iGEM 2024 — Team nau-china

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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NOTEBOOK

* Wet Lab
* HP
* Model
* Art
* Wiki

Wet Lab

Mar.

* Set up NAU-CHINA 2024 team!

Apr.

* Brainstormed and read literature in different fields.
* Found several directions interested in.
* Consulted relevant experts and selected our project.
* Registered our team and determined our team members.
* Organized Yangtze River Conference.
* Participated in SBC: Synthetic Biology Conference & Exhibition.

May.

* Determined the project: SAMUS.
* Determine *E.coli* as engineered bacteria.
* Read papers related to our project.
* Laboratory safety training.

Jun.

* Built the gene circuit.
* Designed our project poster.
* Experiment training.

* Optimized the design of the gene circuit.
* Verified the function of the gene circuit.
* Interviewed relevant experts for experiment problems.
* Attended 11th CCiC.

Jul.

* Communicated with NJMU-China face-to-face.
* Online meeting with iGEM Kyoto.
* Communicated with Westlake face to face.
* Optimized the expression of cmRNA and fusion proteins.
* Completed the contribution.

Aug.

* Verified the overall gene circuit and results.
* Completed the improvement part.
* Completed safety form.

Sept.

* Edited wiki.

Oct.

HP

Mar.

* Analyzed cases from previous successful iGEM teams, drawing inspiration from all teams striving for excellence.
* Designed the ShePower Interview series.
* Set up a "Calibration" Model for our integrated Human Practices.

Apr.

* Launched our WeChat Official Account and Instagram.
* Mass survey on the awareness of hepatitis and synthetic biology.
* Designed and put iGEMBTI initiative into reality.
* PEER Interview with Zou Yuntong.
* An interview with Prof. Zhang Keyun.
* Host Yangtze River Conference, established Partnerships with WHU-China, HUBU-China, NJU-China, NJTech-China, and NJTECH-CHINA-A.
* Designed the ICll platform, and invited other teams to participate.
* An interview with Prof. Qiao Wenjing.
* Attended SBC: Synthetic Biology Conference & Exhibition.
* Got in touch with China Daily-21 Century TEENS editor Xu Zhaolin.

May.

* An interview with Prof. Chen Xi.
* Recorded a SynBio education outreach video for DaGang Oilfield Third High School.
* Launched "Aromatic Encounters: TCM Sachet DIY" in Kindergarten Affiliated to NUAA
* Launched "Science & Art Integration Activity at Science and Technology Activity Week" at Nanjing Agricultural University.
* Established relationships with the Tu Changwang Lecture Team from Nanjing University.
* Assessed stakeholders of our project.
* Assessed the barriers the underrepresented groups face in STEM fields, and established the "EMBRACE" work model.

Jun.

* Made preparations for CCiC.

* Attended 11th CCiC.
* Established relationships with HNU-China, BUCT-China, NNU-CHINA, SCU-China, PekingHSC, etc.
* Had a collaborative discussion with NJMU-CHINA.
* An interview with Prof. Liu Weizhi.
* An interview with Prof. Zhang Lei.

Jul.

* Volunteer teaching in Anyang town, Guangxi with Tu Changwang Lecture Team from NJU.
* Visited Miyako Ecology Center to have a better grasp of SDGs.
* Had a collaborative discussion with iGEM Kyoto.
* Had a collaborative discussion with Westlake.
* Education outreach in Jinling Library.
* An interview with Jiang Zhengri.
* Launched ICII platform, shot a Citywalk vlog video for our team.

Aug.

* Published "Playing with DNA" on "Science Study, TEENS Senior 2".
* Visited Xiaolingwei Middle School.
* Visited the NJAU community.
* Recorded a podcast and co-wrote a journal of Genetic Whismy with BUCT-China
* Visited Nanjing Zhonghua High School.
* Campus activity of iGEM on Yulan Road and carried out the recruitment promotion for college students, on-campus publicity meetings.
* Summarized Human Practices Content and seek advice from Prof. Wang Jufang, prepare mini iGEM in our school.

Sept.

* Edited wiki.

Oct.

Model

Apr.

* Learned relevant knowledge of synthetic biology, related models and previous projects.

May.

* Determined the direction of modeling.

Jun.

* Selected the models initially and determined the model processing method.

* Completed the preliminary establishment.

Jul.

* Adjusted and improved the models.

Aug.

* Completed basic solving of the models.

Sept.

* Completed all models and wiki edits.

Oct.

Art

May.

* Designed the logo of the team.

Jun.

* Design illustrations related to the team and project.

Jul.

* Made posters for our project.

* Designed the wiki of ICII.
* Directed and edited the Promotion Video.
* Designed ICII online poster.

Aug.

* Composed the wiki and illustrated the principle of the project.
* Designed and made goods and uniforms.

Sept.

* Polished wiki.

Oct.

Wiki

Jun.

* Edited the wiki of description.

Aug.

* Edited the wiki of ICII.
* Constructed the framework of the wiki.

Sept.

* Edited the wiki.

Jun.

* Edited wiki ＆ celebrated wiki freeze!

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ENGINEERING

* [![图片1](https://static.igem.wiki/teams/5398/icon/description.svg)

  Description](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27description%27%29%7D%7D)
* [![图片1](https://static.igem.wiki/teams/5398/icon/design.svg)

  Design](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27design%27%29%7D%7D)
* [![图片1](https://static.igem.wiki/teams/5398/icon/engineering.svg)

  Engineering](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27engineering%27%29%7D%7D)
* [![图片1](https://static.igem.wiki/teams/5398/icon/protocol.svg)

  Protocol](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27protocol%27%29%7D%7D)
* [![图片1](https://static.igem.wiki/teams/5398/icon/safety.svg)

  Safety](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27safety%27%29%7D%7D)
* [![图片1](https://static.igem.wiki/teams/5398/icon/implementation.svg)

  Implementation](%7B%7B%20url_for%28%27pages%27%2Cpage%3D%27implementation%27%29%7D%7D)

* Self-healing Module
* Loop1
* Loop2
* Loop3
* Adhesive Module
* Loop1
* Loop2
* Loop3
* Loop4
* Tyrosinase Catalysis System
* Loop1
* Loop2
* Loop3

Self-healing Module

LOOP 1

Design

Soft robots are known for their intelligence, flexibility, and adaptability, showing
great potential in the field of
ocean exploration. However, these robots are easily damaged in the complex and changeable marine
environment. If not
repaired in time, it can greatly decrease their lifespan and increase salvage and repair costs. However,
current
self-healing materials have shortcomings that limit their practical application, such as severe
requirements for the
healing process, long healing times (hours), and are not environmentally friendly. Considering the
existing problems,
our project tried to develop a new type of self-healing material for underwater soft robots.

