and 0.100 mmol/L to find
the optimal inducer concentration; we conducted fermentation at 16°C and 25°C to determine the
most suitable fermentation temperature; and we tested fermentation durations of 24 h and 48 h to
identify the optimal fermentation time.

# Test

We used *p*NPB as the substrate to test the catalytic activity of the fusion protein obtained
from fermentation under conditions of 60°C and pH 8.0.

![](https://static.igem.wiki/teams/5157/wikimages/project/engineering/3.png)

Fig. 2
Optimization of fermentation
conditions for 50-4-N-G4S-PETase

(a). Enzymatic activity of
50-4-N-G4S-PETase in different
culture media. (b).
Enzymatic activity of 50-4-N-G4S-PETase at various
inducer concentrations. (c). Enzymatic activity of 50-4-N-G4S-PETase at different fermentation
temperatures. (d). Enzymatic activity of
50-4-N-G4S-PETase at different fermentation times.

# Learn

The experimental results indicate that using TB medium with an IPTG final concentration of
0.05 mmol/L, fermentation at 16°C for 48 h is most beneficial for the expression of the fusion
protein. Therefore, we used this shake flask fermentation conditions in subsequent experiments.

Cycle4:
Identification of PET-binding Peptides

# Design

We designed to use G4S to connect 40-1, 40-2, 40-3, 40-4, 50-1, 50-3, 50-4, 50-5, 60-1, 60-2, 60-3,
60-4, 70-1, 70-2, 70-3, 70-4 to the C-terminus and N-terminus of PETase, respectively, to
construct a fusion protein prokaryotic expression system.

# Build

We linked the fusion protein gene to pET-21b to construct recombinant plasmids. The
correctly verified recombinant plasmids ([BBa\_K5157051~BBa\_K5157069](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D#part3)) were then
transformed into *E. coli* BL21 (DE3) for
shake-flask fermentation to obtain the fusion proteins.

# Test

We used *p*NPB as the substrate to test the catalytic activity of the fusion protein under
conditions of 60°C and pH 8.0, while also assessing the degradation capability of the purified
fusion protein on PET microplastics.

![](https://static.igem.wiki/teams/5157/wikimages/project/engineering/4.png)

Fig. 3   Identification of PET-binding
peptides

(a). Enzymatic activity of C-terminal PET-binding peptides.
(b).
Enzymatic
activity of N-terminal PET-binding peptides. (c). Degradation
of PET microplastics by C-terminal PET-binding peptides. (d). Degradation of PET microplastics by
N-terminal PET-binding peptides.

# Learn

The experimental results indicated that 19 fusion proteins were successfully expressed and
50-4-N-G4S-PETase ([BBa\_K5157051](https://parts.igem.org/Part%3ABBa_K5157051)),
PETase-G4S-50-5-C ([BBa\_K5157056](https://parts.igem.org/Part%3ABBa_K5157056)),
and 60-2-N-G4S-PETase ([BBa\_K5157067](https://parts.igem.org/Part%3ABBa_K5157067)) exhibited both excellent
expression and PET degradation capabilities. We confirmed the feasibility of peptides in enhancing
microplastic degradation, but the binding capacity still requires further investigation.

Cycle5:
Verification of the Binding Capability
of PET-binding
Peptides with PET Microplastics

# Design

To further verify whether the obtained PET-binding peptides can effectively bind to PET
microplastics, we designed fusion proteins by linking these aforementioned peptides with good
degradation effects, 50-4, 50-5, and 60-2, to enhanced green fluorescent protein (eGFP) through
the linker G4S.

# Build

We linked the fusion protein gene to pET-20b to construct
recombinant plasmids ([BBa\_K5157070~BBa\_K5157072](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D#part3)).
The
correctly verified recombinant plasmids were then transformed into
 *E. coli* BL21 (DE3) for
shake-flask fermentation to obtain the fusion proteins.

# Test

The eGFP solution and eGFP fusion protein solution with equal fluorescence intensity were
taken and incubated with PET thin film, protected from light at 37°C for 24 h. Then, free proteins
were washed and eluted, the PET film was observed under a fluorescence microscope, and the
remaining fluorescence adsorption intensity of PET films was measured at 488 nm excitation
wave.

![](https://static.igem.wiki/teams/5157/wikimages/project/engineering/5.png)

Fig. 4   Fluorescence microscopy results
of
50-4-N-G4S-eGFP, 50-5-C -G4S-eGFP,
60-2-N-G4S-eGFP, and their controls

(a). eGFP as a control for 50-4-N-G4S-eGFP. (b). eGFP as a
control for
50-5-C-G4S-eGFP. (c). eGFP as a control for 60-2-N-G4S-eGFP. (d). Binding of 50-4-N-G4S-eGFP to PET
film. (e). Binding of 50-5-C-G4S-eGFP to PET film. (f). Binding
of 60-2-N-G4S-eGFP to PET
film.

