iGEM 2024 — Team njtech-china

{% extends "layout.html" %}
{% 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 %}
{% block page\_content %}

![Project Image](https://static.igem.wiki/teams/5322/top-head-new3.png)

Notebook

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one
iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{%
endblock %}
{% block page\_content %}

![Project Image](https://static.igem.wiki/teams/5322/top-head-new3.png)

Engineering Success

* [SOD ▼](#SOD)
  + [Cycle 1 ▼](#Cycle-1)
    - [Design](#Cycle1-D)
    - [Build](#Cycle1-B)
    - [Learn](#Cycle1-L)
  + [Cycle 2 ▼](#Cycle-2)
    - [Design](#Cycle2-D)
    - [Build](#Cycle2-B)
    - [Test](#Cycle2-T)
    - [Learn](#Cycle2-L)
  + [Cycle 3 ▼](#Cycle-3)
    - [Design](#Cycle3-D)
    - [Build](#Cycle3-B)
    - [Test](#Cycle3-T)
    - [Learn](#Cycle3-L)
  + [Cycle 4 ▼](#Cycle-4)
    - [Design](#Cycle4-D)
    - [Build](#Cycle4-B)
    - [Test](#Cycle4-T)
    - [Learn](#Cycle4-L)
  + [Cycle 5 ▼](#Cycle-5)
    - [Design](#Cycle5-D)
    - [Build](#Cycle5-B)
    - [Test](#Cycle5-T)
    - [Learn](#Cycle5-L)
* [Mfp ▼](#Mfp)
  + [Cycle 6 ▼](#Cycle-6)
    - [Design](#Cycle6-D)
    - [Build](#Cycle6-B)
    - [Test](#Cycle6-T)
    - [Learn](#Cycle6-L)
  + [Cycle 7 ▼](#Cycle-7)
    - [Design](#Cycle7-D)
    - [Build](#Cycle7-B)
    - [Test](#Cycle7-T)
    - [Learn](#Cycle7-L)
  + [Cycle 8 ▼](#Cycle-8)
    - [Design](#Cycle8-D)
    - [Build](#Cycle8-B)
    - [Test](#Cycle8-T)
    - [Learn](#Cycle8-L)
  + [Cycle 9 ▼](#Cycle-9)
    - [Design](#Cycle9-D)
    - [Build](#Cycle9-B)
    - [Test](#Cycle9-T)
    - [Learn](#Cycle9-L)* [PD-L1
    ▼](#PD-L1)
    + [Cycle 10 ▼](#Cycle-10)
      - [Design](#Cycle10-D)
      - [Build](#Cycle10-B)
      - [Learn](#Cycle10-L)
    + [Cycle 11 ▼](#Cycle-11)
      - [Design](#Cycle11-D)
      - [Build](#Cycle11-B)
      - [Test](#Cycle11-T)
      - [Learn](#Cycle11-L)
    + [Cycle 12 ▼](#Cycle-12)
      - [Design](#Cycle12-D)
      - [Build](#Cycle12-B)
      - [Test](#Cycle12-T)
      - [Learn](#Cycle12-L)
  * [Reference](#Reference)

# Antioxidant Module-Superoxide Dismutase SOD

# Cycle 1

### Design

Reactive oxygen species (ROS) and nitric oxide (NO) can cause tissue damage. Among the known antioxidant
enzymes, superoxide dismutase (SOD) is considered to play a central role due to its ability to scavenge
superoxide anions. Copper and zinc-binding superoxide dismutase-1 (SOD-1) is one of three SODs responsible for
eliminating superoxide free radicals in the body. It catalyzes the conversion of superoxide radicals
(O2−)
into hydrogen peroxide, which can then be further reduced to water and oxygen. We constructed an antioxidant
module using SOD-1 to aid in the clearance of ROS and mitigate oxidative damage. Our team obtained the SOD-1
sequence from [BBa\_K2215003](https://parts.igem.org/Part%3ABBa_K2215003), optimized its codons, and
had the relevant gene fragment synthesized by a company,
designing the plasmid pET29a-J23119-RBS-(SOD-1)-T7, as shown in the accompanying figure.

![](https://static.igem.wiki/teams/5322/t-imgs/1-pet29a-j23119-rbs-sod-1-t7.png)

##### Figure 1-pET29a-J23119-RBS-(SOD-1)-T7

### Build

We performed PCR amplification of the target fragment and the pET29a vector, followed by gel electrophoresis
(30 minutes at 120 V) and purification. After this, we conducted homologous recombination and transformed the
plasmid into  *Escherichia coli* DH5α, constructing the plasmid pET29a-J23119-RBS-(SOD-1)-T7. The culture
was
incubated inverted at 37°C for 16 hours, after which colony PCR was conducted on single colonies.

Plasmids with correctly positioned bands were sent to GENEWIZ for sequencing. However, the sequencing results
indicated that there were random base deletions in the promoter region. We repeated the experiment three
times, but similar issues persisted.

![](https://static.igem.wiki/teams/5322/t-imgs/2-pet-29-a-j23119-sod-1-his-t7-map1.png)

### Learn

Due to the recurrence of similar issues in the three experiments, our team began to reflect on whether the
introduction of the foreign plasmid was toxic to *Escherichia coli*, potentially triggering its
protective
mechanisms. Literature reviews indicated that excessive exogenous SOD can lead to the production of excessive
H2O2 in *E. coli*, reaching toxic levels that cause DNA damage and resulting in
numerous random mutations in the
sequence [1,2]. Considering that BL21 (DE3) generally exhibits higher protein expression than other strains
like DH5α and EcN, which may lead to random mutations in DNA sequences, we decided to use the DH5α strain for
all subsequent SOD experiments to avoid this possibility.

# Cycle 2

### Design

Considering the potential damage that excessive H2O2 can cause to DNA, we chose to use
the lac promoter with
lower strength for the expression of SOD in *E. coli*, induced by IPTG. Consequently, our team redesigned
the
plasmid pET-29(a)-pT7-lac operator-(SOD-1+His)-T7（([BBa\_K5322011](https://parts.igem.org/Part%3ABBa_K5322011))

![](https://static.igem.wiki/teams/5322/t-imgs/3-pet-29-a-pt7-lac-operator-sod-1-his-t7.png)

##### Figure 3 pET-29(a)-pT7-lac operator-(SOD-1+His)-T7 plasmid map

### Build

1. Our team designed primers that added BamHⅠ and EcoRⅠ restriction sites to both ends of the SOD fragment.
We performed digestion using BamHⅠ and EcoRⅠ restriction endonucleases and repeated the Build 1 steps. We then performed colony PCR on the isolated colonies and sent the plasmids with the correct bands to GENEWIZ for sequencing. The sequencing results confirmed the successful construction of the strain.

![]( https://static.igem.wiki/teams/5322/t-imgs/4-pet-29-a-lac-operator-sod-1-his-t72-map.png)

##### Figure 4 - The sequencing results of pET-29(a)-pT7-lac operator-(SOD-1+His)-T7 indicate that the recombinant plasmid has been successfully assembled.

### Test

We took 2 mL of overnight induced bacterial culture and used Beyotime™ bacterial protein extraction reagent
to extract the protein. The SOD activity was measured using the Beyotime™ SOD activity assay kit, with
absorbance measured at A450. The initial data indicated that the inhibition rate of induced SOD was
approximately 46%, while the inhibition rate for DH5α was about 38%, confirming successful expression of the
SOD protein.

The WST-8 can react with superoxide anions generated by xanthine oxidase to produce a water-soluble formazan
dye, which SOD can inhibit. The enzyme activity of SOD can be calculated through colorimetric analysis of the
WST-8 product.

The calculation of the inhibition percentage is as follows:
Inhibition percentage = [(A blank control 1 - A blank control 2) - (A sample - A blank control 3)] / (A blank
control 1 - A blank control 2) × 100%

The definition of one unit of SOD enzyme activity is the activity defined in the reaction system when the
inhibition percentage reaches 50%.

The formula for calculating SOD enzyme activity is:
SOD enzyme activity units in the sample = SOD enzyme activity units in the detection system = inhibition
percentage / (1 - inhibition percentage) units.

![](https://static.igem.wiki/teams/5322/t-imgs/5-data.png)

##### Figure 5-the inhibition rate of DH5αand SOD

### Learn

In recent years, advancements in biotechnology have facilitated the development of directed evolution,
introducing random mutations into enzyme engineering. However, directed evolution is often labor-intensive,
has technical limitations that restrict its applicability, and relies on costly high-throughput screening
systems. Exploring vast sequence spaces through random mutagenesis is fundamentally challenging. Therefore,
strategically introducing mutations to enhance enzyme activity prior to designing directed evolution can
significantly increase the success rate of improving enzyme activity. In this project, we aim to enhance the
activity, solubility, and thermal stability of SOD through computer predictions, employing large-primer
site-directed mutagenesis and high-throughput screening strategies to identify superior SOD variants.

# Cycle 3

### Design

With the completion of the DNA sequencing phase of the Human Genome Project and the anticipated explosion of
protein structures in structural genomics, methods for analyzing receptor-ligand interactions are urgently
needed. Mutations of specific bases in proteins have proven valuable for probing the contributions of
individual amino acid side chains to protein properties. Alanine (side chain R = methyl) lacks unusual
backbone dihedral angle preferences; for instance, glycine (R = H) renders the side chain ineffective but can
introduce conformational flexibility into the protein backbone. The ability of alanine scanning mutagenesis to
provide critical biological insights has been demonstrated through significant early examples. Combining
alanine scanning with the convenience of combinatorial libraries offers a powerful approach. Therefore, we
explored virtual amino acid mutations, using FoldX to perform interaction-based virtual amino acid mutations
on a protein-ligand complex, ranking the changes in Gibbs free energy (ΔG) related to protein folding in
descending order. This allowed us to identify 28 key amino acids in the active site and potential mutations
that could enhance affinity, aiming to improve SOD enzyme activity.