Then, we noticed squid ring teeth (SRT) proteins which have high elastic modulus and
toughness due to their special
sequence. We optimized the tandem repeat polypeptides with n repetitions (TRn) derived from squid ring
teeth proteins
and make them self-assemble into supramolecular β-sheet-stabilized networks (Fig. 1), enhancing their
hydrogen-bonded
nanostructure and network morphology and giving their excellent healing properties (2–23 MPa strength
after 1 s of
healing). Such healing performance creates new opportunities for bioinspired materials design, especially
in
self-healing materials for soft robotics and personal protective equipment.

![](https://static.igem.wiki/teams/5398/design/design-new/fig2.png)

Fig. 1 | The sequence and structure of tandem repeat polypeptides with n repetitions (TRn)
derived from squid ring teeth
proteins.

In our project, we used the biosythetic protein TRn5 as a component of special materials
to realize self-healing. We
anticipated the engineered bacteria could successfully express TRn5 and make it into materials with
self-healing ability.

**Build**

First, we used AlphaFold3 to predict the structure of TRn5. Then, we constructed
pET-29a(+)-TRn5 (Fig. 2a) and
respectively amplified TRn5 and pET-29a(+) vector using PCR. The PCR produced the pET-29a(+)-TRn5 parts
ready for
In-fusion Cloning (Fig. 2b,c).

![](https://static.igem.wiki/teams/5398/cmrna/engineering/2.png)

Fig. 2 | The plasmid map of pET-29a(+)-TRn5 and 1% agarose gel electrophoresis of the PCR
amplified pET-29a(+)-TRn5
parts.

a. The plasmid map of pET-29a(+)-TRn5.
b. 1% agarose gel electrophoresis of the PCR amplified TRn5 (K5398001) (527 bp).
c. 1% agarose gel electrophoresis of the PCR amplified pET-29a(+) plasmid (5170 bp).

Next, we made an In-fusion Cloning of purified PCR amplified TRn5 and the pET-29a(+)
vector parts and transferred the
recombinant plasmid into *E.coli* DH5α. According to the results of colony PCR and Sanger sequencing,
we concluded
that the pET-29a(+)-TRn5 plasmid was constructed successfully (Fig. 3).

![](https://static.igem.wiki/teams/5398/cmrna/engineering/3.png)

Fig. 3 | Verification of recombinant plamid pET-29a(+)-TRn5.

a. 1% agarose gel electrophoresis of colony PCR of using T7 and T7 ter primers.
b. The result of sequencing the TRn5 of the recombinant plasmid.

We expressed the protein in *E.coli* BL21(DE3) using LB medium and incubated the
cells at 23℃ for 16
h and 37℃
for 5 h respectively. Then, we denatured TRn5 with 8 M urea for 12 h and renatured it by dialysis.

**Test**

We found
the TRn5 predicted by AlphaFold had β-sheet composed of five β-strands and were connected by flexible
chains as
we expected (Fig. 4). This provided solid foundation for our next experiment.

![](https://static.igem.wiki/teams/5398/trn5/20240928-124228.webp)

Fig. 4 | TRn5 predicted by AlphaFold3.

Using SDS-PAGE to assess the expression of TRn5, we found that most TRn5 (17.58 kDa)
existed in precipitate as stated in
previous research and the TRn5 expression level at two temperatures had little difference (Fig. 5).

![](https://static.igem.wiki/teams/5398/cmrna/engineering/5.png)

Fig. 5 | SDS-PAGE of expression products of TRn5.

Lane 1: marker; Lanes 2-4: whole-cell lysate, supernatant and pellet from uninduced cells
at 23℃, respectively; Lanes
5-7: whole-cell lysate, supernatant and pellet from induced cells at 23℃, respectively; Lanes 8-10:
whole-cell lysate,
supernatant and pellet from uninduced cells at 37℃, respectively; Lanes 11-13: whole-cell lysate,
supernatant and pellet
from induced cells at 37℃, respectively.

As a result, when we purified TRn5 by Immobilized Metal Affinity Chromatography (IMAC),
we found a great protein loss as
shown in SDS-PAGE because the TRn5 expression level was too low to verify (Fig. 6).

![](https://static.igem.wiki/teams/5398/cmrna/engineering/6.png)

Fig. 6 | SDS-PAGE of expression products of TRn5 purified by IMAC.

Lane 1: marker; Lanes 2-11: induced cell samples at 23℃; Lane 2: pellet; lane 3: sample
washed with denaturing buffer
with 8 M urea; Lane 4: sample after dialysis overnight; Lane 5: sample after being bound to Ni-NTA resin;
Lane 6: sample
eluted with 20 mM Tris-HCl; Lanes 7-11: samples eluted with 20, 50, 150, 300, 500 mM imidazoles.

**Learn**

Based on the results of production of TRn5, we learned that incubation at 37℃ for 5 h was
an efficient method to express
TRn5. At the same time, due to the failure of purification of TRn5 by IMAC, we concluded TRn5 was not
suitable for this
purification strategy. Therefore, after a comprehensive review of the existing literature, we found that
the protonation
of His residued in the sequence of TRn was important for solubility and the acidification of TRn resulted in
conformational switch, causing the protein to self-assemble and facilitating self-healing ability.
Therefore, we
attampted to use a new protocol to purify TRn5.

