iGEM 2025 — Team suis-pinghe

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{% set table\_data = [{
"title": "",
"content": '
'
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{% block title %}Notebook{% endblock %}
{% block lead %}

Notebook

{% endblock %}

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

The objective of our project is to integrate mussel
foot proteins, antimicrobial peptides, and zwitterionic peptides to produce surface coatings for medical devices,
aiming to address the issue of medical-related infections in clinical settings. To achieve this goal, we initially
selected and screened mussel foot proteins Mfp3 and Mfp5, which exhibit adhesive properties, as the adhesion units of
our coating proteins. Meanwhile, we evaluated antimicrobial peptides (D51-P11K, Melittin, Tet213), among which
D51-P11K is a novel modified peptide that demonstrates excellent antimicrobial activity. The zwitterionic peptide
Poly(KE)15 Peptide, composed of 15 lysine and glycine residues, exhibits certain anti-fouling properties due to its
ionic characteristics.

Based on this, we followed the DBTL engineering logic
to construct, express, and test the activities of the adhesion proteins, antimicrobial peptides, and zwitterionic
peptides, to verify their significant potential in the application of antibacterial and anti-fouling coatings for
medical devices and further improve the solution of healthcare related infection.

![图形用户界面, 文本, 应用程序, 聊天或短信  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/engineer/1.webp)

Fig. 1 DBTL cycle of the project

'
}, {
"title": "Cycle 1: Mfp3/Mfp5 construction and
functional test",
"content": "",
"children": [{
"title": "Design",
"content": '

We decide to use proteins from mussel byssus which
are strongly adhesive on multiple materials. Mfp3 and Mfp5 are our final options of the project because they mostly
appear at the surface of byssus. Our purpose is to identify which protein has stronger stickiness. We use pET-21a
plasmid that has T7 promotor, lac operator and 6xHis tag. We use engineered E. coli to produce the target proteins.
The gene fragments and primers were designed and optimized using SnapGene software, and the codon optimization and
synthesis were carried out by Tsingke Biotechnology.

![文本  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/engineer/2.webp)

Fig. 2 Construct design of pET21a-Mfp3 and
pET21a-Mfp5 in Snapgene

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

We linearize target protein DNAs and vectors, amplify
the quantity of target Mfp3 and Mfp5 gene fragments and combine them together to construct the plasmids. Then we
transplant the plasmids to BL21 (DE3) competent cell. We successfully constructed and obtained positive clone strains
of BL21-pET21a-Mfp3 and BL21-pET21a-Mfp5 (Fig. 3).

![图片包含 黑暗, 窗户, 桌子, 小  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/engineer/3.webp)

Fig. 3 DNA electrophoresis identification, separation
and purification of Mfp3, Mfp5, and the pET21a plasmid backbone fragments.

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

First, we performed colony PCR on the constructed
engineered strains to verify the correctness of the plasmid construction.

![图片包含 图形用户界面  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/engineer/4.webp)

Fig. 4 Colony PCR verification of Mfp3 and Mfp5.

At the same time, we further confirmed the
correctness of the plasmid construction through Sanger sequencing and expanded the positive clone bacteria culture to
obtain the seed solution of the engineered strain.

For the positive colony, we make a scale culture of
the seed bacterial. In the process we induce protein generation by adding IPTG molecules to the bacteria. After break
the bacteria walls and extract proteins, we purify the proteins using His-Tag. During the purification process, we
used a gradient concentration of imidazole solution to wash away the impurities and elute the target protein. Next we
perform SDS- PAGE electrophoretic analysis to identify the protein bands of Mfp3 and Mfp5.

![图片包含 游戏机, 吉他  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/engineer/5.webp)

Fig. 5 SDS-PAGE of the purified Mfp3 and Mfp5 protein
by His-Tag purification

Then, for the obtained target proteins Mfp3 and Mfp5,
we conducted three tests to verify their functionality. The first aimed to enhance the adhesion capacity of the
proteins; we performed a hydroxylation reaction on the target protein solution using mushroom-derived tyrosinase,
while the effects of the hydroxylation reaction were identified using the NBT staining method and an amino acid
analyzer. The second test involved detecting the adhesion effects of different protein groups on NC membranes using
Coomassie Brilliant Blue staining. The third test further validated the macroscopic adhesion effects of the target
proteins through adhesion tests with laboratory pipette tips and centrifuge tube lids. Detailed experimental results
can be found in the [Results](%27%2B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%2B%27) section.

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

Through the above plasmid construction work, we
successfully obtained engineered Escherichia coli capable of expressing and producing Mfp3 and Mfp5 proteins. During
the construction, it was necessary to design primers to amplify the core region of the pET21a plasmid. Due to the
presence of repeated sequences in the His tag (caccaccaccaccaccac), we found that there would be instances of
incomplete matching at the beginning of primer design. Therefore, we shifted the selection area of the homologous
recombination sequence (tgagatccggctgctaacaa) to avoid the repeated His tag sequences, which increased the specificity
of the primers and ensured successful amplification of the target fragment. Additionally, during protein purification,
based on our results, we found that low concentrations of imidazole wash buffer (10mM, 20mM) also had some effect on
washing away nonspecific proteins. Thus, in the future, we can first use low concentrations for multiple washes and
then increase the imidazole concentration. We can also appropriately extend the incubation time of the His tag
chelator with the target protein solution to further enhance the purification efficiency.

By conducting hydroxylation reaction effect detection
and adhesion analysis, we found that the Mfp5 protein exhibited superior adhesion properties. Therefore, we chose Mfp5
as the adhesion module for subsequent fusion proteins to construct the final coating protein.

'
}]
}, {
"title": "Cycle 2: Antimicrobial peptide
characterization",
"content": "",
"children": [{
"title": "Design",
"content": '

In order to achieve the antibacterial and
antimicrobial functions of the coating protein, our main objective in this round is to screen suitable antimicrobial
peptides through antimicrobial experiments. Therefore, we identified three antimicrobial peptides, D51, Tet213, and
Melittin, based on the literature. To ensure the stability of the antibacterial activity testing, we chose to obtain
these three antimicrobial peptides directly through synthetic methods and conducted antibacterial tests on commonly
used model organisms, Escherichia coli and Bacillus subtilis.

![](https://static.igem.wiki/teams/5738/engineer/6-01.webp)

Fig. 6 Different antimicrobial peptides and their
amino acid sequences.

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

To ensure the activity and quality of the
antimicrobial peptides, we chose to synthesize the target peptides directly from GeneScript and prepare them at the
same concentration. We mix antimicrobial peptides with E. coli and B. subtilis culture solutions and measure the OD600
values of three groups at four different concentrations (0ug/mL, 25ug/mL, 50ug/mL, 100ug/mL, 400ug/mL). We collected
the OD600 absorbance values of the cultures every hour.

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

![](https://static.igem.wiki/teams/5738/engineer/7.webp)

Fig. 7 Antibacterial testing of different
antimicrobial peptides (growth curve method for Escherichia coli and Bacillus subtilis).