![](https://static.igem.wiki/teams/5322/t-img-3/6-ala.png)

##### Figure 6: 28 pairs of mutation sites based on interaction-driven virtual amino acid mutations.

In recent years, methods based on sequence co-evolution analysis have shown great potential in enzyme
engineering, revealing the interactions between amino acid residues and providing a theoretical basis for the
modification of enzyme function and structure. We generated the final mutation SCI scores through the scanner
and sorted them in descending order to identify five key amino acids distant from the active site and amino
acid mutation targets that could enhance stability, with the aim of improving SOD enzyme activity. The
Sequence Co-evolution Index (SCI) is a metric used in enzyme activity engineering based on sequence
co-evolution analysis to assess the potential impact of co-evolution relationships at mutation sites on enzyme
activity. The calculation of the SCI index takes into account the number and strength of co-evolution
relationships between the mutation site and other sites, as well as the distribution of these relationships
within the enzyme sequence. The SCI index increases with the frequency of occurrence of the mutated amino acid
relative to the wild-type (WT) amino acid pair in multiple sequence alignments (MSA). In simple terms, if a
mutation site has strong co-evolution relationships with many other sites in the enzyme sequence and this
relationship is widespread in the sequence, the SCI index for that site will be high, indicating that
mutations at this site may have a significant impact on enzyme activity.

![](https://static.igem.wiki/teams/5322/t-img-3/7-site.png)

##### Figure 7 displays five mutation targets based on thermal stability virtual amino acid mutations, along with their corresponding SCI values.

Therefore, we identified 33 mutation targets that may significantly impact SOD enzyme activity for optimizing
the SOD sequence.

![](https://static.igem.wiki/teams/5322/t-img-3/8-coding.png)

##### Figure 8 33 pairs of mutation targets

### Build

We performed PCR amplification of the target plasmid and conducted gel electrophoresis (30 min at 120 V). In
the first round of site-directed mutagenesis, 16 mutation sites were unsuccessful, so we set up a touchdown
PCR with a temperature range of 62°C to 58°C, which was successful. After purifying the products, we conducted
homologous recombination and transformed them into *E. coli* DH5α, constructing the plasmid
pET-29(a)-pT7-lac
operator-(SOD-1+His)-T7. The cultures were incubated on plates at 37°C for 16 hours, after which we selected
single colonies and inoculated them into 40 mL LB medium with 40 µL Kanamycin. The cultures were grown
overnight at 30°C for 12 hours, followed by streaking for preservation. Afterward, we added 200 µL IPTG to
achieve a final concentration of 0.5 mM and incubated the cultures at 16°C for 24 hours.

### Test

We took 2 mL of the overnight induced bacterial culture and used the Beyotime™ bacterial active protein
extraction reagent to extract the protein. SOD activity was measured using the Beyotime™ SOD activity
detection kit. The enzyme activity of SOD was calculated through colorimetric analysis of the WST-8 product.

![](https://static.igem.wiki/teams/5322/t-img-3/10-first-live.png)

##### Figure SOD inhibition rate of the 1st mutation

### Learn

The results indicated that 27 point mutations in SOD showed enhanced inhibition rates compared to the
unmodified DH5α, while 21 mutations exhibited increased inhibition rates compared to the unmutated SOD. Among
these, the mutations at positions 33 ([BBa\_K5322020](https://parts.igem.org/Part%3ABBa_K5322020)), 30
([BBa\_K5322019](https://parts.igem.org/Part%3ABBa_K5322019)), 24 ([BBa\_K5322017](https://parts.igem.org/Part%3ABBa_K5322017)), 15 ([BBa\_K5322016](https://parts.igem.org/Part%3ABBa_K5322016)),
26 ([BBa\_K5322018](https://parts.igem.org/Part%3ABBa_K5322018)), 2 ([BBa\_K5322014](https://parts.igem.org/Part%3ABBa_K5322014)), and 13 ([BBa\_K5322015](https://parts.igem.org/Part%3ABBa_K5322015)) significantly improved SOD activity, with
inhibition rates of 73.612%, 71.909%, 70.207%, 67.653%, 67.531%, 67.41%, and 66.802%, and enzyme activities of
2.7896 U, 2.5599 U, 2.3565 U, 2.0915 U, 2.0799 U, 2.0684 U, and 2.0122 U, respectively. The best mutation at
position 33 showed an increase in enzyme activity of 1.8410 U compared to unmodified DH5α and 1.5117 U
compared to SOD-modified DH5α. Generally, single-round mutations often do not achieve the desired goals,
necessitating multiple rounds of iterative mutation. Thus, we plan to select the better-performing variants
from the previous round as templates for subsequent iterative mutations.

# Cycle 4

### Design

Screening is a bottleneck in directed evolution for enzyme engineering. Utilizing sequence and structural
information, we selected amino acid residues with direct interactions with the substrate around the enzyme's
catalytic active site through computer simulations, applying rational grouping for single or multiple rounds
of Iterative Saturation Mutagenesis (ISM). To specifically modify SOD, we selected the seven mutation sites—2,
13, 15, 24, 26, 30, and 33—with the largest increases in activity and determined 21 combinations through
pairwise combinations.

### Build

Same as Build (3)

### Test

Our team extracted proteins from 2 mL of overnight induced bacterial cultures using the Beyotime™ Bacterial
Active Protein Extraction Kit, followed by the calculation of protein concentration based on a standard curve
generated with the Vazyme™ TMBCA Protein Concentration Assay Kit. Since the inhibition rate exceeded 70% in
the first round of mutations, we diluted the sample to 30 μg/μL for subsequent testing. The SOD activity was
measured using the Beyotime™ SOD Activity Assay Kit, and the SOD inhibition rate was visualized using a heat
map, where lighter background colors represent lower inhibition rates.

![](https://static.igem.wiki/teams/5322/t-img-3/11-hotpicture.png)

##### Figure SOD inhibition rate of the 2nd mutation

### Learn

The results indicate that, compared to the original seven mutations, most enzyme activities did not show
significant improvement. However, the combinations 2+33 ([BBa\_K5322021](https://parts.igem.org/Part%3ABBa_K5322021)), 26+33 ([BBa\_K5322024](https://parts.igem.org/Part%3ABBa_K5322024)), 15+24
([BBa\_K5322022](https://parts.igem.org/Part%3ABBa_K5322022)), and 13+30 ([BBa\_K5322023](https://parts.igem.org/Part%3ABBa_K5322023)) exhibited higher inhibition rates of
70.944%, 56.3422%, 55.6047%, and
53.2448%, respectively. The corresponding enzyme activities were 1.1388 U, 2.4416 U, 1.2525 U, and 1.2905 U.
Therefore, we decided to perform triple mutations based on these findings.

# Cycle 5

### Design

At the seven mutation sites 2, 13, 15, 24, 26, 30, and 33, combinations of three at a time yield a total of
35 mutation groups. Considering cost and time constraints, we selected four optimal pairs of mutations—13+30,
2+33,
15+24, and 26+33—based on the results from the double mutations as templates, resulting in the following 20
combinations:

![](https://static.igem.wiki/teams/5322/t-img-3/12-zuhe.png)

##### Figure 20 groups of three-point mutations

### Build

Same as Build (4)

### Test

Same as Test (4)

![](https://static.igem.wiki/teams/5322/20241118154605.webp)

##### Figure SOD inhibition rate of the 3rd mutation

### Learn

The results showed that after three-point mutations, four sets of mutated SOD demonstrated an increased
inhibition rate compared to the unmodified DH5α. Additionally, these four sets of mutated SOD exhibited
enhanced inhibition rates when compared to the unmutated SOD. Notably, the combination of mutations 15+24+13
([BBa\_K5322025](https://parts.igem.org/Part%3ABBa_K5322025)) had an inhibition rate of 58.57% and an
enzyme activity of 1.4136 U, which represents an
increase of 0.5399 U over the unmutated SOD.

To ensure that our SOD is expressed only at the site of intestinal inflammation, we utilized the
characteristic high levels of NO in the inflamed region and regulated the expression with the NO-responsive
promoter SoxR/SoxS. We designed the following gene circuit: pET29a-J23119-RBS-SoxR-T7-pSoxS-RBS-(SOD-1)-T7.

![](https://static.igem.wiki/teams/5322/t-img-3/14-pet29a-j23119-rbs-soxr-t7-psoxs-rbs-sod-1-t7.png)

##### Figure 14-pET29a-J23119-RBS-SoxR-T7-pSoxS-RBS-(SOD-1)-T7 gene line

The team adopted an NO-responsive system in which the transcription factor SoxR serves as a sensor for
oxidative stress and nitric oxide. The presence of NO at the site of intestinal inflammation will activate the
transcription of pSoxS, thereby initiating the expression of downstream SOD. By expressing SOD, excessive ROS
in the intestine can be eliminated, creating a more favorable environment for subsequent treatments. Based on
these design principles, we used Snapgene software to design the plasmid p-SoxR-T-pSoxS-RBS-SOD-T7
([BBa\_K5322024](https://parts.igem.org/Part%3ABBa_K5322024)). Due to time constraints, we have only
completed the plasmid design and plan to test its
performance in the future.