LOOP 2

Design and Build

We set 37℃ for 5 h as our incubation condition and implemented a novel protocol
containing 5% acetic acid for the
purification of TRn5. We then obtained protein samples of TRn5 by freezedrying 24 h, dissolved protein
samples in 5%
acetic acid again and dried them to get TRn5 protein films.

Test

We separated TRn5 by SDS-PAGE, and distinct and clear bands of TRn5 (17.58 kDa) were observed (Fig. 7).

![](https://static.igem.wiki/teams/5398/cmrna/engineering/7.png)

Fig. 7 | SDS-PAGE of expression products of TRn5 using a new protocol.

Lane 1: marker; Lanes 2-4: whole-cell lysate, supernatant and pellet from induced cells
at 37℃, respectively; Lane 5:
sample washed with 5% acetic acid.

We obtained protein samples of TRn5, whose final yield was about 150.4 mg/L bacterial
culture (Fig. 8).

![](https://static.igem.wiki/teams/5398/cmrna/engineering/8.png)

Fig. 8 | The protein sample freeze-dried by a lyophilizer.

To test the self-healing ability of TRn5 protein film, we did a punctured simulation.
After putting the punctured film
at room temperature for 12 h, we saw the hole defect healing (Fig. 9). So it was proved that the materials
made of TRn5
have self-healing properties to some extent. However, the self-healing efficiency of TRn5 could not meet
the
requirements of damaged underwater soft robots, which required higher self-healing efficiency.

![](https://static.igem.wiki/teams/5398/cmrna/engineering/9.png)

Fig. 9 | Self-healing of TRn5 protein film after puncture damage.

**a.** A hole defect was left by a needle through the film. **b.** Puncture damage was
healed.

Learn

To pursue more excellent self-healing materials, we deeply studied literature on TRn and
squid ring teeth protein. We
found there exists a positive correlation between the number of repeat units and self-healing properties
of
squid-inspired proteins in an article, which means the more repeat units the proteins have, the better
self-healing
properties it will be. Therefore, we need to produce the squid-inspired biosynthetic proteins with various
long tandem
repeats (TRn). However, it would lead to excess burden of chassis and low yield finally. To solve these
problems above,
we learnt the *td* intron, also called RNA cyclase ribozyme, which can form a circular mRNA, allowing
ribosomes to
repeatedly translate the sequence derived from squid ring teeth protein and produce proteins with
different repeat
numbers. Thus, we adopted the cmRNA strategy to obtain materials with exceptional self-healing properties.

LOOP 3

Design

We designed a circular mRNA on which the ORF of TRn5 is between the 3' and 5' intron of
*td* gene from T4 phage. The
ribosome binding sequence (RBS) and start codon ATG were placed downstream of TRn5. Consequently, the
regulatory
sequences were located upstream of TRn5 only after circularization of the mRNA. To purify the mixed TRn
protein which
repeated for different times, a His tag was incorporated into the TRn5. If the mRNA is circularized, the
ribosome could
circle the cmRNA, producing proteins with different repeats (Fig. 10).

![](https://static.igem.wiki/teams/5398/design/design-new/fig-3-new.png)

**Fig. 10 | Design of a circular mRNA based on *td* flanking introns.**

Build

We synthesized plasmid pET-29a(+)-cmRNA(TRn5) and transferred it into *E.coli* BL21(DE3) to express and obtain TRn through the successful methods that TRn5 previously used before. At the same time, we built some mathematical and biological models to examine the stability of the proteins.

Test

After the protein samples were subjected to SDS-PAGE (Fig. 11), we found that TRn sample
was a mixture of proteins of
various sizes that formed a ladder on the gel due to uncertainty of the round number that the ribosome
traveled.
According to the protein marker, we supposed that the size of the proteins ranged from about 8 to 96 kDa,
indicating
that the ribosome could travel along the cmRNA at most 6 rounds.

![](https://static.igem.wiki/teams/5398/cmrna/engineering/11.png)

**Fig. 11 | SDS-PAGE of cmRNA expressed at 37℃ and 16℃.**

Lane 1: marker; Lanes 2-5: whole-cell lysate, supernatant, pellet and diluted pellet from
induced cells at 37℃,
respectively; Lane 6: marker; Lanes 7-9: whole-cell lysate, supernatant and pellet from induced cells at
16℃,
respectively.

We did a punctured simulation and tested the self-healing ability of materials. We
clearly saw the hole defect healing
at a quick pace (Fig. 12). Compared with the self-healing effciency of TRn5 protein film (12 h of
healing), this
self-healing efficiency of TRn produced by RNA cyclase ribozyme mechanism is higher because nearly 90%
healing were
completed in 1 h. Meanwhile, it should be noticed that the hole defect of TRn protein film is bigger than
that of TRn5
protein film.

![](https://static.igem.wiki/teams/5398/cmrna/engineering/12.png)

**Fig. 12 | Self-healing of TRn protein films after puncture damage.**

Using mathematical modeling to simulate the individual hydrogen bond network (Fig. 13 and
Fig. 14), we can see that as
the number of repetitions increases, the point-line ratio which can reflect the stability of the network
structure
gradually decreases, indicating that the network gradually changes from loose to tight. In microscopic
protein
structures with self-healing functional materials, networks with dense lines can often recover faster and
show stronger
self-healing ability. Therefore, the more times circular mRNA is translated, the higher the biological
activity and
functional stability of the protein will be.

![](https://static.igem.wiki/teams/5398/cmrna/gif1.webp)

**Fig. 13 | Intermolecular hydrogen bond networks in different proteins.**

TRn with n=5, 10, 15, 20, 25 and 30 from top to bottom, left to right.