We assessed the effects of different concentrations
of antimicrobial peptides on the growth of Escherichia coli and Bacillus subtilis. As shown in Figure 7, the D51
antimicrobial peptide can completely inhibit the growth of E. coli at a concentration of 50 µg/mL, while it exhibits
an even better inhibitory effect on the growth of B. subtilis at the same concentration. At a concentration of 400
µg/mL, it can fully inhibit the growth of B. subtilis.

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

Through antimicrobial testing of different
antimicrobial peptides, we successfully screened the desired antimicrobial peptide D51, which will further advance the
project\'s progress. The next step is to combine the antimicrobial peptide with Mfp5 and the dual-ion peptide to
construct a fusion protein, and we will carry out construction and testing work in line with the project objectives.

'
}]
},{
"title": "Cycle 3: Construction and testing fused
product",
"content": '

![图示  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/engineer/8.webp)

Fig. 8 Overview of the construction of zwi-Mfp5-D51
fusion protein

',
"children": [{
"title": "Overview",
"content": '

Based on the previous Engineering Cycles 1 and 2, we
selected Mfp5 and D51 antimicrobial peptides as components of the fusion protein, while also incorporating the Poly KE
(15) dual-ion peptide for the final product engineering construction. First, the fusion gene and the backbone fragment
of the pET21a plasmid vector were obtained, followed by homologous recombination for linkage, and then transformed
into competent Escherichia coli cells, followed by solid plate screening and cultivation. Subsequently, positive clone
bacteria were expanded, induced for expression, and related functional tests were conducted, including hydroxylation
reaction tests, adhesion tests, antimicrobial tests, and anti-fouling tests.

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

We designed the gene sequence and primers for the
fusion protein zwi-Mfp5-D51 using Snapgene software. We selected pET21a as our plasmid vector. Considering that the T7
tag located at the N-terminus of the protein could potentially affect its function, we removed the T7 tag sequence
during the design and retained the Met amino acid as the starting point of the ORF. The entire composite part includes
a T7 promoter, lac operator, RBS, zwitterionic peptide Poly KE(15), Mfp5, and D51-P11K peptide. Additionally, we
included some flexible linkers as connectors between zwitterionic peptide Poly KE(15), Mfp5, and D51-P11K peptide.

![](https://static.igem.wiki/teams/5738/engineer/9.webp)

Fig. 9 Construct design of pET21a-zwi-Mfp5-D51 fusion
gene in Snapgene

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

According to the designed sequences and primers, we
first obtained the linearized vector pET21a through PCR technology, then connected it with the fragment of the fusion
gene through homologous recombination, and transformed it into competent cells to screen for positive clones.

**![](https://static.igem.wiki/teams/5738/engineer/10.webp)**

Fig. 10 DNA electrophoresis of PCR product of fusion
gene fragment and pET21a linear fragment

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

**![图形用户界面, 网站  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/engineer/11.webp)**

Fig. 11 Colony verification of zwi-Mfp5-D51

After the construction was completed, we first
performed PCR analysis on the plate colonies, identifying positive clone colonies. Then, we carried out liquid culture
and scale-up cultivation to obtain seed bacterial solution for subsequent protein production experiments and
verification experiments.

The engineered bacteria induced for expression were
subjected to ultra-sonication and lysozyme lysis, resulting in a crude protein solution. The protein samples from
different collection tubes during the purification process were analyzed using SDS-PAGE gel electrophoresis.

**![电脑屏幕的照片上有文字  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/engineer/12.webp)**

**Fig. 12 SDS-PAGE analysis of zwi-Mfp5-D51 fusion protein**

For the purified protein solution obtained, we
conducted a series of tests for functional verification, which primarily included hydroxylation reactions and NBT
staining, Coomassie Brilliant Blue staining to verify adhesiveness, pipette tip adhesion testing, antimicrobial
testing of the coated protein, and fouling resistance testing of the coated protein. Specific detailed experimental
data can be referenced in the [Results](%27%2B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%2B%27) section.

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

Through this round of engineering cycle design,
construction, and testing, we successfully completed the verification of the project objectives. By utilizing the good
adhesiveness of Mfp proteins and building upon that adhesion, we further enriched the functional characteristics of
the fusion proteins by introducing antimicrobial peptides and dual ionic peptides, laying a solid foundation for the
development of applications in real-world scenarios. In future work, we can further explore and optimize aspects such
as the stability of the coating proteins, adaptation to more types of medical device materials, and coating
preparation processes.

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

Engineering

{% endblock %}

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

After identifying the genes and vectors required for
our project, we constructed different plasmids—pET21a-Mfp3 and pET21a-Mfp5—by designing specific primers and selecting
homologous recombination strategies for each. We amplified relevant fragments via PCR and ligated them into linearized
plasmid vectors. Positive clones were identified and selected using colony PCR and Sanger sequencing. Following liquid
culture and induction of target protein expression, we assessed hydroxylation reactions using the NBT staining method,
adhesion via Coomassie Brilliant Blue staining, and performed pipette tip adhesion tests to determine the final
adhesive proteins.

For antimicrobial peptide screening, we synthesized
corresponding peptides directly based on their amino acid sequences and carried out subsequent antimicrobial activity
assays. Additionally, to accommodate codon usage preferences in the E. coli expression system, we designed and
optimized the nucleic acid sequences.

For the expression of fusion proteins, we constructed
plasmid DNA using homologous recombination, induced expression and purified the fusion proteins, then conducted
functional assays including adhesion tests (Coomassie Brilliant Blue staining and pipette tip adhesion assays),
antifouling tests, and antimicrobial tests.

'
},{
"title": "Culture of bacterial",
"content": '',
"children": [{
"title": "LB Medium",
"content": '

•LB medium was prepared based on standard
composition:

1% NaCl → 4 g

1% tryptone → 4 g

0.5% yeast extract → 2 g

Total volume: 400 mL

For the LB solid culture medium, add 1.5% agar powder

The prepared culture medium was subjected to
high-pressure sterilization at 121 degrees Celsius for 25 minutes.

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

1. Preparation of Sterile Environment:

• Turn on the UV light of the laminar flow hood 30
minutes in advance for sterilization, followed by ventilation to replace the air.

• Place the pipette, tips, and sterilized LB liquid
medium inside the hood for later use.

2. Convert the medium into a resistant form:

• Add 0.2 mL of Ampenicilin antibiotic into 200 mL of
LB broth. The final concentration is 100ug/ml

• Shake the flask until homogeneous.

3. Inoculation of Bacterial Suspension:

• Label sterile culture tubes with right group name.

• Using a pipette, add 4 ml of LB liquid medium to
each labeled tube.

• Then, using the pipette, accurately transfer 40 µl
of the seed culture into the corresponding labeled tube (hold the pipette vertically to avoid tilting, which could
affect the accuracy of the pipetting), completing the inoculation.