# Learn

The experimental results confirmed that these PET-binding peptides could bind to PET films,
suggesting that the PET affinity of these binding peptides led to the improved degradation
efficiency of PETase-PET-binding peptide.

Cycle6: Replacement of the Linker

# Design

As a segment connecting the enzyme and the peptide, the choice and composition of the
linker can influence the property of the fusion protein. Therefore, we took 70-1-C as an example
to attempt replacing the linker G4S with other linkers to optimize the spatial conformation
between the enzyme and the peptide, reduce the mutual interference between the two protein
components, and enhance the substrate catalytic efficiency of the fusion protein.

# Build

We replaced the gene of linker of the fusion protein in pET-21b to construct recombinant
plasmids ([BBa\_K5157073~BBa\_K5157077](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D#part3)), respectively. The
correctly verified recombinant plasmids were then transformed into *E.
coli* BL21 (DE3) for shake-flask fermentation to obtain the fusion proteins, respectively.

# Test

We used *p*NPB as the substrate to test the catalytic activity of the fusion protein under
conditions of 60°C and pH 8.0.

![](https://static.igem.wiki/teams/5157/wikimages/project/engineering/6.png)

Fig. 5   Enzymatic activity of 70-1-C
fusion proteins connected by different linkers

# Learn

The experimental results indicate that the flexible linker G4S and the rigid linker SLE
performed better. In subsequent experiments, we needed to explore the effect of rigid linker SLE in
fusion proteins.

Cycle7: Replacing the Linker
with SLE

# Design

Flexible linkers offer greater flexibility, while rigid linkers emphasize structural stability and
precision. From the previous screening cycle, we obtained a well-performing rigid linker SLE, and
we attempted to replace the flexible linker G4S in all fusion proteins with the rigid linker SLE.

# Build

We replaced the gene of original linkers of the fusion proteins with SLE encoding gene to
pET-21b to construct recombinant plasmids ([BBa\_K5157077~BBa\_K5157095](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D#part3)), respectively. The
correctly verified recombinant
plasmids were then transformed into *E. coli* BL21 (DE3) for shake-flask fermentation to
obtain
the fusion proteins, respectively.

# Test

We used *p*NPB as the substrate to test the catalytic activity of the fusion protein under
conditions of 60°C and pH 8.0, while also assessing the degradation capability of the purified
fusion protein on PET microplastics.

![](https://static.igem.wiki/teams/5157/wikimages/project/engineering/7.png)

Fig. 6   Replacing the linker with SLE

(a). Enzymatic activity of C-terminal PET-binding peptides. (b).
Enzymatic
activity of N-terminal PET-binding peptides. (c). Degradation
of PET microplastics by C-terminal PET-binding peptides. (d). Degradation of PET microplastics by
N-terminal PET-binding peptides.

# Learn

The experimental results indicate that both the enzymatic activity and degradation efficiency
of 60-2-N-SLE-PETase ([BBa\_K5157093](https://parts.igem.org/Part%3ABBa_K5157093)) were excellent.
After the above cycles, we had obtained some fusion proteins with promising results, but they still
fell short of expectations.

Cycle8:
Modification of PET-binding Peptides

# Design

We obtained peptides 50-4-N, 50-5-C, and 60-2-N that effectively enhanced the PETase
degradation efficiency towards PET microplastics; however, there is still room for improvement in
the expression levels and binding ability to PET microplastic of the fusion proteins. Therefore,
using molecular design and structural biology tools, we further modified the sites of these
PET-binding peptides. To enhance the precision and efficiency of the mutations, we introduced
Mutcompute-super model, which can predict the impact of different mutations on protein stability
and microplastic binding capability based on the three-dimensional structure of the protein.
Through this tool, we optimized the protein folding conformation, thereby improving the
expression levels and microplastic substrate binding capability of the fusion proteins.

# Build

Based on the optimal mutation sites predicted by the 3D deconvolution model, we designed
specific primers and introduced site-directed mutations on the existing recombinant plasmids to
construct recombinant plasmids ([BBa\_K5157096~BBa\_K5157114](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D#part3)) containing the
mutated fusion proteins. The correctly verified
recombinant plasmids were then transformed into *E. coli* BL21 (DE3) for shake-flask
fermentation to obtain the fusion proteins.