![](https://static.igem.wiki/teams/5398/model/1jpg.jpg)

**Fig. 14 | Results of intermolecular hydrogen bond network.**

Subsequently, we simulated the overall hydrogen bond network (Fig. 15 and Fig. 16). we
got consistent conclusions. The more times protein repeats, and the more stable the hydrogen bond network between protein molecules will be, which makes the
interacting
protein molecules more resistant to deformation when facing external perturbations, and have higher
self-healing
adaptability.

![](https://static.igem.wiki/teams/5398/cmrna/gif2.webp)

**Fig. 15 | Simulation of hydrogen bond networks between TRn molecule.**

TRn with n=5, 10, 15, 20, 25 and 30 from top to bottom, left to right.

![](https://static.igem.wiki/teams/5398/model/2jpg.jpg)

**Fig. 16 | Results of overall hydrogen bond network.**

To verify the validity of our mathematical modeling above, molecular dynamics were
simulated and the energy in the
system was calculated (Fig. 17). We found that as TRn repetitions increased, the energy of the system became lower and lower, so the protein became more stable, which was consistent with the results we obtained through mathematical modeling, and to a certain extent, it showed the accuracy of the mathematical modeling results.

![](https://static.igem.wiki/teams/5398/model/energy.jpg)

**Fig. 17 | Unit energy of different TRn.**

Learn

We succeeded in producing the tandem repeat polypeptides with n repetitions (TRn) derived
from squid ring teeth proteins
by RNA cyclase ribozyme mechanism. Simultaneously, we verified our materials with excellent self-healing
ability from
function tests and mathematical modeling. In brief, all of these findings indicate that the cmRNA is a
good strategy to
produce squid-inspired biosynthetic proteins with various long tandem repeats.

In the future, we will further research the production of the long tandem repeats by RNA
cyclase ribozyme mechanism and
improve the self-healing ability of our materials. We hope that our materials can finally realize the
self-healing
capability in 1 sec, which we saw in papers before.

If you want to know more about our work in wet lab experiments and modeling, you can
click the links below.

Wet Lab Experiments <https://2024.igem.wiki/nau-china/projectresult>

Modeling <https://2024.igem.wiki/nau-china/model>

Adhesive Module

LOOP 1

Design

The adhesion of material with self-healing capabilities to the substrate material is a
crucial part of our project.
After extensive literature review, we discovered that the tandem repeat polypeptides with 4
repetitions(TRn4) can bind
to the tandem repeat polypeptides with n repetitions(TRn) through hydrogen bonding derived from β-sheets.
Additionally,
the mussel foot protein type 5 (Mfp5), which is rich in tyrosine residues, can be oxidized to
Levodopa(L-DOPA) by
tyrosinase TyrVs and then adheres to the substrate material of the soft robot.

Based on the above, we designed a fusion protein TRn4-mfp5 for the adhesion of
self-healing proteins to the substrate
material of soft robots.

![](https://static.igem.wiki/teams/5398/trn4-mfp5/loop1-learn.webp)

Fig. 18 | The adhesion principle of Mfp5.

Build

We used the pET-21a(+) vector to express the protein in *E. coli* BL21(DE3) strains,
with 0.5 M IPTG induction.

![](https://static.igem.wiki/teams/5398/trn4-mfp5/pet21a-trn4-mfp5-map-white-1.webp)

Fig. 19 | The plasmid map of pET-21a(+)-His-SUMO-TRn4-mfp5.

We planned to express the protein in *E. coli* BL21(DE3) using LB medium. We
conducted incubation at 16°C for 20 h
or at 37°C for 4 h.

Additionally, we used both LB and TB media for cultivation to identify optimal conditions
for protein expression. This
comparison was aimed at determining which medium would support higher yields and better solubility of the
fusion
protein.

Test

We used SDS-PAGE to analyze the protein expression under the conditions of 16°C for 20
h or 37°C for 4 h (Fig.
20).

![](https://static.igem.wiki/teams/5398/trn4-mfp5/16-37-lb-pet21a-new-white.webp)

Fig. 20 | Comparison of fusion protein expression in different temperature.

Lane 1: Protein ladder; Lanes 2-7 (LB 37°C 4 h): Lane 2: Total liquid (IPTG); Lane 3:
Supernatant (IPTG); Lane 4:
Precipitate (IPTG); Lane 5: Total liquid; Lane 6: Supernatant; Lane 7: Precipitate; Lanes 8-13 (TB 16°C 20
h): Lane 8:
Total liquid (IPTG); Lane 9: Supernatant (IPTG); Lane 10: Precipitate (IPTG); Lane 11: Total liquid; Lane
12:
Supernatant; Lane 13: Precipitate; Lane 14: Protein ladder.

We found that the protein expressed better under the 16°C for 20 h condition, as
indicated by the stronger bands in Fig.
20. This suggests that lower temperature incubation may enhance protein solubility and proper folding,
resulting in
improved yield.

This is the SDS-PAGE analysis after cultivation in LB and TB media. (Fig. 21)

![](https://static.igem.wiki/teams/5398/trn4-mfp5/tb-lb-prt21a-new-white.webp)

Fig. 21 | Comparison of fusion protein expression in LB and TB media use vector pET-21a(+).

Lanes 1-6 (LB 16°C 20 h): Lane 1: Total liquid (IPTG); Lane 2: Supernatant (IPTG); Lane
3: Precipitate (IPTG); Lane 4:
Total liquid; Lane 5: Supernatant; Lane 6: Precipitate; Lane 7: Protein ladder; Lanes 8-13 (TB 16°C 20 h):
Lane 8: Total
liquid (IPTG); Lane 9: Supernatant (IPTG); Lane 10: Precipitate (IPTG); Lane 11: Total liquid; Lane 12:
Supernatant;
Lane 13: Precipitate.

In Fig. 21, We found that the bands in the TB medium were indeed thicker than those in the
LB medium, indicating a slight
increase in expression levels.