',
},{
"title": "shaking culture",
"content": '

1. Pre-Culture Documentation:

• Inside the laminar flow hood, take photos of each
inoculated test tube to clearly record the initial state of the bacterial suspension (e.g., liquid level, clarity of
the solution, etc.) for subsequent comparison:

2. Incubation:

• Place the inoculated test tubes into the incubator
shaker and set the appropriate culture parameters (37°C, 220 rpm) to start the overnight culture, preparing the
bacterial culture for subsequent experiments.

'
}]
}, {
"title": "Bacterial plasmid extraction",
"content": '

1. Label the centrifuge tube with the correctname, Transfer the cultured bacterial into 2 ml EP，Place the labelled
   tubes into a centrifuge. Set the centrifuge at appropriate parameters (12000 rpm, 1 min)
2. Remove the supernatant using a 1ml volume pipette leaving a bacterial precipitation inside the labelled tubes.
   Repeat the above steps 1 and 2, and continue until all the bacterial solutions have been centrifuged and collected.
3. Add 250 μL of buffer P1 into labelled tubes using a 500μL volume pipette. And fully suspense the bacterial
   precipitate in buffer P1 until uniform turbid solution (bacterial suspension).
4. Using a 500μL volume pipette, add 250 μL of buffer P2 reagent into each labelled tubes. Gently and attentively
   rotate the tubes perpendicularly 7-10 times and 3-4 minutes.
5. Using a 500μL volume pipette, add 350μL of P3 reagent into labelled tubes. Gently and attentively rotate the tubes
   perpendicularly 7-10 times.
6. Place the labelled tubes into a centrifuge, set at appropriate parameter (12000 rpm 10 min). After centrifuge,
   using a 1ml volume pipette, transfer all the supernatant in the labelled tubes into a Spin Column, then placed
   inside a collection tube. Place the collection tube with a spin column in the centrifuge, set at appropriate
   parameters (12000 rpm, 1 min)
7. After centrifugation, discard the waste liquid in the collection tube. Using a 1mL volume pipette, add 600μL of
   PW2 Wash buffer into the spin column. Centrifugation, at 12,000 rpm, for 1 minute. Repeat this operation once.
8. Centrifuge the centrifugal column tube and collection tube at 12000 revolutions per minute for 1 minute to
   completely remove the corresponding residual liquid. After centrifugation, transfer the column tube to a new
   1.5-milliliter centrifuge tube, open the cap and let it stand at room temperature for 2 minutes to fully evaporate
   the residual liquid.
9. Using a 200μL volume pipette, add 65 μL of Elution buffer on the surface of filtering film of the spin column.
   Centrifuge at 12000 rpm for 2 minutes, and obtain the final plasmid DNA solution.
10. Through Nanodrop analysis, the purity and concentration of the plasmid DNA were determined.

'
}, {
"title": "PCR amplification",
"content": '

1. Take out the required reagents: plamid sample, forward primer, reverse primer, mix dNTP, and ddH2O.
   Place all reagents in an ice box and operate at low temperature.
2. Use a pipette to add samples in the following order: total PCR reaction volume is 50 uL

20 μL ddH2O

25 μL mix dNTP

2 μL forward primer

2 μL reverse primer

3 μL sample plasmid

3. After adding each component, gently pipette up and down to mix thoroughly without creating bubbles. Put the PCR
   tube into the PCR instrument, setting up PCR amplifying condition.

Pre-denaturation: 95 ℃, 5 min;

Denaturation: 95 ℃, 15 s;

Annealing: 55 ℃, 15 s;

Extend: 72 ℃, 1 min

Over extension: 72 ℃， 5min

Incubation/ Store: 4 ℃

4. Start the PCR program and allow it to run until completion

'
},{
"title": "Gel electrophoresis",
"content": '

1. Preparing 1% agarose gel:
2. Weigh out the 2g agarose powder, dissolve into 200mL 1\*TAE buffer in a flask.
3. Heat the misxture in a microwave about 3 minutes until the agarose is completely dissolved. Pause mintues to make
   it cool.
4. After cooling, add 20μL of nucleic acid dye (1:10000) into the cooled agarose solution and gentlely mix.
5. Pour the agarose solution into the gel casting tray with the comb, and wait for it to solidify about 20 minutes.
6. Take each of PCR product and mix with 6× DNA loading buffer, gently mix well.
7. Slowly pipette each sample mixture into the small holes of the gel.
8. Add DNA marker into the leftmost hole as a reference.
9. Set the electrophoresis condition (120V, 30min).
10. After electrophoresis, observe the gel by using an ultraviolet imaging system.

',
},{
"title": "Gel DNA extraction and Purification of
confirmed PCR product",
"content": '

1. Use pipette to transfer the PCR product to a 1.5mL centrifuge tube. Add ddH2O to make the volume reach
   100μL.
2. Add 300μL of buffer GDP, then invert the mixture several times to ensure thorough mixing.
3. Transfer the entire mixture to the spin column.
4. Centrifuge at 12000 rpm for 1minutes.
5. Pour out the waste liquid, keep the column and put it back into the collection tube.
6. Add 700μL wash buffer GW to the column.
7. Centrifuge at 12000 rpm for 1minutes.
8. Pour out the effluent and repeat the washing steps once.
9. Centrifuge again for 2 minutes to ensure that all residual ethanol is removed. Let it stand at room temperature
   for 1minute to fully evaporate the residual ethanol.
10. Transfer the purification column to a new 1.5 mL centrifuge tube.
11. Slowly add 20μL of elution buffer to the column center. Let it stand at room temperature for 2minutes.
12. Centrifuge at 12000 rpm for 1minute and collect the eluent (the purified PCR product).

'
},{
"title": "Homologous recombination and
transformation",
"content": '

1. The DNA seamless cloning technology enables the rapid and precise insertion of fragments into any position on any
   vector. By linearizing the vector in any way, the 5\' ends of the primers used for reverse/forward amplification of
   the insertion fragment are introduced into the terminal sequence of the linearized vector, ensuring that the 5\' and
   3\' ends of the PCR product respectively carry sequences (15 - 20 bp) consistent with the two terminal sequences of
   the linearized vector. After mixing the PCR product with the linearized vector in a certain proportion and catalyzed
   by the recombinase, the transformation can be completed within 5 - 15 minutes at 50°C, thus achieving the targeted
   cloning.

* Prepare the following reaction system on the ice:
* 2\* ClonExpress Mix: 5uL
* Linear vector: 0.03 pmol
* Insert fragment: 0.06pmpl
* ddH2O: to 10 uL

2. Reaction codition:

Use a pipette to gently draw and mix (do not shake
for mixing), and then perform a short centrifugation to collect the reaction solution at the bottom of the tube.