![](https://static.igem.wiki/teams/5322/t-imgs-2/15-p-soxr-t-psoxs-rbs-sod-t.png)

##### Figure 15-p-SoxR-T-pSoxS-RBS-SOD-T plasmid map

# Mfp

# Cycle 6

### Design

In response to the inflammation caused by the reduced thickness of the intestinal mucus layer and the
disruption of microbial communities in patients with intestinal inflammation, we aim to address the disorder
of the mucosal system from the ground up. A natural high-adhesion protein derived from marine mussels—mussel
foot protein (Mfp)—has garnered our attention. Among the six types of mussel proteins, Mfp5 and Mfp3, which
contain the highest levels of dopa, were chosen for expression. Considering that these are animal proteins,
there may be unknown issues associated with expressing them in prokaryotes. Therefore, we first utilized
*Escherichia coli* BL21(DE3) to construct a production factory for Mfp based on efficient bacterial
synthesis,
aimed at enhancing adhesion in the damaged intestine and regulating gut microbiota. Consequently, we designed
the plasmids pET29a-J23119-RBS-Mfp3-T7 ([BBa\_K5322000](https://parts.igem.org/Part%3ABBa_K5322000))
and pET29a-J23119-RBS-Mfp5-T7 ([BBa\_K5322002](https://parts.igem.org/Part%3ABBa_K5322002)). The
plasmid maps are as follows:

![](https://static.igem.wiki/teams/5322/t-imgs-2/20-pet29a-j23119-rbs-mfp3-t7.png)

##### pET29a-J23119-RBS-Mfp3-T7 plasmid map

![](https://static.igem.wiki/teams/5322/t-imgs-2/21-pet29a-j23119-rbs-mfp5-t7.png)

##### pET29a-J23119-RBS-Mfp5-T7 plasmid map

### Build

The construction process was the same as Build-1, and plasmids pET29a-J23119-RBS-Mfp5-T7 and
pET29a-J23119-RBS-Mfp3-T7 were constructed.

### Test

Next, we performed colony PCR and sent the successfully validated plasmids for sequencing. Following
confirmation of the correct sequences, we cultured the corresponding bacteria and conducted Tricine-SDS-PAGE
analysis.

![](https://static.igem.wiki/teams/5322/t-imgs-2/29-sds-mfp1.png)

##### 1: Mfp5 total protein (9.8 kDa) 2: Mfp5 supernatant (9.8 kDa) 3: Mfp5 precipitate (9.8 kDa) 4: Mfp5 after purification (9.8 kDa) Tricine-SDS-PAGE analysis of Mfp5.

![](https://static.igem.wiki/teams/5322/t-imgs-2/30-sds-mfp2.png)

##### 1: Mfp3 total protein (6.5 kDa) 2: Mfp3 supernatant (6.5 kDa) 3: Mfp3 precipitate (6.5 kDa) 4: Mfp3 after purification (6.5 kDa) Tricine-SDS-PAGE analysis of Mfp3.

Due to the inability to determine the expression of Mfp3 and Mfp5 from the protein gel images, we conducted
Western Blot analysis for further validation.

![](https://static.igem.wiki/teams/5322/t-imgs-2/31-wb-mfp1.png)

##### 1: Total protein of Mfp5 (9.8 kDa) 2: Supernatant of Mfp5 (9.8 kDa) 3: Precipitate of Mfp5 (9.8 kDa) 4: Purified Mfp5 (9.8 kDa) Western Blot analysis of Mfp5.

![](https://static.igem.wiki/teams/5322/t-imgs-2/32-wb-mfp2.png)

##### 1: Total protein of Mfp3 (6.5 kDa) 2: Supernatant of Mfp3 (6.5 kDa) 3: Precipitate of Mfp3 (6.5 kDa) 4: Purified Mfp3 (6.5 kDa) Western Blot analysis of Mfp3.

### Learn

Based on the protein characterization results, both Mfp3 and Mfp5 were expressed; however, the observed
protein sizes deviated from the theoretical sizes. The upward shift of the Mfp3 band may indicate the
formation of dimers or multimers, while the shift of the Mfp5 band could be attributed to its higher
viscosity. To achieve stable and efficient expression, we decided to use a flexible protein linker (GGGGS [BBa\_K5322001](https://parts.igem.org/Part%3ABBa_K5322001)) to connect Mfp3 and Mfp5, aiming to obtain
recombinant mussel proteins with enhanced functionality.

# Cycle 7

### Design

Mfp5 and Mfp3 are rich in DOPA residues, which facilitate the formation of hydrogen bonds with various
surfaces, enhancing their adhesive properties. Therefore, we designed a flexible linker composed of a double
copy of GGGGS to fuse the two proteins, aiming to obtain recombinant mussel proteins with improved
functionality. Consequently, we constructed the plasmid pET29a-J23119-RBS-Mfp53-T7 ([BBa\_K5322003](https://parts.igem.org/Part%3ABBa_K5322003)).

The plasmid map is shown below:

![](https://static.igem.wiki/teams/5322/t-imgs-2/33-pet29a-j23119-rbs-mfp53-t7.png)

##### pET29a-J23119-RBS-Mfp53-T7 plasmid map

### Build

The construction process was the same as Build-1 to construct the expression plasmid
pET29a-J23119-RBS-Mfp53-T7.

### Test

We then performed colony PCR and sent the successfully verified plasmid to the company for sequencing, and
the sequencing results were correct.

![](https://static.igem.wiki/teams/5322/t-imgs-2/40-cexu-mfp1.png)

##### pET29a-J23119-RBS-Mfp53-T7 sequencing result diagram

### Learn

We used the pET29a vector and molecular cloning techniques such as PCR and homologous recombination to
successfully construct the recombinant plasmid pET29a-J23119-RBS-Mfp53-T7. Colony PCR validation and
sequencing results indicated the successful construction of the recombinant strains
DH5α-pET29a-J23119-RBS-Mfp53-T7 and BL21-pET29a-J23119-RBS-Mfp53-T7. Next, we plan to upgrade the plasmid to
enable protein expression regulation in the high NO environment of colitis. During an interview with Dr. Wang
Yun, a chief physician at Jiangsu Provincial People's Hospital, we recognized the importance of the safety of
engineered bacteria in the human body. Therefore, we decided to change the chassis to use the non-endotoxin
*Escherichia coli* Nissle 1917.

# Cycle 8

### Design

To achieve better intestinal mucosal repair in the high NO environment associated with colitis, we used the
probiotic *Escherichia coli* Nissle 1917 as a carrier and employed the oxidative stress-responsive
SoxR/SoxS
promoter for targeted therapy at the site of intestinal inflammation. Accordingly, we designed the plasmids
pET29a-J23119-soxR-T-psoxS-RBS-Mfp3-T7 ([BBa\_K5322005](https://parts.igem.org/Part%3ABBa_K5322005))
and pET29a-J23119-soxR-T-psoxS-RBS-Mfp5-T7 ([BBa\_K5322006](https://parts.igem.org/Part%3ABBa_K5322006)). The plasmid maps are shown below:

![](https://static.igem.wiki/teams/5322/t-imgs-2/41-pet29a-j23119-soxr-t-psoxs-rbs-mfp5-t7.png)

##### Plasmid map of pET29a-J23119-soxR-T-psoxS-RBS-Mfp5-T7

![](https://static.igem.wiki/teams/5322/t-imgs-2/42-pet29a-j23119-soxr-t-psoxs-rbs-mfp3-t7.png)

##### Plasmid map of pET29a-J23119-soxR-T-psoxS-RBS-Mfp3-T7

### Build

The construction process is the same as Build-1, resulting in the construction of the plasmid
pET29a-J23119-soxR-T-psoxS-RBS-Mfp3-T7 and pET29a-J23119-soxR-T-psoxS-RBS-Mfp5-T7.

### Test

Sodium Nitroprusside (SNP) Induction

Given that the mussel proteins used in this study are applied in the context of mammalian intestinal
inflammation, we selected 37 °C, the temperature closest to body temperature, for induction. The duration of
induction was identified as a key factor in protein characterization. The recombinant strains
EcN-pET29a-J23119-soxR-T-psoxS-RBS-Mfp3-T7 and EcN-pET29a-J23119-soxR-T-psoxS-RBS-Mfp5-T7 were grown to
OD600= 0.8, then sodium nitroprusside (SNP) was added to a final concentration of 100 μM for induction. The recombinant proteins were
expressed for 16
hours in the EcN strains and subsequently detected using Tricine-SDS-PAGE.

![](https://static.igem.wiki/teams/5322/t-imgs-2/52-pcr-mfp23.png)

##### 1. Induced total protein of Mfp3 2. Induced supernatant of Mfp3 3. Induced precipitate of Mfp3 4. Purified Mfp3 after induction Tricine-SDS-PAGE analysis of recombinant Mfp3 (6.5 kDa) expression.

![](https://static.igem.wiki/teams/5322/t-imgs-2/53-pcr-mfp24.png)

##### 1. Induced total protein of Mfp5 2. Induced supernatant of Mfp5 3. Induced precipitate of Mfp5 4. Purified Mfp5 after induction Tricine-SDS-PAGE analysis of recombinant Mfp5 (9.8 kDa) expression.

Due to the inability to assess the expression of Mfp3 and Mfp5 from the gel image, we performed Western Blot
for further verification.

![](https://static.igem.wiki/teams/5322/t-imgs-2/54-wb-mfp3.png)

##### 1. Induced total protein of Mfp3 2. Induced supernatant of Mfp3 3. Induced precipitate of Mfp3 4. Purified Mfp3 after induction Western Blot analysis of recombinant Mfp3 (6.5 kDa) expression.

![](https://static.igem.wiki/teams/5322/t-imgs-2/55-wb-mfp4.png)

##### 1. Non-induced total protein of Mfp5 2. Non-induced supernatant of Mfp5 3. Non-induced precipitate of Mfp5 4. Purified Mfp5 after non-induction Western Blot analysis of recombinant Mfp5 (9.8 kDa) expression.