However, the theoretical size of the fusion protein TRn4-mfp5 on the pET-21a(+) vector is
21.2 kDa, but the bands in
Fig. 20-21 did not appear in the expected position.

Learn

Based on the result above, we have doubted the expression results of the TRn4-mfp5 fusion protein using
the pET-21a(+) vector. This
discrepancy could be due to unsuitable expression vectors or conditions, or it may be caused by structural
interference
between the TRn4 and Mfp5 domains, leading to an incorrect protein size.

The fusion protein plays a crucial role in our material. It comprises TRn4, Mfp5, and a
GS linker (which links the
terminal proteins). However, the structure data for these proteins are currently absent in common protein
databases such
as RCSB, UniProt, and NCBI.

Consequently, we must predict their structures separately to validate their properties.
Fortunately, we can collect
possible structural characteristics of TRn4 and Mfp5 from the literature, which will guide our predictions
and improve
the accuracy of the modeled structures, allowing us to prioritize predictions for these two proteins.

LOOP 2

Design

The fusion protein plays a crucial role in our material. It comprises TRn4, Mfp5, and a
GS linker (amino acid sequence:
GGGGSGGGGSGGGGS, which links the terminal proteins).

However, the structure data for these proteins are currently absent in common protein
databases such as RCSB, UniProt,
and NCBI. Consequently, we predicted their structures separately to validate their properties.
Fortunately, we can
collect possible structural characteristics of TRn4 and Mfp5 from the literature, which will guide our
predictions and
improve the accuracy of the modeled structures, allowing us to prioritize predictions for these two
proteins.

Given the limitations of SWISS-MODEL in predicting the structure of larger proteins, we
chose to use AlphaFold for
predicting the structure of this fusion protein.

Test

To further validate our design, we conducted molecular dynamics simulations, based on the
AlphaFold prediction results
(Fig. 22), the results of which are shown in Fig. 23. These simulations provided critical insights into the
protein's
stability, as well as potential dynamic behaviors and conformational changes under various environmental
conditions.

To assess whether there was a trend in the root mean square deviation (RMSD) graph, we applied the
Cox-Stuart trend test
during the 2-3 ns stage of the simulation. This non-parametric test helped determine whether the data
exhibited an
upward or downward trend over time, providing key analysis to confirm the stability and overall
suitability of the
fusion protein for real-world applications.

![](https://static.igem.wiki/teams/5398/trn4-mfp5/fusion-protein-pdb.webp)

Fig. 22 | Fusion protein predicted by AlphaFold

![](https://static.igem.wiki/teams/5398/model/beta.gif)

Fig. 23 | Molecular dynamics simulation for fusion protein.

Learn

From the molecular dynamics simulation of the fusion protein, we observed that the
overall conformation remained
relatively stable throughout the simulation, with no significant structural dissociation or major
deformations.

Thus, one of our hypothesis that the fusion protein's unexpectd expression was due to
structural interference has been
resolved, as the model confirmed that the TRn4 and Mfp5 domains do not interfere with each other.

So we tried to change expression vectors to address the problem with protein expression.

Several studies suggest that using the Rosetta strain may be advantageous, as it provides
tRNA genes for rare codons
that are often underrepresented in *E. coli*. This strain is specifically engineered to enhance the
expression of
eukaryotic genes in prokaryotic systems. We anticipated that utilizing Rosetta would significantly improve
protein yield
and overall expression efficiency.

Studies have shown that a SUMO tag can enhance protein solubility and reduce the
formation of inclusion bodies.

LOOP 3

Design

We decided to incorporate a SUMO tag into the plasmid to enhance protein solubility. To
achieve this, we planned to
construct the plasmid using pET-SUMO (Fig. 24) not only in BL21(DE3) but also in Rosetta strains, which
provides tRNA for
rare codons, to improve both the expression and quality of the TRn4-mfp5 protein. Additionally, the
expression
conditions were optimized to further increase protein yield and solubility.

![](https://static.igem.wiki/teams/5398/trn4-mfp5/pet-pc-sumo-trn4-mfp5-map-white.webp)

Fig. 24 | The plasmid map of pET-SUMO-TRn4-mfp5.

Build

We expressed the proteins under the optimized conditions of induction at 37°C for 4 h
using 0.5 mM IPTG. After lysis,
the target protein was primarily found in the pellet, which we denatured with 8 M urea for 12 h. The fusion
protein was
then purified using a Ni-NTA Gravity Column.

Test

The target protein was successfully expressed in both BL21(DE3) and Rosetta *E. coli* strains.
Although the
electrophoresis bands showed slight deviations from the theoretical molecular weight, they remained within
an acceptable
range, likely due to potential modifications. In Fig. 25, we compared the expression efficiency between the
two strains.

![](https://static.igem.wiki/teams/5398/trn4-mfp5/rostta-bl21-de3-trn4-mfp-new-white.webp)

Fig. 25 | Comparison of fusion protein expression in *E. coli* BL21(DE3) and Rosetta.

Lane 1: Protein ladder; Lanes 2-4 (BL21(DE3) LB 37℃ 4 h): Lane 2: Total liquid (IPTG);
Lane 3: Supernatant (IPTG); Lane
4: Precipitate (IPTG); Lanes 5-7 (Rosetta LB 37℃ 4 h) Lane 5: Total liquid (IPTG); Lane 6: Supernatant
(IPTG); Lane 7:
Precipitate (IPTG)

Through SDS-PAGE analysis of the purified protein by using a Ni-NTA Gravity Column, we
observed the target protein in
lanes 4 to 7, with a particularly strong band in the supernatant after denaturation (Fig. 26, Lane 7),
confirming
successful expression.

![](https://static.igem.wiki/teams/5398/trn4-mfp5/purification-trn4-mfp5-new-white.webp)

Fig. 26 | Comparison of fusion protein expression in *E. coli* BL21(DE3) and Rosetta.