3. Single fragment recombination reaction, at 50°C for 5 minutes; then cooled to 4°C or immediately placed on ice for
   cooling.
4. Recombinant product transformation：

Place the chemical competent cells used for cloning
on ice to thaw (e.g: DH5α competent cell or BL21(DE3) Competent cell)

5. Add 10 μl of the recombinant product to 100 μl of the competent cells, gently tap the wall of the tube to mix (do
   not shake for mixing), and let it stand on ice for 30 minutes.
6. After incubation at 42°C for 45 seconds in a water bath, it was immediately placed on ice for cooling for 2 to 3
   minutes.
7. Add 900 μl LB liquid medium (without antibiotics), and shake the bacteria at 37°C for 1 hour (rotation speed 220
   rpm).
8. Preheat the corresponding resistant LB solid culture medium plates in a 37°C incubator.
9. Centrifuge at 5,000 rpm (2,500 × g) for 5 minutes. Discard 900 μl of the supernatant. Resuspend the bacteria in
   the remaining culture medium and gently spread it evenly on a plate containing the appropriate resistance using a
   sterile spreader.
10. In a 37°C incubator, perform inverted culture for 12 - 16 hours.

'
},{
"title": "Colony PCR verification",
"content": '

1. Prepare the PCR system:
   * Prepare the standard PCR reaction system (20 μL), which includes:
   * 2\*PCR Mix buffer: 10 uL
   * Primers (forward and reverse primers): each 1 uL
   * DNA template: 2 uL
   * ddH2O: 6 uL
2. Pick the colony:
3. Gently pick the target colony using a sterile pipette. Stir gently in the PCR tube (containing 10 ul of ddH2O) to
   dissolve it into the reaction mixture, or gently drop the colony onto the bottom of the PCR tube to ensure that a
   small amount of bacterial cells enter the system.
4. Perform the PCR reaction:
5. Conduct the PCR directly (heating at 95°C for 5-10 minutes) to facilitate the lysis of the bacteria and release
   the template DNA. Place it in the PCR machine and run the amplification. The program and cycle conditions are the
   same as those of the conventional PCR, and are set according to the target fragment and primers (such as
   pre-denaturation, denaturation, annealing, extension, number of cycles, etc.).
6. PCR product detection:
7. Take the reaction solution and analyze it in an agarose gel electrophoresis to observe whether there is a target
   band. The detection results are used to identify the positive clones.

'
},{
"title": "Scale-up culture in liquid medium and
expression induction",
"content": '

All operations were performed in a laminar flow
cabinet to ensure aseptic conditions.

Pick the target single positive bacterial colony
using a sterile pipette or toothpick. Inoculate it onto 4 mL of fresh LB liquid medium containing antibiotics (in a
culture tube), and incubate at 37°C, 180 - 220 rpm for 6 - 16 hours (usually overnight) to obtain the seed culture.

For expanding cultivation

Add 100μL Amp+ to each of 100mL LB liquid medium
(final concentration is 50ug/mL). Ensure contactless addition by holding the pipette perpendicular to the bottom of
the conical flask during inoculation.

• For each of the bacterial seed cultures (prepared
culture of the positive colony), inoculate 1mL from both samples and transfer into 100mL LB-Amp. Maintain the upper
portion of the conical flask empty when the bottleneck is exposed.

• It was cultured under shaking at 37°C and at a
speed of 220 rpm until the OD600 reached approximately 0.4 - 0.8 (for the expression-type strain)

\*Notes: Antibiotics will be denatured in high
temperature of steam sterilization, hence adding before preparation of culture instead of preparation of LB.

'
},{
"title": "Absorbance measurement",
"content": '

• Gently swirl the conical flask. Using a pipette,
transfer 300μL seed culture into one well of a 96-well microplate.

• Send the microplate to the microplate reader to
test OD600 every 2 hours to monitor the growth curve of E. coli by measuring OD600 until the culture reaches the
logarithmic phase (OD600 ≈ 0.4–0.8).

**IPTG induction**

1. Weigh 0.61g of isopropyl-β-D-thiogalactoside (IPTG) (with a molar mass Mr of 238.3g/mol).
2. Add 2.5ml of water and the above-weighed 0.61g IPTG (final concentration is 1M) into a centrifuge tube, and mix
   well to dissolve.
3. Solution filtration: Draw the above solution into a syringe, replace the needle with a filter membrane (usually
   with a pore size of 0.22μm), and slowly inject it into a clean small tube to obtain a 1 M IPTG stock solution.
4. Use a pipette with a range of 100-1000 μL to take 0.1 mL of IPTG solution from the stock solution.
5. Add the IPTG solution to the bacterial cultures respectively (1:1000)
6. Place the cultures in a constant temperature shaker at 25°C for overnight (20 hours).

'
},{
"title": "Bacterial collection and protein
extraction",
"content": '

1. After the induction expression is completed, the bacterial culture solution is transferred from the culture bottle
   to a 50-milliliter centrifuge tube that has been pre-cooled in advance.
2. The PBS solution of 500 ml is pre-cooled on ice in advance.
3. The centrifuge is pre-cooled to 4 degrees Celsius in advance to maintain the low-temperature condition during the
   centrifugal collection process.
4. The bacterial cultures were taken from shake flasks and each transferred into two 50 mL collection tubes.
5. The cultures were centrifuged at 6000 rpm for 10 minutes to collect the bacterial pellets.
6. After centrifugation, discard the supernatant. Resuspend the bacterial precipitate in pre-cooled PBS solution and
   centrifuge again at 6000 rpm for 10 minutes. Wash once.

'
},{
"title": "Preparation of Lysis Buffer and Protein
Extraction",
"content": '

1. Lysis buffer preparation: 40 mM Tris-HCl, 80 mM NaCl and 60 mM imidazole, pH = 7.0, with 8 M urea
2. A total of 45 mL binding buffer, 5 mL lysozyme solution (final concentration: 1 mg/mL), and 500 μg protease
   inhibitor cocktail were prepared.
3. The wet weight of the bacterial pellet was measured by weighing the tube before and after adding the pellet.
   Buffer was added at a 1 mg: 15mL ( weight-to-volume ratio). Use the mixer oscillator to thoroughly mix.
4. At a temperature of 4 degrees Celsius, the bacterial suspension was placed in a rotary mixing instrument and
   incubated under rotation for 2 hours.
5. The bacterial suspension was placed on ice to maintain low temperature and prevent protein denaturation.
6. Ultrasonication was performed until the solution became clear and non-viscous, indicating complete lysis.
7. After the liquid becomes clear for 30 minutes, set the power to 40%. Start the ultrasonic disruption for 2
   seconds, then stop for 3 seconds. Continue this cycle until the disruption is complete.
8. 1 mL of whole cell lysate was collected for later analysis (e.g., SDS-PAGE).
9. The remaining lysate was centrifuged at 12000 rpm, 4°C, 20 minutes to remove cell debris. The supernatant,
   containing soluble proteins including the target fusion protein, was collected for purification.