### Learn

According to the results from Tricine-SDS-PAGE and Western Blot, both pET29a-J23119-soxR-T-psoxS-RBS-Mfp5-T7 and
pET29a-J23119-soxR-T-psoxS-RBS-Mfp3-T7 were successfully expressed after induction, indicating their stable
expression in high NO environments. This suggests that our engineered bacteria can produce Mfp5 and Mfp3 in
the high NO conditions of intestinal inflammation, facilitating the repair of intestinal mucosa and regulating
gut microbiota, thereby treating colitis. Next, we decided to design and construct
pET29a-J23119-soxR-T-psoxS-RBS-Mfp53-T7, following the construction of pET29a-J23119-RBS-Mfp53-T7, with the
expectation that it would also express stably in inflammatory environments.

# Cycle 9

### Design

Using the probiotic *Escherichia coli* Nissle 1917 (EcN) as the host, we regulated the transcription and
expression of the downstream high-adhesion proteins Mfp5 and Mfp3 through the oxidative stress-responsive
promoter SoxR/SoxS. Additionally, we fused the expression of Mfp53 using a flexible protein linker ([BBa\_K5322001](https://parts.igem.org/Part%3ABBa_K5322001)) to
achieve improved therapeutic effects. Consequently, we designed the plasmid
pET29a-J23119-soxR-T-psoxS-RBS-Mfp53-T7 ([BBa\_K5322007](https://parts.igem.org/Part%3ABBa_K5322007)).

The plasmid map is shown below:

![](https://static.igem.wiki/teams/5322/t-imgs-2/56-pet29a-j23119-soxr-t-psoxs-rbs-mfp53-t7.png)

##### pET29a-J23119-soxR-T-psoxS-RBS-Mfp53-T7

### Build

The construction process follows the same procedure as Build-1, resulting in the plasmid
pET29a-J23119-soxR-T-psoxS-RBS-Mfp53-T7.

### Test

Next, we performed colony PCR and sent the successfully validated plasmids for sequencing at a company. The
sequencing results were confirmed to be correct.

![](https://static.igem.wiki/teams/5322/t-imgs-2/62-cexu-mfp2.png)

The sequencing results for pET29a-J23119-soxR-T-psoxS-RBS-Mfp53-T7 are illustrated in the figure below.

### Learn

Using pET29a as the vector, we successfully constructed the recombinant plasmid
pET29a-J23119-soxR-T-psoxS-RBS-Mfp53-T7 through molecular cloning methods such as PCR and homologous
recombination. Colony PCR validation and sequencing results indicated the successful construction of the
recombinant strains DH5α-pET29a-J23119-soxR-T-psoxS-RBS-Mfp53-T7 and
EcN-pET29a-J23119-soxR-T-psoxS-RBS-Mfp53-T7. This demonstrates our ability to utilize the probiotic
*Escherichia coli* Nissle 1917 (EcN) as a host to regulate the transcription and expression of the
downstream
adhesion protein Mfp53 via the oxidative stress-responsive promoter SoxR/SoxS. The intestinal inflammatory
factor NO serves as a signal for its accumulation at tissue injury sites, aiding in the repair of intestinal
mucosa, regulating gut microbiota, and inhibiting the growth of pathogenic bacteria.

# PD-L1

# Cycle 10

### Design

In regions of intestinal inflammation, the overactive immune response of CD8+ T cells necessitates
the
modulation and downregulation of immune activity. To address this, we plan to utilize the PD-1/PD-L1 immune
checkpoint to inhibit the activity of CD8+ cytotoxic T cells. For the convenience of future experiments, we
selected mouse PD-L1 ([BBa\_K5322033](https://parts.igem.org/Part%3ABBa_K5322033)) and performed codon
optimization to design the plasmid
pET29a-J23119-RBS-PD-L1 (Mus)-T7, as shown in the figure below.

![](https://static.igem.wiki/teams/5322/t-img-3/pet29a-j23119-rbs-pd-l1-mus-t7-map.png)

### Build

The sequence J23119-RBS-PD-L1 (Mus)-T7 was sent to a company for synthesis. After two weeks, the results
indicated that random mutations occurred during the recombination of the fragment with the vector in
*Escherichia coli*. Considering the uncertainties introduced by these random mutations, we decided to
abandon
this approach.

### Learn

We noted that mouse PD-L1, as an animal protein, has a complex structure and a large molecular weight,
making its expression in the prokaryotic system of *E. coli* challenging. This design presents issues,
necessitating the exploration of better methods for expressing PD-L1 protein.

# Cycle 11

### Design

According to the descriptions of mouse PD-L1 in the NCBI database (ADK70951.1, ADK70950.1, Q9NZQ7.1,
AAH66841.1), the amino acids from 20 to 130 in the 290 aa sequence correspond to the functional domain of
PD-L1, specifically the immunoglobulin variable (IgV) domain (CD20947). To ensure that the functionality of
the truncated protein is not compromised, we retained the first 1-19 aa at the N-terminus and the last 131-150
aa at the C-terminus, aiming to preserve the integrity of the IgV domain as much as possible. Subsequently, we
utilized AlphaFold2, developed by Google DeepMind, to predict the protein structures of both the full-length
and truncated PD-L1, yielding the following prediction results.

![](https://static.igem.wiki/teams/5322/t-img-3/pd-l1-all.jpg)
![](https://static.igem.wiki/teams/5322/t-img-3/pd-l1.jpg)

Based on the prediction results from AlphaFold 2, it is evident that the main functional domain of the
truncated PD-L1 remains unaffected. Accordingly, we designed an expression system for the PD-L1 functional
domain:

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5322/gif/alphafold2-pd-l1-model1.gif) | ![](https://static.igem.wiki/teams/5322/gif/full/alphafold2-pd-l1-model1.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/alphafold2-pd-l1-model2.gif) | ![](https://static.igem.wiki/teams/5322/gif/full/alphafold2-pd-l1-model2.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/alphafold2-pd-l1-model3.gif) | ![](https://static.igem.wiki/teams/5322/gif/full/alphafold2-pd-l1-model3.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/alphafold2-pd-l1-model4.gif) | ![](https://static.igem.wiki/teams/5322/gif/full/alphafold2-pd-l1-model4.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/alphafold2-pd-l1-model5.gif) | ![](https://static.igem.wiki/teams/5322/gif/full/alphafold2-pd-l1-model5.gif) |

pET29a-J23119-RBS-PD-L1 (functional domain)-T7. The plasmid diagram is shown in the figure below.

![](https://static.igem.wiki/teams/5322/t-img-3/pet29a-j23119-rbs-pd-l1-functional-domain-t7-map.png)

Subsequently, Google launched AlphaFold3, which we used to predict the protein structure once again. We also
performed molecular docking to demonstrate that the truncated PD-L1 can successfully bind to PD-1 and exert
its function. The results are shown in the figure below.

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5322/gif/pdl1/full/pd-l1-alpha3-model1.gif) | ![](https://static.igem.wiki/teams/5322/gif/full/alphafold2-pd-l1-model1.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/pdl1/full/pd-l1-alpha3-model2.gif) | ![](https://static.igem.wiki/teams/5322/gif/pdl1/half/pd-l1-alpha3-model2.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/pdl1/full/pd-l1-alpha3-model3.gif) | ![](https://static.igem.wiki/teams/5322/gif/pdl1/half/pd-l1-alpha3-model3.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/pdl1/full/pd-l1-alpha3-model4.gif) | ![](https://static.igem.wiki/teams/5322/gif/pdl1/half/pd-l1-alpha3-model4.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/pdl1/full/pd-l1-alpha3-model5.gif) | ![](https://static.igem.wiki/teams/5322/gif/pdl1/half/pd-l1-alpha3-model5.gif) |

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5322/half-mouse/pd-l1-pd-1-model1.gif) | ![](https://static.igem.wiki/teams/5322/whole-mouse/pd-l1-pd-1-model1.gif) |
| ![](https://static.igem.wiki/teams/5322/half-mouse/pd-l1-pd-1-model2.gif) | ![](https://static.igem.wiki/teams/5322/whole-mouse/pd-l1-pd-1-model2.gif) |
| ![](https://static.igem.wiki/teams/5322/half-mouse/pd-l1-pd-1-model3.gif) | ![](https://static.igem.wiki/teams/5322/whole-mouse/pd-l1-pd-1-model3.gif) |
| ![](https://static.igem.wiki/teams/5322/half-mouse/pd-l1-pd-1-model4.gif) | ![](https://static.igem.wiki/teams/5322/whole-mouse/pd-l1-pd-1-model4.gif) |
| ![](https://static.igem.wiki/teams/5322/half-mouse/pd-l1-pd-1-model5.gif) | ![](https://static.igem.wiki/teams/5322/whole-mouse/pd-l1-pd-1-model5.gif) |

### Build

We ordered the synthetic gene fragment J23119-RBS-PD-L1 (functional domain)-T7 from a company. Upon receipt,
we performed PCR to amplify the target fragment and the pET29a vector. The PCR products were analyzed by gel
electrophoresis (30 min, 120 V), as shown in the figure below. After purifying the products, we conducted
homologous recombination and transformed *Escherichia coli* DH5α. The cultures were incubated on inverted
plates
at 37°C, and after 16 hours, colony PCR was performed on single colonies, with the results displayed in the
figure.

![](https://static.igem.wiki/teams/5322/t-imgs/pcr-1.png)
![](https://static.igem.wiki/teams/5322/t-imgs/pcr-2.png)

### Test

Successful colonies from PCR were inoculated and cultured at 37°C, 220 rpm for 12-16 hours. Plasmids
were then extracted and sent for sequencing, with the sequencing results shown in the figure below.