# Test

We used *p*NPB as the substrate to test the catalytic activity of the fusion protein under
conditions of 60°C and pH 8.0. At the same time, we utilized experiments to verify the effects of
the predicted mutations from the 3D deconvolution model and assessed the degradation capability
of the purified fusion protein on PET microplastics.

![](https://static.igem.wiki/teams/5157/wikimages/project/engineering/8.png)

Fig. 7   The expression and PET
degradation
results of fusion protein mutants

(a). Enzymatic activity of the mutants 50-4-N-G4S-PETase. (b).
Enzymatic
activity of the mutants PETase-G4S-50-5-C. (c). Enzymatic
activity of the mutants 60-2-N-SLE-PETase. (d). Degradation of PET microplastics by the mutants
50-4-N-G4S-PETase. (e). Degradation
of PET microplastics by the mutants PETase-G4S-50-5-C. (f). Degradation of PET microplastics by the
mutants 60-2-N-SLE-PETase.

# Learn

The experimental results indicate that the mutants V19I ([BBa\_K5157098](https://parts.igem.org/Part%3ABBa_K5157098)), L23K,
and S61A of
50-4-N-G4S-PETase exhibit good PET microplastic degradation effects, while the mutants
R287V ([BBa\_K5157103](https://parts.igem.org/Part%3ABBa_K5157103)), R301V,
and A325S of PETase-G4S-50-5-C also show favorable PET microplastic degradation. Additionally, the
mutants A13S, N29D, and L60I ([BBa\_K5157114](https://parts.igem.org/Part%3ABBa_K5157114)) of 60-2-N-SLE-PETase
demonstrate effective
PET microplastic degradation.

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Part1: Validation of Highly Scored
PET-binding Peptides

1.1 Construction of Plasmids

1.2 Enzyme Activity

1.3 PET Degradation

Part2: Optimization of Fermentation
Conditions and Linker Replacement

2.1 Optimization of Fermentation Conditions

2.2 Linker Replacement

Part3: Binding Capacity Characterization

3.1 Construction of Plasmids

3.2 Characterization

Part4: Modification of
PET-binding peptides

4.1 Construction of Plasmids

4.2 Enzyme Activity

4.3 PET Degradation

Part1: Validation of Highly Scored
PET-binding
Peptides

1.1 Construction of Plasmids

The pET-21b was selected as the backbone vector, and the composition of
respective DNA fragments were
amplified by
Flash Super-Fidelity DNA Polymerase and constructed through one-step cloning method. The recombinant
plasmid
was
transformed into *Escherichia coli* BL21 (DE3) for expression.

1.2 Enzyme Activity

The catalytic activity of the fusion protein was determined in the 1.5 mL pH 8.0 0.1 M
phosphate buffer system at 60°C, using *p*NPB as the substrate. And our preliminary results
have revealed that the enzyme activities were significantly
different when the PET-binding peptides were connected to the -NH2 terminus or the -COOH
terminus of PETase. Therefore, we tried to construct 32 fusion proteins by assembled these
16 peptides at the -NH2 and -COOH terminus of PETase, respectively. Finally, 19 fusion
proteins
were successfully expressed and 60-1-fusion protein and 70-2-fusion protein were
not expressed at either the C-terminus or the N-terminus. Enzyme activity of expressible fusion
protein is shown in Fig. 1a. The results showed that the enzyme activities obtained from the
expression of PETase-G4S-50-1-C, PETase-G4S-50-5-C, and 60-2-N-G4S-PETase were relatively high.

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/results/g4s2.png)

Fig. 1   Enzyme
activity
and PET degradation products release of fusion proteins with G4S as linker
(a) . Enzyme activity
of fusion proteins. (b). Release
of PET
degradation products after 24 h.

1.3 PET Degradation

A total of 35 nmol enzyme solution was purified and reacted with 25 mg PET microplastics at 60°C in
8.75 mL
of
Gly-NaOH buffer (pH 9.0), at the end of which the products were analyzed using High Performance
Liquid
Chromatography (HPLC) and calculated the total amount of TPA and MHET released (Fig. 2). The result
showed
that
50-3-N-G4S-PETase, 50-4-N-G4S-PETase, PETase-G4S-50-5-C, 60-2-N-G4S-PETase, and 70-1-N-G4S-PETase
showed
improved degradation efficiency of PET microplastics compared to PETase, with products releases of
139.48%,
144.67%, 126.22%, 126.80%, and 155.62% of PETase, respectively (Fig. 1b).