'
},{
"title": "SDS-PAGE:",
"content": "",
"children": [{
"title": "Gel preparation",
"content": '

1. Assemble the gel-making mold properly. First, prepare
the lower layer gel (1.50 mm thick mini gel);

2. Take 4 mL of the lower layer gel solution (2X) and
the lower layer gel buffer solution (2X) of equal volume, and mix them in the gel-making cup thoroughly;

3. Add 80 μl of the modified coagulant to the mixed
solution in step 2, gently stir to mix well, avoiding the formation of bubbles;

4. Add the mixed lower layer gel solution in step 3
to the gel-making mold, making the liquid surface 1.5 cm away from the upper edge of the glass plate. Then cover the
lower layer gel solution with a layer of water or alcohol to keep the gel surface flat and avoid the formation of
bubbles;

5. Let it stand at room temperature (25°C) for 6~10
minutes. When a boundary appears between the lower gel and the covering phase, it indicates that the gel has
solidified;

6. Prepare the upper layer gel (1.50 mm thick mini
gel);

7. Slowly pour out the covering phase, take 1.0 mL of
the upper layer gel solution (2X) and the colored upper layer gel buffer solution (2X) of equal volume, and mix them
in a new gel-making cup thoroughly;

8. Add 20 μl of the modified coagulant to the mixed
solution in step 7, gently stir to mix well, avoiding the formation of bubbles;

9. Add the upper layer gel solution to the upper
layer of the lower gel until the gel solution reaches the top of the glass plate. Insert the comb slowly into the gel
to avoid the formation of bubbles.

10. Let it stand for 10~15 minutes. Wait for the
upper layer gel to solidify. Carefully remove the comb, use a pipette or syringe to aspirate the electrophoresis
buffer to rinse the sample wells clean, and then proceed with the SDS-PAGE electrophoresis operation.

'
},{
"title": "Protein denaturation",
"content": '

1. Use 5\* protein loading buffer, take 40 microliters of protein sample, add 10 microliters of protein loading
   buffer, and then mix thoroughly.
2. Using Dry Bath Incubator: Place the protein sample tubes then heat at 100℃ for 5 minutes.
3. After heating, perform centrifugation at a speed of 12,000 revolutions per minute.

'
},{
"title": "Protein Electrophoresis",
"content": '

1. Add 20 microliters to the protein electrophoresis. When the solution is in the spacer gel, the voltage is 100V;
   when it is in the separation gel, the voltage is 180V. The total time is 40 minutes.
2. After the electrophoresis is completed. Add the staining solution and the decolorization solution

'
}]
},{
"title": "Protein purification (nickel column affinity chromatography) for large volume purification and SDS-PAGE verification",
"content": '

* The purification reagent we use is HisSep Ni-NTA Agarose Resin (Yeasen). This resin is based on cross-linked 6%
  agarose gel as the matrix. It is chemically coupled with tetra-coordinated nickel tri-acetate (NTA) as the ligand.
  After chelating with nickel ions (Ni2+), a very stable octahedral structure is formed, with the nickel ions at the
  center of the octahedron. Such a structure can protect the nickel ions from being attacked by small molecules,
  making it more stable and capable of withstanding certain concentrations of reducing agents, denaturants, or
  coupling agents and other harsh conditions.
* Based on the amount of the purified sample, add 1 mL of HisSep Ni-NTA Agarose Resin to the centrifuge tube.
  Centrifuge at 1000 rpm for 1 minute, then discard the supernatant.
* Add 5 times the volume of Lysis Buffer to the centrifuge tube to wash the filler. Centrifuge at 1000 rpm for 1
  minute, then discard the supernatant; repeat this process twice or more.
* Add the sample, seal the centrifuge tube, and incubate at 4℃ for 3 hours or at 37℃ for 1 hour.
* After incubation, perform a 1000 rpm centrifugation for 1 minute, then aspirate the supernatant. The supernatant
  is retained and used as the flow-through for electrophoretic analysis.
* Use 5 times the volume of Wash Buffer to wash the filler, centrifuge at 1000 rpm for 1 minute to remove the
  supernatant (be careful not to suck up the filler), repeat this process 3-5 times. During the process, replace the
  new centrifuge tube to collect different washing solutions. At the same time, adjust the concentration of imidazole
  for impurity removal (10 mM, 20 mM, 50 mM, 100 mM, 150 mM, 500 mM).
* Add 3 times the column volume of Elution Buffer (500 mM imidazole) for elution, incubate at room temperature for
  10-15 minutes, centrifuge at 1000 rpm for 1 minute each time (1 ml), repeat 3 times.

'
},{
"title": "Hydroxylation reaction",
"content": '

1. Create a buffer that blending 0.34g sodium acetate with 0.38g sodium borate and 0.2g ascorbic acid. Maintain the
   pH level around 5.5.
2. Combine the elution liquid of target proteins with 1ml buffer, add 50 U tyrosinase from mushroom, leave the
   solution in 37 degrees Celsius for 30min, then add 5% acetic acid to finish the reaction. Repeat the process on all
   proteins.

Note: leave one sample of each protein as contract in
testing

'
},{
"title": "NBT staining",
"content": '

1. Prepare the NBT staining solution using deionized water, with a concentration of 10 mg/ml. Store it as a stock
   solution at -20 degrees Celsius in a dark place.
2. Dip 50 microliters of the adhesion protein sample onto a nitrocellulose membrane and let it dry overnight. Wash
   the membrane with PBS buffer to remove impurities.
3. The membrane of the sample protein solution was incubated in 20 milliliters of freshly prepared 1 milligram per
   milliliter NBT solution at room temperature in the dark (covered with aluminum foil) for 45 minutes. Then, the
   membrane was washed twice with 10 milliliters of 0.16M sodium borate solution and soaked in another 20 milliliters
   of sodium borate solution overnight. The colored film is recorded as an image through photography.

'
},{
"title": "Adhesiveness testing：Coomassie brilliant blue
staining",
"content": '

1. Add 10μl protein on PVDF layer.
2. Wait until the liquid is dried, then add another round of 10μl protein.
3. After the new round is dried, add 30μl protein and dry the sample for an entire night.
4. Drop 50μl onto each of the five different substrate surfaces: PE, PV, PS, stainless steel, and silicone
5. Repeat the process on all proteins.
6. The next day, place differerent materials in different tubes containing purified water, and shake in the shaking
   incubator for 2 hours to wash off all of the unbound proteins.
7. Then dry at room temperature until no more liquids are left on the surface.
8. After that is done, stain the material with Coomassie Brilliant Blue for 30 minutes .
9. Upon completion, use ImageJ software to analyze the intensity of the stained spots.

'
},{
"title": "Adhesiveness testing：Pipette tip adhesion
test",
"content": '

1. Another way we used to test the adhesive of the proteins is by using PTFE (polytetrafluoroethylene) pipette tips.
   Where PTFE (polytetrafluoroethylene) pipette tips of the same specifications are dipped into 1 mg/mL solutions of
   modified and unmodified recombinant proteins, BSA, and Cell-Tak™ separately.
2. Subsequently, attach each tip to the polypropylene centrifuge tube caps. Remember to be extra careful during this
   part, as you don’t want to knock over any pipette tips. Let the tips stand at room temperature for 1 hour to allow
   them to stick to the caps.
3. Finally, observe the macroscopic adhesion of the sample proteins by turning the centrifuge tube caps upside down
   and seeing if the pipette tips fall off.