![](https://static.igem.wiki/teams/5322/t-imgs/cexu-pet29a-j23119-rbs-pd-l1-functional-domain-t7-map.png)

Based on the sequencing results, we confirmed the successful construction of the plasmid, which was then
transformed into EcN, completing the expression system. We are now preparing to conduct protein function
tests.

### Learn

During the project, we considered that PD-L1 may be encapsulated by mussel foot protein Mfp during its
expression and release, which could potentially reduce the therapeutic efficacy.

# Cycle 12

### Design

To address the issues previously mentioned, we considered utilizing the surface display system Lpp-OmpA to
present our target protein PD-L1 on the outer membrane of *Escherichia coli*. This approach facilitates
easier
and more effective binding with PD-1. Additionally, to confirm the surface display in *E. coli*, we
introduced a
flexible protein linker and a recognition and cleavage site for Tobacco Etch Virus protease (TEV site) between
Lpp-OmpA and the PD-L1 functional domain. By incubating the cells with TEV protease, we can cleave PD-L1 from
OmpA. If the presence of the PD-L1 functional domain is detected in the supernatant, this would indirectly
demonstrate the success of the surface display. The plasmid design is illustrated in the figure.

![](https://static.igem.wiki/teams/5322/t-img-3/pet29a-j23119-lpp-ompa-giss-tevsite-pd-l1-functional-domain-t7-map.png)

### Build

Similar to Cycle 11, construct the plasmid.

![](https://static.igem.wiki/teams/5322/t-imgs/pcr-3.png)

Successful colonies from PCR were inoculated and cultured at 37°C, 220 rpm for 12-16 hours. Plasmids were
then extracted and sent for sequencing, with the sequencing results shown in the figure below.

![](https://static.igem.wiki/teams/5322/t-imgs/cexu-pet29a-j23119-lpp-ompa-giss-tevsite-pd-l1-functional-domain-t7-map.png)

Based on the sequencing results, we confirmed the successful construction of the plasmid, which was then
transformed into EcN, completing the expression system. We are now prepared to conduct protein function tests.

### Test

We performed expression tests on the constructed system. After culturing in a 250 mL shaking flask for 16
hours, we conducted ultrasonic cell disruption, followed by nickel column purification and protein
purification. The results of the SDS-PAGE analysis are shown below.

![](https://static.igem.wiki/teams/5322/t-imgs/sds-1.png)

##### 1: Total protein; 2: Supernatant; 3: Precipitate; 4: After purification; 5: Eluate from nickel column; 6: Before ultrafiltration; 7: After ultrafiltration; M: Sewell pre-stained protein marker.

Validation through protein gel electrophoresis alone was insufficient to confirm protein expression.
Therefore, after reviewing the literature, we hypothesized that the large amount of Lpp-OmpA-PD-L1 protein and
the low bacterial expression levels might be contributing factors. To address this issue, we opted for Western
Blot, which offers higher sensitivity and can detect proteins at the ng level. After a period of study, we
mastered the WB techniques and key considerations. We initially performed WB on the total protein, and the
results are shown in the figure below.

![](https://static.igem.wiki/teams/5322/t-imgs/wb-1.png)

After confirming the presence of bands in the total protein, we demonstrated the correct expression of the
target protein. We then proceeded with purification and ultrafiltration of the protein, followed by direct
verification using WB. The results are shown in the figure below.

![](https://static.igem.wiki/teams/5322/t-imgs/wb-2.png)

##### 1: Total protein; 2: Supernatant; 3: Precipitate; 4: After purification; 5: Eluate from nickel column; 6: Before ultrafiltration; 7: After ultrafiltration; M: Sewell pre-stained protein marker.

From the WB results, it is clear that a significant portion of Lpp-OmpA, due to its hydrophobic nature, is
present in the precipitate, which aligns with theoretical expectations. However, we also observed multiple
bands in the blot. Upon analysis, a possible explanation is that the membrane protein is degraded at 100°C due
to its inherent characteristics, leading to protein fragmentation. Therefore, we designed experiments based on
previous studies to control the temperature of protein denaturation to investigate the optimal temperature for
WB experiments involving membrane proteins. The results are shown below.

![](https://static.igem.wiki/teams/5322/t-imgs/wb-3.png)

According to the WB result graph, we can find that under the deformation condition of 30℃ and 30min, the
34kDa protein is less damaged, so this denaturation condition is determined to be the preferred condition. So
far, we have proved the expression of the target protein, and the next step is to prove the success of the
surface membrane display system.

200mL of bacterial solution cultured for 12-16h and 50U TEV enzyme were incubated at 4℃ for 24h, and the
supernatant was taken after centrifugation for ultrafiltration and concentration, and the bacteria were broken
for standby. The two samples were subjected to the deformation condition of 30℃ and 30min for WB experimental
verification. According to the WB results, PD-L1 (functional domain) exists in the incubation supernatant,
which proves that the surface membrane display is successful.

![](https://static.igem.wiki/teams/5322/t-imgs/wb-4.png)

# References

1. Sunny Park, Xiaojun You, and James A. Imlay.Substantial DNA damage from submicromolar intracellular
hydrogen peroxide detected in Hpxmutants of Escherichia coli.Department of Microbiology, University of
Illinois, Urbana, IL 61801:1.
2. J.F. Ward, J.W. Evans, C.L. Limoli & P.M. Calabro-Jones.Radiation and hydrogen peroxide induced free
radical damage to DNA.Division of Radiation Biology, Department of Radiology, University of California at San
Diego, MO1O, La Jolla, California,92093, USA.
3. Combinatorial alanine-scanning Kim L Morrison and Gregory A Weiss.Department of Chemistry, University of
California, Irvine,CA 92697-2025, USA.
4. Ge Qu, Jing Zhao, elt. Recent advances in directed evolution. Chinese Journal of Biotechnology. Jan. 25,
2018, 34(1): 1−11

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

![Project Image](https://static.igem.wiki/teams/5322/top-head-new3.png)

Experiments

* [SOD](#page1)
* [Mfp](#page5)
* [PD-L1](#page13)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}
![Project Image](https://static.igem.wiki/teams/5322/top-head-new3.png)

Results

* [SOD](#SOD)
* [Mfp](#MFP)
* [PD-L1](#PD-L1)
* [Cracking module and safety module](#Crackingmoduleandsafetymodule)

# SOD

1. We designed the plasmid pET29a-J23119-RBS-(SOD-1)-T7, and primers for the homologous arms
were designed
to perform PCR on the vectors pET-29(a), J23119+RBS, and SOD.

![](https://static.igem.wiki/teams/5322/t-imgs-2/101-pet-29-a-j23119-sod.png)

Using homologous recombination, we obtained the target plasmid and subsequently transformed it into DH5α
competent cells. Single colonies were selected from the transformed plates, and PCR validation was performed
on these colonies.

![](https://static.igem.wiki/teams/5322/t-img-3/102.png)

After verifying the correct bands through nucleic acid electrophoresis, we transferred the corresponding
single colonies with the correct bands to LB (Kana) liquid culture medium. Subsequently, we extracted the
plasmids and sent them to GENEWIZ for sequencing. The sequencing results indicated that there was a base
deletion in the promoter region, as shown in the figure.

![](https://static.igem.wiki/teams/5322/t-imgs/2-pet-29-a-j23119-sod-1-his-t7-map1.png)

We selected single colonies and performed PCR validation on these colonies.

![](https://static.igem.wiki/teams/5322/t-img-3/103-pcr-2.png)

We transferred the corresponding single colonies with the correct bands to LB (Kana) liquid culture medium.
Subsequently, we extracted the plasmids for sequencing, and both sequencing results indicated a base deletion
in the promoter region (as shown in the figure).

![](https://static.igem.wiki/teams/5322/t-img-3/104-2.png)

![](https://static.igem.wiki/teams/5322/t-img-3/105-3.png)

Considering the potential damage that excess H2O2 could cause to DNA, we opted to use
the lac promoter, which
has a lower strength, and induced the expression of SOD in *Escherichia coli* with IPTG. Therefore, we
redesigned the plasmid to pET-29(a)-lac operator-SOD-1(+His)-T7 (as shown in the figure) and designed primers
to add BamHⅠ and EcoRⅠ restriction sites to both ends of the SOD fragment.

We then performed digestion using BamHⅠ and EcoRⅠ restriction endonucleases, repeated the aforementioned
steps, and the colony PCR results (shown in the figure) as well as the sequencing results (shown in the
figure) confirmed the successful construction of the plasmid.

![](https://static.igem.wiki/teams/5322/t-img-3/108-sod-pcr.png)

![](https://static.igem.wiki/teams/5322/t-img-3/107.png)

We took 2 mL of overnight induced bacterial culture and extracted the protein using the Beyotime™ bacterial
active protein extraction reagent. The SOD enzyme activity was measured using the Beyotime™ SOD enzyme
activity assay kit, and the absorbance at 450 nm (A450) was determined using a microplate reader. The original
data is shown in the table below, and the calculated inhibition rate of the induced SOD enzyme was
approximately 46%, while the inhibition rate for DH5α was about 38%. This confirms the successful expression
of the SOD protein.

![](https://static.igem.wiki/teams/5322/t-imgs/5-data.png)

##### the inhibition rate of DH5αand SOD

2. Using the plasmid pET-29(a)-lac operator-SOD-1(+His)-T7 as a template, we designed 33
pairs of primers
for site-directed mutagenesis PCR. After completion, we took 10 µL of the reaction mix for nucleic acid gel
electrophoresis verification (as shown in the figure).