Lane 1: Protein-Binding buffer; Lane 2: 20 mM imidazole and 8 M urea elution; Lane 3: 50
mM imidazole and 8 M urea
elution; Lane 4: 150 mM imidazole and 8 M urea elution; Lane 5: 300 mM imidazole and 8 M urea elution;
Lane 6: 500 mM
imidazole and 8 M urea elution; Lane 7: Supernatant; Lane 8: Impurities; Lane 9: Protein ladder.

To precisely confirm the expression of TRn4-mfp5, we performed a western blotting
analysis, which provided clear and
definitive results, verifying the successful expression of the TRn4-mfp5 protein under the aforementioned
conditions
(Fig. 27).

![](https://static.igem.wiki/teams/5398/trn4-mfp5/wb-new-white.webp)

Fig. 27 | Western blotting of purified fusion protein TRn4-mfp5(35.4 kDa) expressed by vector
pET-SUMO.

**a.** Western blotting of the pre-expressed protein. Lane 1: Total liquid (IPTG);
Lane 2: Supernatant (IPTG); Lane
3: Precipitate (IPTG), **b.** Western blotting after column purification of the supernatant following
denaturation.
Lane 1: Supernatant; Lane 2: 20 mM imidazole and 8 M urea elution; Lane 3: 50 mM imidazole and 8 M urea
elution; Lane 4:
150 mM imidazole and 8 M urea elution; Lane 5: 300 mM imidazole and 8 M urea elution; Lane 6: 500 mM
imidazole and 8 M
urea elution.

We obtained protein samples of TRn4-mfp5 by freezedrying 24 h (Fig. 28). We finally got
about 25 mg protein from 1 L bacterial
culture.

![](https://static.igem.wiki/teams/5398/trn4-mfp5/protein-freeze-actual-picture-new.webp)

Fig. 28 | The protein sample freeze-dried by a lyophilizer.

Learn

The above addressed the expression issues of the fusion protein TRn4-mfp5. In the following steps, we
planned to conduct
experiments co-expressing it with tyrosinase to verify that the tyrosine residues in mfp5 need to be
converted into
L-DOPA, as highlighted in our design, for it to exhibit adhesive properties.

LOOP 4

Design

We aimed to ensure that the tyrosine residues in the Mfp5 portion of TRn4-mfp5 can be
catalyzed by tyrosinase to form
L-DOPA, thereby enabling its adhesive functionality.

To achieve the co-expression of Mfp5 and tyrosinase, we designed a system utilizing two
separate plasmids. Tyrosinase
was cloned into the pET-SUMO vector to ensure proper folding and solubility, while the fusion protein
(TRn4-mfp5) was
inserted into the pET-28a(+) vector. This strategic design aims to enable the simultaneous expression of
both proteins
in the same host cell, allowing for the efficient oxidation of tyrosine residues in Mfp5 during the
expression process,
thus enhancing its adhesive properties.

Build

Both plasmids were co-transformed into BL21(DE3) cells for simultaneous expression. This
was chosen to make the tyrosine
residues in Mfp5 oxidized into L-DOPA during the expression process, thereby enhancing its adhesive
properties.

Test

To confirm the successful co-expression of tyrosinase and the fusion protein (TRn4-mfp5),
we performed a western
blotting analysis using an anti-His antibody. Since the fusion protein (TRn4-mfp5) was tagged with a
His-tag, this
allowed us to specifically detect its expression. The results showed a clear band at the expected
molecular weight of
TRn4-mfp5, confirming that the fusion protein was successfully expressed. Additionally, a band
corresponding to
tyrosinase was also detected, suggesting that the tyrosinase may have been expressed with some affinity
for the His-tag,
likely due to interactions between the proteins during the expression process. The WB image below shows
these results.

![](https://static.igem.wiki/teams/5398/trn4-mfp5/coexpression-tyrvs-trn4-mfp5-wb-white.webp)

Fig. 29 | Western blotting analysis of tyrosinase and TRn4-mfp5 co-expression.

Lane 1: total liquid (IPTG);
Lane 2: supernatant (IPTG);
Lane 3: precipitate (IPTG).

We dissolved protein samples in Buffer A (10 mL 20 mM Tris pH = 8) to reach 0.3 mg/mL,
and conduct adhesive ability
tests on the fusion protein(Fig. 30). 20 μL of the protein solution was applied, and the
pipette tip was
placed on a plastic Petri dish lid. After incubation at 37°C for 4 h, the pipette tip successfully
adhered.

We found that when tyrosinase is added, the tyrosine residues in Mfp5 are successfully
converted to L-DOPA, allowing the
fusion protein to adhere effectively. However, without the addition of tyrosinase, the fusion protein
fails to exhibit
adhesive properties.

![](https://static.igem.wiki/teams/5398/trn4-mfp5/part-fig10.webp )

Fig. 30 | Adhesive ability test of fusion protein on plastic surface.

**Viscosity Calculations**

**Surface Area Calculation:**

The surface area for the annular region of the pipette tip is calculated as:

**S** = π × (router2 - rinner2)

Where:
router = 3 mm = 0.3 cm
rinner = 1.85 mm = 0.185 cm

Substituting these values, we get:

**S** = π × (0.32 - 0.1852) = π × (0.09 - 0.034225) = π × 0.055775 ≈ 0.1753 cm2

Force Calculation:

The total force is calculated as:

**F** = (5.951 + 0.448 × 15) g × 9.8 N/kg = 12.671 g × 9.8 N/kg ≈ 0.12418 N

**Adhesive Force Calculation:**

The adhesive force produced by the protein is:

**P** = **F** / **S** = 0.12418 N / 0.1753 cm2 ≈ 0.708 N/cm2 = 7.08 kPa

**Adhesive Force per Milligram of Protein:**

The adhesive force per milligram of protein is:

**P'** = **P** / m = 7.08 kPa / 1 mg = 7.08 kPa/mg

Learn

From this experiment, we learned that tyrosinase successfully oxidized the tyrosine
residues in Mfp5, which is crucial
for enhancing its adhesive properties. We also recognized the potential issue of L-DOPA being further
oxidized into
dopaquinone, which could negatively impact the adhesive performance of Mfp5. To address this, we planned
to add Mfp6 in
the co-expression system, as it has the ability to reduce DOPA-quinone back to DOPA, maintaining the
adhesive
functionality of Mfp5.