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/results/hplc.png)

Fig. 2
HPLC results of catalytic products

Part2: Optimization of Fermentation
Conditions
and
Linker
Replacement

2.1 Optimization of Fermentation Conditions

The comprehensive results of the previous experiments revealed that the PET degradation efficiency of
50-4-N-G4S-PETase
was high, but its expression level was low. Therefore, we took it as an example to optimized type of
fermentation medium, concentration of IPTG, fermentation temperature, and fermentation time at the
shaking
flask-level to enhance the protein
expression.

TB, LB, and ZYM were selected as culture media to investigate the optimal medium type (Fig. 3a); IPTG
concentrations of
0.025 mmol/L, 0.050 mmol/L, 0.075 mmol/L, and 0.100 mmol/L were selected to investigate the optimal
IPTG
concentration (Fig. 3b); fermentation was carried out at 16°C and 25°C to investigate the optimal
fermentation
temperature
(Fig. 3c); 24 h and 48 h were selected to investigate the optimal fermentation time (Fig. 3d).

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/results/fajiaoyouhua.png)

Fig. 3
50-4-N-G4S-PETase fermentation conditions optimization results

(a) . Fusion protein enzyme activity in different media. (b). Fusion protein
enzyme
induced by different concentrations of
IPTG. (c). Fusion protein enzyme activity at different fermentation temperatures. (d). Fusion
protein enzyme
activity at
different fermentation times.

The results showed that fermentation with TB as the medium, addition of
IPTG at the concentration of
0.05 mM,
and
fermentation at 16°C for 48 h were the optimal fermentation conditions.

2.2 Linker Replacement

Taking PETase-G4S-70-1-C as an example, we replaced the flexible linker G4S with EAAAK, AG, Tr, 10A,
and SLE,
respectively. Then, we constructed recombinant plasmids
PETase-EAAAK-70-1-C, PETase-AG-70-1-C, PETase-Tr-70-1-C, PETase-10A-70-1-C, PETase-SLE-70-1-C and
transformed them
into *E. coli* BL21 (DE3) to express fusion proteins, respectively (Fig. 4). The enzyme
activity of the
fusion
proteins
was determined (Fig. 5). The results showed that except for G4S, PETase-SLE-70-1-C had highest
enzyme
activities.

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/results/goujian.png)

Fig. 4   Construction of
50-4-N-SLE-PETase fusion protein

(a). PCR validation results. (b). Plasmid map of pET-21b-50-4-N-SLE-PETase.

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/results/linker.png)

Fig. 5   Fusion protein enzyme activity
with different linkers

We attempted to replace the linker of the 50-4-N fusion protein, followed by protein structure
simulations using AlphaFold2 with (G4S)3, (EAAAK)3, (SLE)5, Tr, (AG)5, and 10A linkers, respectively
(Fig. 6).

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/results/alphafold2.png)

Fig. 6   AlphaFold2 structure simulation
results

Molecular dynamics simulations showed that the G4S, Tr, and AG linkers are flexible in the fusion
protein, while the SLE, EAK,
and 10A linkers function as rigid linkers in the fusion protein (Fig. 7).

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/results/fenzidonglixue.png)

Fig. 7   Molecular dynamics simulations

(a). RMSF analog. (b). RMSD analog.

Then, we replaced all the linkers in the 14 heterologously expressible recombinant plasmids with SLE
and transformed
them into *E. coli* BL21 (DE3) for expression. We determined the enzyme activity (Fig. 8a)
and PET degradation ability (Fig. 8b) of the fusion proteins.

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/results/sle-gai.png)

Fig. 8   Enzyme activity and PET
degradation products release of fusion proteins with SLE as linker
(a). Enzyme activity of fusion
proteins. (b). Release of PET degradation products after
24 h.

The results showed that the enzyme activity of the fusion proteins
PETase-SLE-50-5-C, 60-2-N-SLE-PETase, PETase-SLE-70-1-C was relatively high. And the fusion proteins
40-1-N-SLE-PETase, 50-1-N-SLE-PETase, 60-2-N-SLE-PETase, and 70-1-N-SLE-PETase showed improved
degradation efficiency of PET microplastics compared to PETase, with the
products releases 103.75%, 146.97%, 176.37%, and 135.45% of PETase, respectively.

Part3: Binding Capacity Characterization

3.1 Construction of Plasmids

Combining the results of the previous experiments, we found that the PET degradation effect and
enzyme
activity of the
fusion proteins 50-4-N-G4S-PETase, PETase-G4S-50-5-C, and 60-2-N-SLE-PETase were both high at
expression-level and degradation-level. Thus, in order to verify the binding effect of the above three short peptides
with PET
microplastics, we
constructed recombinant plasmids by assembling these three peptides with eGFP with pET-20b as vector
and G4S
as linker,
respectively, and transformed them into *E. coli* BL21 (DE3) for expression.