![](https://static.igem.wiki/teams/5322/t-imgs-2/16-1.png)

The results indicated that some of the site-directed mutations were unsuccessful; therefore, we switched to
drop-off PCR, setting a gradient annealing temperature from 62°C to 58°C. This approach successfully achieved
the remaining mutations.

![](https://static.igem.wiki/teams/5322/t-imgs-2/17-2-png.jpeg)

After recovering the remaining 40 µL of the reaction mixture, we took 20 µL for DpnI digestion to remove
methylated templates. Subsequently, we proceeded with the recombination and transformed the resulting mixture
into DH5α competent cells.

![](https://static.igem.wiki/teams/5322/t-imgs-2/112-png.png)

We selected single colonies from the transformed plates and transferred them to LB (Kana) liquid medium,
incubating overnight at 37°C for 12 hours. Afterward, we inoculated this culture into 40 mL of fresh medium
and added 200 µL of IPTG to achieve a final concentration of 0.5 mM, followed by incubation at 16°C for 24
hours. We then extracted the protein using the Beyotime™ bacterial active protein extraction reagent and used
the Novagen TMBCA protein concentration determination kit to generate a protein standard curve, allowing us to
calculate the protein concentration. The samples were then diluted to 65 µg/µL.

![](https://static.igem.wiki/teams/5322/t-img-3/101-protein-standard-curve1.png)

##### protein standard curve1

SOD enzyme activity was measured using the Beyotime™ SOD enzyme activity assay kit. The results indicated
that among the 27 site-directed mutants, the SOD variants exhibited enhanced inhibition rates compared to the
unmodified DH5α strain, while 21 of the mutants demonstrated increased inhibition rates compared to the
unmutated SOD. Notably, the mutations at sites 33, 30, 24, 15, 26, 2, and 13 significantly enhanced SOD enzyme
activity, with increases of 73.612%, 71.909%, 70.207%, 67.653%, 67.531%, 67.41%, and 66.802%, corresponding to
enzyme activities of 2.7896 U, 2.5599 U, 2.3565 U, 2.0915 U, 2.0799 U, 2.0684 U, and 2.0122 U, respectively.
The optimal variant at site 33 exhibited an increase of 1.8410 U in enzyme activity compared to the unmodified
DH5α strain, and a 1.5117 U increase compared to the SOD-modified DH5α strain.

![](https://static.igem.wiki/teams/5322/t-img-3/10-first-live.png)

##### SOD inhibition rate of the 1st mutation

3. We used templates from samples 2, 13, 15, 24, 26, 30, and 33 to conduct 21 sets of
site-directed
mutagenesis. After completing the PCR, we took 10 µL of the reaction mix for nucleic acid gel electrophoresis
verification (as shown in the figure).

![](https://static.igem.wiki/teams/5322/t-img-3/109.png)

After recovering the remaining 40 µL of the reaction mixture, we took 20 µL for DpnI digestion to remove
methylated templates. We then proceeded with the recombination and transformed the resulting mixture into DH5α
competent cells.

![](https://static.igem.wiki/teams/5322/t-img-3/110.png)

We selected single colonies from the transformed plates and transferred them to LB (Kana) liquid medium,
incubating overnight at 37°C for 12 hours. Afterward, we inoculated this culture into 40 mL of fresh medium
and added 200 µL of IPTG to achieve a final concentration of 0.5 mM. The culture was then incubated at 16°C
for 24 hours. We extracted the protein using the Beyotime™ bacterial active protein extraction reagent and
utilized the Novagen TMBCA protein concentration determination kit to create a protein standard curve,
allowing us to calculate the protein concentration. The samples were then diluted to 30 µg/µL.

![](https://static.igem.wiki/teams/5322/t-img-3/111-protein-standard-curve-png.png)

##### protein standard curve2.png

Using the Beyotime™ SOD activity detection kit, SOD can inhibit the production of water-soluble
methylthiazole dye. The enzyme activity of SOD can be calculated through colorimetric analysis of the WST-8
product. The results revealed that, compared to the original seven mutations, most enzyme activity
enhancements were not significant; however, the combinations of 2+33, 26+33, 15+24, and 13+30 exhibited higher
inhibition rates of 70.944%, 56.3422%, 55.6047%, and 53.2448%, corresponding to enzyme activities of 1.1388 U,
2.4416 U, 1.2525 U, and 1.2905 U, respectively. A heatmap was utilized to represent enzyme activity, with
lighter background colors indicating better inhibition effects, higher inhibition rates, and increased enzyme
activity.

![](https://static.igem.wiki/teams/5322/t-img-3/11-hotpicture.png)

##### SOD inhibition rate of the 2nd mutation

We used templates from combinations of 13+30, 2+33, 15+24, and 26+33 to form 20 pairs of combinations. After
completion, we took 10 µL of each reaction mix for nucleic acid gel electrophoresis verification, and it was
found that 10 of the reactions were unsuccessful in PCR amplification.

![](https://static.igem.wiki/teams/5322/t-img-3/113.png)

After recovering the remaining 40 µL of the reaction mixture, we took 20 µL for DpnI digestion to remove
methylated templates. We then proceeded with the recombination and transformed the resulting mixture into DH5α
competent cells (as shown in the figure).

![](https://static.igem.wiki/teams/5322/t-img-3/114.png)

We selected single colonies from the transformed plates and transferred them to LB (Kana) liquid medium,
incubating overnight at 37°C for 12 hours. Following this, we inoculated the culture into 40 mL of fresh
medium and added 200 µL of IPTG to achieve a final concentration of 0.5 mM. The culture was then incubated at
16°C for 24 hours. We extracted the protein using the Beyotime™ bacterial active protein extraction reagent
and utilized the Novagen TMBCA protein concentration determination kit to create a protein standard curve,
allowing us to calculate the protein concentration. The samples were then diluted to 30 µg/µL.

![](https://static.igem.wiki/teams/5322/t-img-3/115-protein-standard-curve3.png)

##### protein standard curve3.png

Using the Beyotime™ SOD enzyme activity assay kit, we measured the enzyme activity. The results indicated
that after three point mutations, four groups of SOD with triple mutations showed increased inhibition rates
compared to the unmodified DH5α. Among these, the inhibition rate for the 15+24+13 combination was 58.57%,
with an enzyme activity of 1.4136 U, representing an increase of 0.5399 U compared to the unmutated SOD.

![](https://static.igem.wiki/teams/5322/t-img-3/13-live.png)

##### SOD inhibition rate of the 3rd mutation

# Mfp

1. The constitutive plasmids pET29a-J23119-RBS-Mfp3-T7 and pET29a-J23119-RBS-Mfp5-T7 have
been constructed.

We obtained the mussel foot proteins (Mfp3/Mfp5) [BBa\_K4854000](https://parts.igem.org/Part%3ABBa_K4854000) and [BBa\_K1583002](https://parts.igem.org/Part%3ABBa_K1583002) from the iGEM Parts Registry and
synthesized the gene sequences for Mfp3 and Mfp5. We selected the J23119 strong constitutive promoter and BL21
(DE3) as the promoter element and chassis cells, respectively, to facilitate sustained and efficient
expression of Mfp. Using SnapGene software, we designed the plasmids pET29a-J23119-RBS-Mfp3-T7 and
pET29a-J23119-RBS-Mfp5-T7. Through homologous recombination, we constructed the plasmids
pET29a-J23119-RBS-Mfp3-T7 and pET29a-J23119-RBS-Mfp5-T7, utilizing *E. coli* BL21 (DE3) as the chassis
and *E.
coli* DH5α for auxiliary construction. After selecting several single colonies from the transformation
plates,
we extracted the plasmids and performed PCR validation, ultimately confirming the correct sequencing results.
The plasmids pET29a-J23119-RBS-Mfp3-T7 and pET29a-J23119-RBS-Mfp5-T7 were successfully constructed.

![](https://static.igem.wiki/teams/5322/t-imgs-2/28-pcr-mfp7.png)

##### M：DL2000 Plus DNA Marker(Vazyme)

##### 1-4： BL21 Mfp5 Single colony(512bp)；5-7： BL21 Mfp3 Single colony(422bp) BL21-pET29a-J23119-RBS-Mfp5/3-T7 Colony PCR gel image

![](https://static.igem.wiki/teams/5322/t-imgs-2/95-cexu-5.png)

##### Sequencing map of plasmid pET29a-J23119-RBS-Mfp5-T7

![](https://static.igem.wiki/teams/5322/t-imgs-2/93-cexu-3.png)

##### Sequencing map of plasmid pET29a-J23119-RBS-Mfp3-T7

2. Construction of constitutive plasmid pET29a-J23119-RBS-Mfp53-T7

To obtain a recombinant mussel foot protein with enhanced functionality, we designed a flexible linker
composed of a double repeat of GGGGS to fuse Mfp3 and Mfp5, resulting in the construction of the plasmid
pET29a-J23119-RBS-Mfp53-T7. We used *Escherichia coli* BL21 (DE3) as the chassis cell, with
*E. coli* DH5α
assisting in the construction. The final sequencing results confirmed the successful construction of the
plasmid pET29a-J23119-RBS-Mfp53-T7.