**Tyrosinase Catalysis System**

LOOP 1

Design

Tyrosinase can oxidize tyrosine residues in TRn4-mfp5 to L-DOPA, thereby imparting
adhesive properties. After reviewing
relevant literature, we selected the tyrosinase TyrVs from *Verrucomicrobium spinosum*, which
exhibits high
expression efficiency in heterologous hosts and possesses high monophenolase activity. It
effectively converts
L-tyrosine into L-DOPA, with greater specificity for L-tyrosine and lower consumption of L-DOPA
compared to other
bacterial tyrosinases. Based on the reasons above, we chose to express the tyrosinase TyrVs in
*E. coli.*

![](https://static.igem.wiki/teams/5398/mfp6-picture/20241002-115707.png)

Fig. 31 | Synthesis scheme of L-DOPA and further oxidized product L-dopaquinone.

Build

We first synthesized the plasmid pETDuet-1-TyrVs-mfp6 and transformed it into *E.
coli* BL21(DE3).

![](https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/duet-map.webp)

Fig. 32 | Expression plasmids of the co-expression system to obtain in vivo-modified
TRn4-mfp5.

We tested the parts we constructed through Sanger sequencing. The results showed
that we had successfully constructed
the plasmid. The *E. coli* BL21(DE3) carrying the pETDuet-1-TyrVs-mfp6 were grown at 16°C for
20 h, and a
separate batch was grown at 24°C for 20 h in Luria-Bertani (LB) medium.

Test

After protein extraction, different proteins were separated by SDS-PAGE and stained
with Coomassie Brilliant Blue (Fig.
33). The results revealed that the expression levels of TyrVs (38 kDa) were low.

![](https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/duet-1-tyrvs-expression.webp)

Fig. 33 | Expression of recombinant TyrVs in *E. coli* BL21(DE3) with
pETDuet-1-TyrVs-mfp6.

**a.** SDS-PAGE analysis for the expression of TyrVs cultured for 20 h at 16℃.
Lane 1: marker; Lanes 2-4: whole-cell
lysate, supernatant and pellet from induced cells with 0.5 mM IPTG respectively; Lanes 5-7:
whole-cell lysate,
supernatant and pellet from induced cells respectively.**b.** SDS-PAGE analysis for the
expression of TyrVs cultured
for 20 h at 24℃. Lane 1: marker; Lanes 2-4: whole-cell lysate, supernatant and pellet from induced
cells with 0.5 mM
IPTG respectively; Lanes 5-7: whole-cell lysate, supernatant and pellet from induced cells
respectively.

Learn

We considered that the low protein expression might be due to the weak strength of
the promoter in the pETDuet-1
vector, and certain sequences in the vector may also affect the expression of TyrVs.

LOOP 2

Design

We cloned TyrVs into other high-copy vectors with a strong promoter and further
reduced the protein expression
temperature in an attempt to obtain soluble TyrVs.

Build

We cloned TyrVs into pET-29a(+) (Fig. 34)and pET-SUMO (Fig. 35). We tested the parts
we constructed through Sanger
sequencing. The results showed that we had successfully constructed plasmids.

![](https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/29a-tyrvs-gel-new.png)

Fig. 34 | Plasmid pET-29a(+)-TyrVs construction results.

**a.** Expression plasmids of TyrVs. **b.** PCR results of pET-29a(+)-TyrVs. Lane
1: Marker; Lanes 2,3: Vector;
Lanes 4,5: Gene.

![](https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/tyrvs-gel-new.png)

Fig. 35 | Plasmid pET-SUMO-TyrVs construction results.

**a.** Expression plasmids of TyrVs. **b.**PCR results of pET-SUMO-TyrVs.
Lane 1: Marker; Lanes 2,3: Vector; Lanes
4,5: Gene.

The engineered bacteria were further incubated at 16°C for 20 h and 37℃ for 6 h.

Test

After protein extraction, different proteins were separated by SDS-PAGE and stained
with Coomassie Brilliant Blue. The
Coomassie Brilliant Blue staining results showed whether the cultivation temperature is 16℃ or 37 ℃,
*E. coli* BL21(DE3) carrying pET-29a(+) did not exhibit significant expression of TyrVs.

![](https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/29a-tyrvs.webp)

Fig. 36 | Expression of recombinant TyrVs in *E. coli* BL21(DE3) with
pET-29a(+)-TyrVs.

**a.** SDS-PAGE analysis for the expression of TyrVs cultured for 20 h at
16℃. Lane 1: marker; lanes 2-4:
whole-cell lysate, supernatant and pellet from induced cells with 0.5 mM IPTG respectively; Lanes
5-7: whole-cell
lysate, supernatant and pellet from induced cells respectively. **b.** SDS-PAGE analysis for the
expression of TyrVs
cultured for 6 h at 37℃. Lane 1: marker; Lanes 2-4: whole-cell lysate, supernatant and pellet
from induced cells
with 0.5 mM IPTG respectively.

The *E. coli* BL21(DE3) carrying pET-SUMO-TyrVs cultured at 16°C were
successfully expressed SUMO-TyrVs (52.2 kDa)
and it was primarily present in the supernatant, indicating that it was expressed in a soluble form.

![](https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/tyrvs-pre-expression.webp)

Fig. 37 | Expression of recombinant TyrVs in *E. coli* BL21(DE3) with
pET-SUMO-TyrVs.

Lane 1: Marker; Lanes 2-4: whole-cell lysate, supernatant and pellet from induced
cells with 0.5 mM IPTG respectively;
Lanes 5-7: whole-cell lysate, supernatant and pellet from induced cells respectively.