'
},{
"title": "Testing the antibacterial properties of
different antimicrobial peptides",
"content": '

1. We selected the model organisms Escherichia coli
and Bacillus subtilis as the objects for the antibacterial peptide activity test.

2. The growing and active Escherichia coli and
Bacillus subtilis were re-inoculated into fresh culture medium. Corresponding amounts of antibacterial peptide stock
solution were added to the freshly inoculated bacterial solution according to different antibacterial peptide
concentrations (0 ug/mL, 25 ug/mL, 50 ug/mL, 100 ug/mL, 400 ug/mL).

3. The OD600 value was measured every 1 hour and the
detection was conducted for 24 hours.

4. Based on the measured OD600 absorbance, the
bacterial growth curves of different experimental groups were plotted.

'
},{
"title": "Testing the anti-microbial properties of the fusion protein",
"content": '

1. To investigate the antibacterial effect of the fusion protein coating, the bacterial solutions of Escherichia coli
   and Bacillus subtilis were diluted to 1×106 CFU/mL.
2. Glass slides and stainless steel plates coated with the protein were added to a 12-well plate, with each well
   containing 1 mL of the bacterial solution.
3. The plates were incubated at 37°C with a speed of 220 rpm for 20 hours.

'
},{
"title": 'Test of the anti-fouling property of the fusion protein',
"content": '

1. Firstly, cultivate E. coli and B. subtilis in a 48-well plate until their OD600 reaches 0.6.
2. Then, place the coated glass sheet (a 10mm glass sheet covering 18 micrograms of recombinant fusion protein) into
   the plate and incubate it at a speed of 60 rpm for 120 minutes.
3. After that, remove the glass sheet, wash the coating 3 times with 1 mL of sterile 0.9% NaCl solution.
4. Next, use an ultrasonic cleaning machine (for 5 minutes) to wash off the bacteria on the coating in 1 mL of
   sterile 0.9% sodium chloride solution.
5. Finally, spread 100 microliters of the bacterial suspension on the LB plate, incubate it at 37°C for 24 hours, and
   observe the growth of the colonies.

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

Experiments

{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "1. Construction of module for the Mfp3 and Mfp5",
"content": '',
"children":[{
"title": "1.1 The construction of plasmid pET21a-Mfp3,
pET21a-Mfp5",
"content":'

We constructed Mfp3 and Mfp5 using homologous
recombinant. To generate sufficient quantity of DNA fragments for downstream cloning, we amplified the target DNA
fragments (Mfp3, Mfp5) and linearized empty vector (pET21a) through PCR, and verified successful amplification by
agarose gel electrophoresis on a 1% agarose gel. The result of gel electrophoresis (Fig. 1) shows a distinct band of
the empty vector at expected size of 5300bp~. Both bands of Mfp3 and Mfp5 samples also appear distinctly at expected
sizes (Mfp3: 141bp, Mfp5: 231bp). The DL5000 DNA marker was included for size reference. The result confirmed our
successful PCR amplification of linearized empty vector, Mfp3 and Mfp5 DNA fragments without DNA degradation.

![](https://static.igem.wiki/teams/5738/results/1.webp)

Fig. 1. The gel electrophoresis validation of
linearized empty vector, Mfp3 and Mfp5 fragments.

'
},{
"title": "1.2 Homologoud recombination and colony PCR
verification",
"content":'

After Gel extraction of the target fragments and
purification, we ligated the PCR product of Mfp3, Mfp5 and the linearized vevtor. We transformed the plasmids Mfp3 and
Mfp5 into DH5α, and Fig. 2A showes the growth of single clones, compared to the control group which had no Amp
resistance. We selected clones 1-4 of Mfp3 and Mfp5 respectively to perform PCR verification. Fig.2B showes bands that
matched the expected size, indicating successful construction and transformation.

![](https://static.igem.wiki/teams/5738/results/2.webp)

Fig. 2. The monoclonal antibody validation of Mfp3
and Mfp5.

Note:

A. The plate images of respectively Mfp3 and Mfp5
transformed into E.Coil DH5α cells, and the control groups.

B. The images showing the verification of single
colony PCR of Mfp3 and Mfp5.

'
},{
"title":"1.3 Sanger sequencing validation",
"content": '

![模糊的照片上写着字  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/results/3.webp)

Fig. 3 Sanger sequencing of pET21a-Mfp3, pET21a-Mfp5

We confirmed the correctness of the final constructed plasmid through Sanger sequencing. The results are shown in the figure. The sequencing signal was stable and matched the theoretical designed sequence.

'
},{
"title": "1.4 induction and expression of Mfp3 and Mfp5",
"content": '

In order to assess the effectiveness of bacterial
disruption and solubility of our target protein in supernatants (SUP), we performed SDS-PAGE using whole-cell lysates
(WL) derived from E. coli harboring recombinant plasmids (Mfp3, Mfp5) and supernatant (SUP) obtained after
centrifugation as samples. For Mfp3 and Mfp5 in Fig. 4, the existence of the bands of WL and Sup with similar
intensity indicates that both samples expressed target protein, hence indicates successful cell lysis and confirms
that the target protein is soluble in supernatant.

![模糊的照片上写着字  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/results/4.webp)

Fig. 4. The SDS-PAGE protein gel of WL and SUP of
Mfp3 and Mfp5.

WL: whole cell lysis; SUP: supernatant

'
},{
"title": "1.5 His-tag purification of Mfp3 and Mfp5",
"content":'

To evaluate the purity of target protein Mfp3 and Mfp5(Fig. 5) after purifying the protein-containing Sup by nickel column affinity chromatography, we use
flow-through, wash fractions of imidazole in different concentrations (10mM, 20mM, 50mM, 100mM, 150mM) and elution
fraction of imidazole concentration 500mM as samples to run SDS-PAGE.

As concentration of imidazole increases, the single
band of target protein existed., hence showing that purification of target protein increases. Elution fraction shows
higher intensity, hence higher concentration as well as higher purification in contrast to adjacent bands.

![](https://static.igem.wiki/teams/5738/results/5.webp)

Fig. 5. The SDS-PAGE protein gel of different
fractions of Mfp3 and Mfp5

'
},{
"title": "1.6 BCA characterization of purified protein",
"content": '

We use Bicinchoninic Acid (BCA) Assay to determine
the concentration of Mfp3 and Mfp5 in each wash fractions and elution fraction with bovine serum albumin (BSA)
standards (0-2000 μg/mL) labeled as A-I. Standards A-D (conc. = 2000-750 μg/mL) were excluded as outliers in
the construction of standard curve due to the inconsistent OD value of standard D, where concentrations of all samples
to be tested were included in the linear range of 0-500 μg/mL (OD562 = 0.1243-0.7809, R² = 0.9998). This improved the
linearity of the optimal standard curve.

'
},{
"title": "1.7 Hydroxylation of mfp3 and mfp5",
"content": '

We extracted mfp3 and mfp5 proteins and performed
hydroxylation treatment on both. To evaluate their DOPA characterization and adhesive properties, we conducted both
macroscopic tests and staining-based visualization assays, aiming to identify which protein exhibits the best adhesion
after modification.