![](https://static.igem.wiki/teams/5322/t-imgs-2/39-pcr-mfp13.png)

##### M：DL2000 DNA Marker 1-3：J23119-Mfp53(680bp)

##### BL21 pET29a-J23119-RBS-Mfp53-T7 Colony PCR gel image

![](https://static.igem.wiki/teams/5322/t-imgs-2/40-cexu-mfp1.png)

##### Sequencing map of plasmid pET29a-J23119-RBS-Mfp53-T7

3. Construction of NO-inducible plasmids pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp5-T7 and
pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp3-T7

*Escherichia coli* possesses a superoxide stress system composed of the SoxR gene, which naturally
responds to
redox signals. In the high NO environment associated with enteritis, constitutively expressed SoxR can
activate the SoxS promoter, thereby regulating the transcription and expression of downstream genes.
Consequently, we decided to use the oxidative stress-inducible promoter SoxR/SoxS as a regulatory element to
achieve targeted induction of engineered bacteria in high NO concentrations typical of enteritis, facilitating
the expression of therapeutic agents. Additionally, since *E. coli* Nissle 1917 is endotoxin-free, we
opted to
change the chassis to this strain. Thus, we designed and constructed the plasmids
pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp5-T7 and pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp3-T7, utilizing *E. coli*
Nissle
1917 as the chassis and *E. coli* DH5α for auxiliary construction. The final sequencing results confirmed
the
successful construction of the plasmids pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp5-T7 and
pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp3-T7.

![](https://static.igem.wiki/teams/5322/t-imgs-2/51-pcr-mfp22.png)

##### M：DL2000Plus DNA Marker 1-4：EcN-Mfp3 Colony(1075bp)； EcN-pET29a-J23119-soxR-T-psoxS-RBS-Mfp3-T7 Colony PCR gel image

![](https://static.igem.wiki/teams/5322/t-imgs-2/50-pcr-mfp21.png)

##### M：DL2000Plus DNA Marker 1-4：EcN-Mfp5 Colony(1165bp)； EcN-pET29a-J23119-soxR-T-psoxS-RBS-Mfp5-T7Colony PCR gel image

![](https://static.igem.wiki/teams/5322/t-imgs-2/96-cexu-6.png)

##### EcN-pET29a-J23119-soxR-T-psoxS-RBS-Mfp3-T7 Sequencing results

![](https://static.igem.wiki/teams/5322/t-imgs-2/97-cexu-7.png)

##### EcN-pET29a-J23119-soxR-T-psoxS-RBS-Mfp5-T7 Sequencing results

4. Construction of NO-inducible plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7

Following the construction of the constitutive plasmid pET29a-J23119-RBS-Mfp53-T7, we fused the NO-inducible
Mfp3 and Mfp5 using a double repeat of GGGGS as a flexible linker. We selected the SoxR/SoxS promoter and
*Escherichia coli* Nissle 1917 as the promoter element and chassis cell, respectively. This led to the
design
and construction of the plasmid pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7. We used *Escherichia coli*
Nissle 1917
as the chassis cell, with *Escherichia coli* DH5α assisting in the construction. The final sequencing
results
confirmed the successful construction of pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp53-T7.

![](https://static.igem.wiki/teams/5322/t-imgs-2/61-pcr-mfp28.png)

##### M：DL5000 DNA Marker 1-7：EcN Mfp53 Colony(1333bp) EcN pET29a-J23119-soxR-T-psoxS-RBS-Mfp53-T7 Colony PCR gel image

![](https://static.igem.wiki/teams/5322/t-imgs-2/62-cexu-mfp2.png)

##### pET29a-J23119-soxR-T-psoxS-RBS-Mfp53-T7 Sequencing results

5. Protein characterization of constitutive plasmids pET29a-J23119-RBS-Mfp3-T7 and
pET29a-J23119-RBS-Mfp5-T7

The bacteria that have been successfully verified by colony PCR were sent to the company for sequencing, and
the bacteria that were sequenced correctly were cultured and subjected to Tricine-SDS-Page detection.

![](https://static.igem.wiki/teams/5322/t-imgs-2/29-sds-mfp1.png)

##### 1:Mfp5 Total Protein（9.8kDa） 2:Mfp5 Supernatant（9.8kDa） 3:Mfp5 precipitation（9.8kDa） 4:Mfp5 After purification（9.8kDa） Tricine-SDS-Page analysis of Mfp5

![](https://static.igem.wiki/teams/5322/t-imgs-2/30-sds-mfp2.png)

##### 1:Mfp3 Total Protein（6.5kDa） 2:Mfp3 Supernatant（6.5kDa） 3:Mfp3 precipitation（6.5kDa） 4:Mfp3 After purification（6.5kDa） Tricine-SDS-Page analysis of Mfp3

Due to the inability to determine the expression of Mfp3 and Mfp5 from the protein gel image, we conducted a
Western Blot for further verification.

![](https://static.igem.wiki/teams/5322/t-imgs-2/31-wb-mfp1.png)

##### 1:Mfp5 Total Protein（9.8kDa） 2:Mfp5 Supernatant（9.8kDa） 3:Mfp5 precipitation（9.8kDa） 4:Mfp5 After purification（9.8kDa） Western blot analysis of Mfp5

![](https://static.igem.wiki/teams/5322/t-imgs-2/32-wb-mfp2.png)

##### 1:Mfp3 Total Protein（6.5kDa） 2:Mfp3 Supernatant（6.5kDa） 3:Mfp3 precipitation（6.5kDa） 4:Mfp3 After purification（6.5kDa） Western blot analysis of Mfp3

The experimental results indicated that both Mfp3 and Mfp5 were expressed; however, the observed protein
sizes deviated from the theoretical values. The band for Mfp3 appeared higher, which may suggest the formation
of dimers or multimers. The band for Mfp5 also shifted upwards, likely due to its higher viscosity, which
slows the protein's mobility during electrophoresis.

6. Protein characterization of inducible plasmids pET29a-J23119-soxR-T-psoxS-RBS-Mfp3-T7 and
pET29a-J23119-soxR-T-psoxS-RBS-Mfp5-T7

Considering that the mussel proteins are applied in the field of mammalian intestinal inflammation, we
selected 37 °C, the temperature closest to body temperature, then sodium nitroprusside (SNP) was added to a final concentration of 100 μM for induction. The duration of induction emerged
as a critical factor for protein characterization. The recombinant strains
EcN-pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp3-T7 and EcN-pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp5-T7 were cultured to
OD600 = 0.8, after which 100 μM SNP was added for induction. Expression was induced for 16 hours in
the EcN
strains, and the proteins were analyzed using Tricine-SDS-PAGE.

![](https://static.igem.wiki/teams/5322/t-imgs-2/52-pcr-mfp23.png)

##### 1、After induction Mfp3 Total Protein 2、After induction Mfp3 Supernatant 3. After induction Mfp3 precipitation 4、After induction Mfp3 After purification Tricine-SDS-PAGE analysis of recombinantly expressed Mfp3 (6.5 kDa)

![](https://static.igem.wiki/teams/5322/t-imgs-2/53-pcr-mfp24.png)

##### 1、After induction Mfp5 Total Protein 2、After induction Mfp5 Supernatant 3、After induction Mfp5 precipitation 4、After induction Mfp5 After purification Tricine-SDS-PAGE analysis of recombinantly expressed Mfp5 (9.8 kDa)

The expression of Mfp3 and Mfp5 could not be determined based on the protein gel image, so we performed
Western Blot for further verification.

![](https://static.igem.wiki/teams/5322/t-imgs-2/54-wb-mfp3.png)

##### 1、After induction Mfp3 Total Protein 2、After induction Mfp3 Supernatant 3、After induction Mfp3 precipitation 4、After induction Mfp3 After purification Western blot analysis of recombinantly expressed Mfp3 (6.5 kDa)

![](https://static.igem.wiki/teams/5322/t-imgs-2/55-wb-mfp4.png)

##### 1、Not induced Mfp5 Total Protein 2、Not induced Mfp5 Supernatant 3、Not induced Mfp5 precipitation 4、Not induced Mfp5 After purification WB analysis of recombinant expression of Mfp5 (9.8 kDa)

The results indicated that both pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp5-T7 and
pET29a-J23119-SoxR-T-pSoxS-RBS-Mfp3-T7 were expressed after induction, demonstrating stable expression in an
NO environment. This confirms that our engineered bacteria can produce Mfp5 and Mfp3 in the high NO conditions
associated with intestinal inflammation, thereby repairing intestinal mucosa and regulating gut microbiota to
facilitate the treatment of colitis.

# PD-L1

Based on the descriptions of mouse PD-L1 in the NCBI database (ADK70951.1, ADK70950.1, Q9NZQ7.1, AAH66841.1),
the functional domain of PD-L1 spans from amino acid 20 to amino acid 130 within the 290 amino acid sequence,
corresponding to the immunoglobulin variable (IgV) domain (CD20947). To ensure that the functionality of the
truncated protein is not adversely affected, we retained the first 1-19 amino acids and the last 131-150 amino
acids, aiming to preserve the integrity of the IgV domain as much as possible. Subsequently, we utilized
AlphaFold2, developed by Google DeepMind, to predict the structural models of both the full-length PD-L1 and
the truncated PD-L1 proteins, yielding the following prediction results.

![](https://static.igem.wiki/teams/5322/t-imgs/pd-l1-all.jpg)
![](https://static.igem.wiki/teams/5322/t-imgs/pd-l1.jpg)

Based on the predictions from AlphaFold 2, it was determined that the main functional domain of the truncated
PD-L1 remained unaffected. Consequently, we designed an expression system for the PD-L1 functional domain:
pET29a-J23119-RBS-PD-L1 (functional domain)-T7.