We further confirmed the expression of SUMO-TyrVs through western blotting (WB).

![](https://static.igem.wiki/teams/5398/tyrvs/tyrvs-new/tyrvs-western.webp)

Fig. 38 | Western blotting (WB) analysis of recombinant TyrVs in *E. coli*BL21 (DE3)
with pET-SUMO-TyrVs.

Lanes 1-3: whole-cell lysate, pellet and supernatant from induced cells with 0.5 mM
IPTG respectively.

Subsequently, we conducted enzymatic activity tests and determined that the
Km and Vmax for the
catalysis of tyrosine to L-DOPA are: 456.8 μmol/L and 0.31 μmol·L-1·s-1 ; the
Km and
Vmax for the conversion of L-DOPA to dopaquinone are: 8787 μmol/L and 0.86
μmol·L-1·s-1.

Learn

Through the aforementioned methods and attempts, we successfully achieved the
soluble expression of tyrosinase and
tested its enzymatic activity, laying the foundation for the modification of TRn4-mfp5.

Unfortunately, we found that a portion of L-DOPA is excessively oxidized to
L-dopaquinone, which reduces the adhesion
ability of TRn4-mfp5. Through an extensive literature review, we found that mussel foot protein 6
(Mfp6) is
particularly rich in cysteine, a sulfur-containing amino acid that can reduce the oxidized form of
dopaquinone back to
L-DOPA. Therefore, we decided to express the Mfp6 protein to address the issue of excessive
oxidation.

Loop3

Design

We added the Mfp6 protein into the tyrosinase catalytic system and explored its
reducing capacity. Furthermore, we
learned that the Mfp6 protein is sensitive to high concentrations of IPTG. So, we adjusted the final
IPTG concentration
to 10 μM during the experimental process.

Build

We cloned Mfp6 into the pET-28a(+) vector, constructed the plasmid pET-28a(+)-Mfp6
and selected *E. coli* BL21(DE3)
as the host to express the Mfp6 protein individually.

![](https://static.igem.wiki/teams/5398/mfp6-picture/zhilitupu-pcr-10-2.png)

**Fig. 39 | The results of Plasmid amplification and reconstruction.**

**a.**The plasmid map of pET-28a(+)-Mfp6. **b.**1 % agarose gel
electrophoresis of the PCR amplified Mfp6 and
pET-28a(+) vector. Lane 1: 5000bp DNA Marker；Lanes 2,3: the PCR amplified Mfp6(363 bp); Lanes 4,5:
the PCR amplified
pET-28a(+) vector(5725 bp).

Then, we dialyzed the extracted SUMO-Mfp6 with acetic acid at 16℃ for 12 h.
Finally, we used a Hypur T Ni-NTA 6FF
(His-Tag) Prepacked Chromatographic Column, 1mL for immobilized metal affinity chromatography (IMAC)
purification of the
sample and dialyzed it with acetic acid again.

Test

Through SDS-PAGE and western blotting(WB), we found that TyrVs existed in the
pellet.

![](https://static.igem.wiki/teams/5398/mfp6-picture/yubiaoda-wb-10-2.png)

**Fig. 40 | Expression of pET-28a(+)-Mfp6(28 kDa).**

**a.**SDS-PAGE of
pET-28a(+)-Mfp6(28 kDa). Lane 1: Protein Marker; Lane
2: Mfp6-Whole Cell Lysate (IPTG); Lane 3: Mfp6-Supernatant (IPTG); Lane 4: Mfp6-Pellet-PBS (IPTG);
Lane 5:
Mfp6-Pellet-Extraction Buffer (IPTG); Lane 6: Mfp6-Whole Cell Lysate-1; Lane 7: Mfp6-Supernatant-1;
Lane 8:
Mfp6-Pellet-PBS-1; Lane 9: Mfp6-Pellet-Extraction Buffer-1; Lane 10: Mfp6-Whole Cell Lysate-2; Lane
11:
Mfp6-Supernatant-2; Lane 12: Mfp6-Pellet-PBS-2; Lane 13: Mfp6-Pellet-Extraction Buffer-2.
**b.**Western blotting(WB)
of pET-28a(+)-Mfp6(28 kDa). Lane 1: Mfp6-Whole Cell Lysate (IPTG); Lane 2: Mfp6-Supernatant (IPTG);
Lane 3:
Mfp6-Pellet-PBS (IPTG).

After obtaining active Mfp6 protein through denaturation and renaturation methods,
we adopted two strategies to test the
activity of it.

The first strategy: We tested the activity of Mfp6 at different concentrations. We
found the OD475 in the
experimental group were consistently lower than those in the control group across all tested
concentrations. Besides, an
increasingly obvious colour occurs in experimental group compared to control group along with the
increase of
concentration (Fig. 41). This suggests that Mfp6 indeed facilitated the reduction of some dopaquinone
back to L-DOPA,
with the reduction effect being more significant at higher substrate concentrations.

![](https://static.igem.wiki/teams/5398/mfp6-picture/nongdu-od-96-10-2.png)

**Fig. 41 | Result of activity analysis.**

**a.**Mfp6 activity analysis at different substrate concentrations.
**b.**Mfp6 activity analysis on a 96-Well
Plate.

The second strategy: We tested the activity of Mfp6 at different reaction time. We
knew the OD475 in the
experimental group gradually decreased, while those of the control group remained virtually
unchanged (Fig. 42). This
indicates that Mfp6 progressively reduced dopaquinone back to L-DOPA as the reaction progressed.

![](https://static.igem.wiki/teams/5398/mfp6-picture/shijian-od-10-2.png)

**Fig. 42 | Mfp6 activity analysis at different reaction time.**

Learn

In summary, although the experimental process was very arduous, we ultimately
verified the feasibility of this project
in the experimental environment. In the future, we will continue to optimize this system and strive
to expand it to
other application areas as soon as possible.

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