Hydroxylation reaction and NBT Staining Assays:

Mfps is rich in polyphenyl groups and positively
charged amino acids. The polyphenyl groups not only have powerful anti-inflammatory and antioxidant functions but also
endow Mfps with strong underwater adhesion, enabling it to effectively adhere to the surface of medical device
implants in body fluids.

To enhance the adhesive properties of the protein, we
first performed the hydroxylation reaction. After that, we performed the NBT staining and analyzed the DOPA conversion
rates to verify the success of the hydroxylation reaction.

![手机屏幕的截图  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/results/6.webp)

Fig 6. NBT staining for the identification of protein
hydroxylation.

From left to right: negative control (BSA),
Post-Hydroxylation (dMfp3,dMfp5), Pre-Hydroxylation (Mfp3, Mfp5), positive control (Cell-Tak)

From the staining result, we observe that the
post-hydroxylation sample showed significantly stronger staining compared to the pre-hydroxylation sample. This
indicates that the hydroxylation process was successful.

|  |  |
| --- | --- |
| Recombinant protein | DOPA conversion rate (%) |
| Mfp3 | 22.2 |
| Mfp5 | 26.7 |

Table 1. Amino Acid Analyzer: DOPA conversion rates
of different recombinant proteins

The DOPA conversion rates measured by the amino acid
analyzer confirmed the success of the hydroxylation reaction. Moreover, the conversion rate of mfp3 was lower than
that of mfp5 (22.2% ＜26.7%), indicating that mfp5 underwent more efficient hydroxylation.

'
},{
"title": "1.8 Coomassie Brilliant Blue Staining",
"content": '

To further evaluate and compare the adhesive
performance between mfp3 and mfp5, we performed the Coomassie Brilliant Blue Staining:

![图片包含 图形用户界面  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/results/fig-7.avif)

Fig .7 Coomassie brilliant blue staining of different groups of protein and Image J analysis.

To evaluate the relative adhesion capacity of Mfp3
and Mfp5 after hydroxylation, we conducted a grayscale intensity analysis of Coomassie blue staining using ImageJ. The integrated gray values were
extracted for each protein as an measure of protein abundance, where a lower gray value corresponds to a light stain,
indicating weak staining and thus a lower protein adhension.

According to ImageJ analysis, the gray value of
dMfp3 was a little lower than that of dMfp5. At the same time, we can see thay hydroxylated group are all significantly higher than that of non-hydroxylation group (Mfp3,Mfp5). The higher gray value of dMfp5 indicates stronger staining and thus
higher protein retention, suggesting superior adhesive capacity.

Therefore, combined with the result of amino acid
analyzer, Mfp5 was incorporated into the final fused protein due to its strong adhesive properties.

'
},{
"title": "1.9 Macroscopic Assay (Pipette Tip Inversion
Test)",
"content": '

We used a pipette tip adhesion test to further assess
adhesive strength macroscopically. The lids of 50-milliliter centrifuge tubes (PP, Polypropylene) and the tips of
10-microliter pipettes (PP, Polypropylene) were respectively used. On the surface of four test tube caps, we applied
equal amounts of four sample groups:

1. Negative Control (BSA),
2. Untreated Mfp5 Protein (non-hydroxylated),
3. Hydroxylated Protein Mfp5
4. Positive Control (commercial protein production for cell adhension culture, Corning® Cell-Tak™ Cell and Tissue
   Adhesive (354240) ).

After applying pipette tips onto each sample, the
caps were incubated at 37°C until completely dry. Then, we inverted the test tubes. As expected, the pipette tips in
the negative control and non-hydroxylated groups fell off, whereas the hydroxylated samples and positive control
remained adhered, indicating that our modified Mfp3 proteins possess adhesive capabilities after hydroxylation.

Mfp5：

![图片包含 图形用户界面  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/results/8.webp)

Fig .8 Adhesion test of Mfp5 using pipette tips

A: Befor Hydroxylated and positive control remained;
B: after rotation

C: Hydroxylated Mfp5 remained sticky for one day

By comparing the results of different group, we
observe that mfp5’s adhesion is sufficient validated. The hydroxylated sample stayed adhered, whereas the untreated
mfp5 group detached, indicating that hydroxylation enhances the adhesive properties of mfp5, and its overall adhesion
strength is significantly obvious. So for the future construction and final functional test, we chose the Mfp5 as our
adhensive module to combined with antimicrobial peptide and zwitterionic peptide.

'
}]
},{
"title": "2. Antimicrobial peptide assay for verification of different peptide",
"content": '',
"children": [{
"title": "2.1 Antimicrobial Peptide Screening – D51,
Tet213, and Melittin",
"content": '

Through literature review, we found that in the
research of 2006 (Loose et al., 2006), D51 is an antimicrobial peptide containing 20 amino acids, which was designed
based on natural antimicrobial peptides through a language model. D51 only contains hydrophobic and cationic amino
acids (sequence FLFRVASKVFPALIGKFKKK).

Tet213, as a broad-spectrum antibacterial peptide
with 10 amino acids, plays a significant role in promoting the repair of infected wounds and can be used in
combination with collagen to exert its function (Sequence KRWWKWWRRC).

Melittin is the main component and active substance
of bee venom. As a cationic linear peptide, it contains 26 amino acids: GIGAVLKVLTTGLPALISWIKRKRQQ. Its primary
sequence is amphiphilic. Studies have shown that melittin has significant antibacterial activity against bacteria,
fungi and mold spores.

To evaluate the antibacterial efficacy of different
antimicrobial peptides (AMPs), we chose three distinct peptides for comparison: D51, Tet213 and Melittin. We obtained
these three antibacterial agents respectively through synthesis, and tested their bactericidal activity effects on two
bacterial species: Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis).

For each peptide, five experimental groups were
prepared: one control group (no peptide added), and four treatment groups with peptide concentrations ranging from 25
μg/ml to 400 μg/ml.

Each bacterial strain was cultured to an optimal
density (OD600), and measurements were taken hourly over 24 hours to monitor bacterial growth. These measurements were
then compared to the baseline values recorded at time=0.

![](https://static.igem.wiki/teams/5738/results/9.webp)

Fig .9 Antimicrobial test of synthetic peptide (D51,
Tet213, Melittin)

From the graph above, we observe that D51 exhibits
the strongest antibacterial efficacy against both E. coli and B. subtilis compared to Tet213 and Melittin.

At the same concentrations, D51 consistently shows
the lowest growth curve slope, indicating the most effective inhibition of bacterial growth. This effect is
particularly displayed on E.coli, where at 50, 100 and 400 μg/ml, the bacterial growth is almost completely
suppressed.

Therefore, we choose D51 as our antimicrobial peptide
for the construction and functional verification of composite fusion protein.