3.2 Characterization

The eGFP solution and eGFP-fusion protein solution with equal fluorescence intensity were taken and
incubated
with PET
film, protected from light at 37°C for 24 h, respectively. Then, free proteins were eluted, and the
PET film
was observed
under a fluorescence microscope at 488 nm excitation wave (Fig. 9).

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/results/5.png)

Fig. 9
Fluorescence
characterization of 50-4-N, 50-5-C, 60-2-N linked eGFP fusion proteins.

(a) . eGFP of equal fluorescence intensity to 50-4-N-G4S-eGFP binding
PET.(b).
50-4-N-G4S-eGFP binding PET. (c). eGFP of equal fluorescence intensity to 50-5-C-G4S-eGFP binding
PET. (d).
50-5-C-G4S-eGFP binding PET. (e). eGFP of
equal fluorescence intensity to 60-2-N-G4S-eGFP binding PET. (f). 60-2-N-G4S-eGFP-binding PET.

Comparing with the eGFP, 50-4-N-G4S-eGFP, 50-5-C-G4S-eGFP, and
60-2-N-G4S-eGFP showed different
degrees of
enhanced fluorescence signals under the microscope. The results demonstrated that all three
peptides,
50-4-N, 50-5-C, and
60-2-N, could bind to PET efficiently, thus enhancing the degradation of PET of
PETase-linker-peptide fusion
proteins.

Part4: Modification of PET-binding Peptides

4.1 Construction of Plasmids

Based on the multiple mutation sites of 50-4-N, 50-5-C, and 60-2-N
predicted by the dry lab, we
successfully
constructed
19 recombinant plasmids carrying respective mutants using MEGAWHOP cloning method. And these
plasmids were
transformed into *E. coli* BL21 (DE3) for expression, respectively.

4.2 Enzyme Activity

The results showed that the enzyme activity of 50-4-N-Y33F-G4S-PETase was higher than that of its
wild type
(Fig. 10a), which was 175.25% of the wild type.

The enzyme activities of the mutants of PETase-G4S-50-5-C were all higher than that of the wild type
(Fig.
10c), among
which, the enzyme activities of PETase-G4S-50-5-C-R287V and PETase-G4S-50-5-C-C293V were much higher
than
that
of the wild type, which were respectively 184.67%, and 181.39% of wild type.

The 60-2-N-SLE-PETase mutants all had lower enzyme activities than the wild type (Fig. 10e).

![](https://static.igem.wiki/teams/5157/wikimages/wetlab/results/tubian.png)

Fig. 10   Enzyme
activity
and PET degradation products release of single mutants of fusion proteins
(a) . Enzyme activity
of PETase-G4S-50-4-N and its mutants. (b). PET
degradation
products release from
PETase-G4S-50-4-N and mutants after 24 h. (c). Enzyme activity of PETase-G4S-50-5-C and its mutants.
(d).
PET
degradation products release of PETase-G4S-50-5-C and its mutants after 24 h. (e). Enzyme activity
of
PETase-SLE-60-2-N
and its mutants. (f). PET degradation products release from PETase-SLE-60-2-N and its mutants after
24 h.

4.3 PET Degradation

The results showed that product release of 50-4-N-S14A-G4S-PETase, 50-4-N-V19I-G4S-PETase,
50-4-N-L23K-G4S-PETase, 50-4-N-Y33F-G4S-PETase, 50-4-N-S61A-G4S-PETase were all higher than the wild
type
(Fig. 10b), 103.59%, 133.47%, 117.53%, 115.94%, and 126.10% of the wild type, respectively.

The
product
release of all mutants of PETase-G4S-50-5-C were higher than that of the wild type, in which the PET
degradation efficiency of the PETase-G4S-50-5-C-R287V was the most efficient (Fig. 10d), which
was
164.38% of that
of the wild type.

The product release of 60-2-N-N29D-SLE-PETase and 60-2-N-L60I-SLE-PETase was higher than that of the
wild
type (Fig. 10f), which was 102.94%, and 106.21% of the wild type, respectively.

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# Project

[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
[Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)
[Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
[Safety](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27safety%27%29%20%7D%7D)

# Model

[Introduction & Related work](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
[Methology](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27methology%27%29%20%7D%7D)
[Experiment & Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiment_results%27%29%20%7D%7D)

# Wet lab

[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)
[Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
[Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)

# Human Practices

[Integrated Human Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
[Education & Communication](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27education%27%29%20%7D%7D)

# Team

[Team-members](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%20%7D%7D)
[Attributions](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D)

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The repository used to create this website is available at
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