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5322/gif/alphafold2-pd-l1-model1.gif) | ![](https://static.igem.wiki/teams/5322/gif/full/alphafold2-pd-l1-model1.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/alphafold2-pd-l1-model2.gif) | ![](https://static.igem.wiki/teams/5322/gif/full/alphafold2-pd-l1-model2.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/alphafold2-pd-l1-model3.gif) | ![](https://static.igem.wiki/teams/5322/gif/full/alphafold2-pd-l1-model3.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/alphafold2-pd-l1-model4.gif) | ![](https://static.igem.wiki/teams/5322/gif/full/alphafold2-pd-l1-model4.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/alphafold2-pd-l1-model5.gif) | ![](https://static.igem.wiki/teams/5322/gif/full/alphafold2-pd-l1-model5.gif) |

Subsequently, Google released AlphaFold 3, which we utilized to further predict the protein structure. We
also conducted molecular docking to demonstrate that the truncated PD-L1 can successfully bind to PD-1 and
exert its effects. The results are shown in the following figure.

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5322/gif/pdl1/full/pd-l1-alpha3-model1.gif) | ![](https://static.igem.wiki/teams/5322/gif/full/alphafold2-pd-l1-model1.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/pdl1/full/pd-l1-alpha3-model2.gif) | ![](https://static.igem.wiki/teams/5322/gif/pdl1/half/pd-l1-alpha3-model2.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/pdl1/full/pd-l1-alpha3-model3.gif) | ![](https://static.igem.wiki/teams/5322/gif/pdl1/half/pd-l1-alpha3-model3.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/pdl1/full/pd-l1-alpha3-model4.gif) | ![](https://static.igem.wiki/teams/5322/gif/pdl1/half/pd-l1-alpha3-model4.gif) |
| ![](https://static.igem.wiki/teams/5322/gif/pdl1/full/pd-l1-alpha3-model5.gif) | ![](https://static.igem.wiki/teams/5322/gif/pdl1/half/pd-l1-alpha3-model5.gif) |

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5322/half-mouse/pd-l1-pd-1-model1.gif) | ![](https://static.igem.wiki/teams/5322/whole-mouse/pd-l1-pd-1-model1.gif) |
| ![](https://static.igem.wiki/teams/5322/half-mouse/pd-l1-pd-1-model2.gif) | ![](https://static.igem.wiki/teams/5322/whole-mouse/pd-l1-pd-1-model2.gif) |
| ![](https://static.igem.wiki/teams/5322/half-mouse/pd-l1-pd-1-model3.gif) | ![](https://static.igem.wiki/teams/5322/whole-mouse/pd-l1-pd-1-model3.gif) |
| ![](https://static.igem.wiki/teams/5322/half-mouse/pd-l1-pd-1-model4.gif) | ![](https://static.igem.wiki/teams/5322/whole-mouse/pd-l1-pd-1-model4.gif) |
| ![](https://static.igem.wiki/teams/5322/half-mouse/pd-l1-pd-1-model5.gif) | ![](https://static.igem.wiki/teams/5322/whole-mouse/pd-l1-pd-1-model5.gif) |

For the successful colony PCR results, we proceeded to inoculate the bacteria and cultured them at 37 °C, 220
rpm for 12-16 hours. Following this, plasmids were extracted and sent for sequencing. The sequencing results
are shown in the following figure.

![](https://static.igem.wiki/teams/5322/t-imgs/cexu-pet29a-j23119-rbs-pd-l1-functional-domain-t7-map.png)

According to the sequencing results, the plasmid construction was confirmed to be successful, and the plasmid
was transformed into EcN, completing the expression system setup in preparation for protein functionality
testing.

During the project, we considered that PD-L1 might be encapsulated by the mussel foot protein Mfp during its
expression and release, which could potentially reduce the therapeutic efficacy.

To address this issue, we proposed utilizing the surface display system Lpp-OmpA to present our passenger
protein PD-L1 on the outer membrane of *Escherichia coli*. This approach would facilitate easier binding
with
PD-1 and enhance the effectiveness of the interaction. Additionally, to confirm the surface display of PD-L1
on *E. coli*, we introduced a flexible protein linker between Lpp-OmpA and the PD-L1 functional domain,
as well
as a recognition and cleavage site for Tobacco Etch Virus (TEV) protease. By incubating the bacterial cells
with TEV protease, PD-L1 can be cleaved from OmpA. If the PD-L1 functional domain is detected in the
supernatant, this would indirectly demonstrate the success of the surface display.

![]( https://static.igem.wiki/teams/5322/t-img-3/1.png )

![]( https://static.igem.wiki/teams/5322/t-img-3/2.png )

We amplified the target fragment and vector, purified the products, and performed homologous recombination.
The mixture was then transformed into *Escherichia coli* DH5α and incubated at 37 °C on inverted plates
for 16
hours. Subsequently, colony PCR was conducted on single colonies, with the results shown in the following
figure.

![](https://static.igem.wiki/teams/5322/t-imgs/pcr-3.png)

For the successful colony PCR results, we inoculated the bacteria and cultured them at 37 °C, 220 rpm for
12-16 hours. Following this, plasmids were extracted and sent for sequencing. The sequencing results are
displayed in the following figure.

![](https://static.igem.wiki/teams/5322/t-imgs/cexu-pet29a-j23119-lpp-ompa-giss-tevsite-pd-l1-functional-domain-t7-map.png)

The sequencing results confirmed the successful construction of the plasmid, which was then transformed into
EcN, completing the expression system setup. To evaluate the expression system, we cultured the samples in a
250 mL shake flask for 16 hours, followed by ultrasonic lysis, passage through a nickel column, and protein
purification. The samples were analyzed using SDS-PAGE, with the results shown below.

![](https://static.igem.wiki/teams/5322/t-imgs/sds-1.png)

'

##### 1: total protein; 2: supernatant; 3: precipitate; 4: after purification; 5: discarded after passing through nickel column; 6: before ultrafiltration; 7: after ultrafiltration; M: Seville pre-stained protein marker

Protein gel validation did not confirm protein expression; therefore, we hypothesized that the possible
reason for this could be the relatively high molecular weight of the Lpp-OmpA-PD-L1 protein, resulting in low
expression levels in the bacteria. To address this issue, we opted to use Western Blot (WB), which has higher
sensitivity and can detect proteins at the ng level. After a period of study, we mastered the operation and
precautions associated with WB.

We first performed WB experiments directly on total protein, and the results are shown in the following
figure.

![](https://static.igem.wiki/teams/5322/t-imgs/wb-1.png)

After confirming the presence of bands in the total protein, we validated the correct expression of the
target protein. The protein was then purified and subjected to ultrafiltration, followed by direct Western
Blot verification. The results are shown in the following figure.

![](https://static.igem.wiki/teams/5322/t-imgs/wb-2.png)

##### 1: total protein; 2: supernatant; 3: precipitate; 4: after purification; 5: discarded after passing through nickel column; 6: before ultrafiltration; 7: after ultrafiltration; M: Seville pre-stained protein marker

From the WB results, it is clear that a significant portion of Lpp-OmpA is present in the pellet due to its
hydrophobic nature, which aligns with theoretical expectations. However, we also observed the occurrence of
multiple bands in the blot. Upon analysis, we speculated that this might be due to the degradation of membrane
proteins at 100°C, leading to protein fragmentation.

To investigate this further, we designed an experiment based on previous studies, aiming to determine the
optimal temperature for membrane protein denaturation in WB assays. The results are presented below.

![](https://static.igem.wiki/teams/5322/t-imgs/wb-3.png)

Based on the WB results, we observed that under the denaturation conditions of 30°C for 30 minutes, the 34
kDa protein was minimally degraded. Therefore, we determined this denaturation condition as the optimal
choice. With this, we have confirmed the expression of the target protein, and the next step is to validate
the success of the surface display system.

We incubated 200 mL of bacterial culture grown for 12-16 hours with 50 U of TEV protease at 4°C for 24 hours.
After centrifugation, the supernatant was collected for ultrafiltration concentration, and the bacterial cells
were lysed for later use. We subjected both samples to WB experiments under the denaturation conditions of
30°C for 30 minutes for verification.

![](https://static.igem.wiki/teams/5322/t-imgs/wb-4.png)

# Cracking module and safety module

To achieve optimal results in bacterial therapy, we designed a pLuxI-regulated PhiX174E lysis system,
pLuxI-RBS-PhiX174E-rrnB T1. When the engineered bacteria detect NO, they synthesize the mussel foot protein
Mfp, facilitating the attachment of the probiotic EcN to the site of enteritis, thereby establishing a
dominant microbial community. However, since the therapeutic protein SOD cannot cross membranes, we employed
the principle of quorum sensing among bacteria. As the population density of EcN increases at the site of
enteritis, this triggers the quorum sensing system to express the PhiX174E protein, ultimately leading to
bacterial lysis and the release of the therapeutic protein, thereby achieving optimal therapeutic efficacy.

![](https://static.igem.wiki/teams/5322/t-img-3/4.png)

##### pLuxⅠ-RBS-PhiX174E-rrnB T1

Biosafety has always been a major concern for biologists. To protect biological safety and avoid potential
issues such as gene contamination, we designed the pDawn-MazF safety module. pDawn is a blue-light-inducible
promoter that can activate the expression of downstream genes under natural light or a single blue light
source. MazF is an RNAse that effectively degrades bacterial mRNA, thereby inhibiting bacterial growth and
other life activities.

To implement this, we designed the plasmid pET29a-LuxI-PhiX174E-pDawn-MazF, as illustrated in the
accompanying figure.

![]( https://static.igem.wiki/teams/5322/t-imgs/luxi-phix174e-pdawn-mazf-map.png)

The plasmid was constructed to achieve the effects of lysis and suicide. We successfully constructed the
plasmid. At the same time, in order to explore the performance of the group response system, we measured the
group response growth curve to further prove the feasibility of the project. The results are shown in the
figure below.

![]( https://static.igem.wiki/teams/5322/t-imgs/phix174e.png)

{% endblock %}