'
}]
}, {
"title": "3. Construction and test of fusion protein: zwi-Mfp5-D51",
"content": "",
"children": [{
"title": "3.1 construction of pET21a-zwi-mfp5-D51 and
verification by colony PCR and Sanger sequencing",
"content": '

We constructed zwi-mfp5-D51 fused plasmid using
homologous recombinant. To generate sufficient quantity of DNA fragments for downstream cloning, we amplified the
target DNA fragments (zwi-mfp5-D51 fused) through PCR, and verified successful amplification by agarose gel
electrophoresis. The result of gel electrophoresis shows a distinct band of the fused fragment which matches the
expected size of 420bp. The result confirmed our successful PCR amplification of the fused DNA fragments without DNA
degradation.

![黑暗中的灯光  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/results/10.webp)

Fig. 10. The gel electrophoresis validation of the
zwi-mfp5-D51 fused fragment.

We transformed the fused plasmids into DH5α, and
Fig. 11A showed the growth of single clony, compared to the control group which had no Amp resistance. We selected
clony 1-4 of colonies harboring fused plasmids respectively to perform colony PCR verification. Fig. 11B showed
bands that matched the expected size of 420bp, indicating successful liagtion and transformation. Then we choose the
positive colony for Sanger sequencing to further verify the construction.

![图形用户界面, 网站  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/results/11.webp)

Fig. 11 Colony PCR verification of the pET21a-
zwi-mfp5-D51

![图形用户界面, 网站  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/results/12.webp)

Fig. 12 Sanger sequencing of pET28a-zwi-Mfp5-D51

We confirmed the correctness of the final constructed plasmid of pET28a-zwi-Mfp5-D51 through Sanger sequencing. The results are shown in the Fig. 12. The sequencing signal was stable and matched the theoretical designed sequence.

'
},{
"title": "3.2 Induction and expression of zwi-mfp5-D51
fused protein",
"content": '

In order to assess the effectiveness of bacterial
disruption and solubility of our target protein in Sup, we performed SDS-PAGE using WL derived from E. coli harboring
recombinant zwi-mfp5-D51 fused plasmids and Sup obtained after centrifugation as samples. Similarity between WL and
Sup indicates our successful ultrasonic disruption, and our target protein are soluble in supernatant.

![图片包含 游戏机, 食物  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/results/13.webp)

Fig. 13. The SDS-PAGE protein gel of WL and Sup of
Mfp3 and Mfp5.

To evaluate the purity of target protein zwi-mfp5-D51
after purifying the protein-containing Sup by nickel column affinity chromatography, we use flow-through, wash
fractions of imidazole in different concentrations (20mM, 50mM, 100mM, 150mM) and elution fraction of imidazole
concentration 500mM as samples to run SDS-PAGE.

As concentration of imidazole increases, the band of
target protein stood out, hence showing that purification of target protein increases. Elution fraction shows higher
intensity, hence higher concentration as well as higher purification in contrast to adjacent bands.

![一些文字和图案  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/results/14.webp)

Fig. 14. The SDS-PAGE protein gel of different
fractions of fused protein.

'
},{
"title": "3.3 Hydroxylation and test of
zwi-Mfp5-D51：NBT for the verification of hydroxylation effect",
"content": '

![](https://static.igem.wiki/teams/5738/results/15.webp)

Fig. 15 NBT staining of fusion protein zwi-Mfp5-D51

To enhance the adhesion of the protein, mushroom
tyrosinase was used for the in vitro modification of the target protein, converting the tyrosine residues into
dopamine (Dopa) under the conditions of 37°C for 30 minutes. In an alkaline environment, when potassium glycine was
used as a reducing agent, nitroblue tetrazolium (NBT) could react with DOPA, producing a blue-purple color. NBT
staining indicates that the in vitro modification of Dopa in the fusion protein was successful.

'
},{
"title": "3.4 Adhesiveness 1: Detection of the
adhesiveness of zwl-Mfp5-D51 composite protein using Coomassie Brilliant Blue staining",
"content": '

![屏幕上有字  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/results/16.webp)

Fig. 16 Coomassie brilliant blue staing of
zwi-Mfp5-D51

Take 5 microliters each of 0.3 mg/mL BSA, commercial
mussel foot protein Cell-TakTM, and hydroxylated/non-hydroxylated recombinant protein samples, and place them on the
surfaces of clean polystyrene plates and glass slides. Dry at room temperature for 12 hours, shake in pure water for
0.5 hours, dry at room temperature, and stain with Coomassie Brilliant Blue G-250 for 10 minutes. Compare the
adsorption capacity of the protein samples on different substrate surfaces based on the staining intensity.

The hydroxylated fusion protein shows good adsorption
capacity on the surfaces of polystyrene and glass slides.

'
},{
"title": "3.5 Adhesiveness 2: Adhesion Test of
zwi-Mfp5-D51 (Pipette Tip)",
"content": '

![穿蓝色上衣的人躺在地上  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/results/17.webp)

Fig. 17 Adhession test of zwi-Mfp5-D51 by pipette
tips. a) BSA, b) Cell-Tak, c) Fusion protein (Hydroxylation), d) Fusion protein (Non-hydroxylation)

For the fused protein, we observed that after
inverting the sample, only the hydroxylated group remained attached, while all other groups detached from the surface
of the lid, indicating that the hydroxylated fused protein exhibits strong adhesive properties.

'
},{
"title": "3.6 Anti-fouling test: glass material and
stainless steel material",
"content": '

![](https://static.igem.wiki/teams/5738/results/18.webp)

Fig. 18 Anti-fouling test of the coated glass and
stainless steel by zwi-Mfp5-D51

After preparing coatings by combining the fusion
protein zwi-Mfp5-D51 with glass and stainless steel, we established co-culture systems with Escherichia coli and
Bacillus subtilis for both coating materials. After incubation, we plated the bacteria that adhered to the washed
coatings. As shown in the above image, we observed that the number of colonies with coatings on both materials was
significantly lower than that of the uncoated material group. The above results indicate that the dual ion peptide in
the fusion protein meets the project\'s design goals and can achieve a certain level of anti-fouling effect.

'
},{
"title": "3.7 Antibacterial testing; Bacillus subtilis
and Escherichia coli, glass and stainless steel",
"content": '

![图片包含 游戏机, 眼镜, 食物, 红酒  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/results/19.webp)

![图形用户界面, 图表  AI 生成的内容可能不正确。](https://static.igem.wiki/teams/5738/results/20.webp)

Fig. 19 Antibacterial testing of fusion proteins by
bacterial growth curve method

In a co-culture system, glass and stainless steel
material samples coated with fusion proteins were continuously incubated on a porous plate with Escherichia coli and
Bacillus subtilis. Monitoring the bacterial growth through changes in turbidity and dynamic measurements of OD600
absorbance over time revealed that the fusion protein coating on the glass surface exhibited a significantly stronger
antibacterial effect compared to that on stainless steel. Notably, at a coating concentration of 3 mg/mL, the growth
of both bacterial strains was markedly inhibited. These results indicate that glass materials demonstrate superior
compatibility for fusion protein coatings in terms of adhesion and antibacterial efficacy.

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

Results

{% endblock %}