iGEM 2025 — Team lzu


{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}**Protecting Ancient Murals from Microbial Destruction** - Team LZU 2025{% endblock %}
{% block styles %}
{% endblock %}
{% block page\_content %}

[Antibacterial Module](#sec-antibacterial)
[Pigment Degradation Module](#sec-pigment)
[pH-Responsive Switch](#sec-ph)
[Suicide Module](#sec-suicide)

# Engineering

## Antibacterial Module

### Design

To ensure that our project can inhibit the growth of fungi on murals, we attempted to find an antimicrobial
peptide with antifungal properties. We found the antimicrobial peptide Pn-AMP1 (AP00270) in the
Antimicrobial Peptide Database. The database indicates that Pn-AMP1 is extracted from *Pharbitis nil*
and may disrupt the cell membranes of fungi, having inhibitory effects on *Fusarium oxysporum* and
*Saccharomyces cerevisiae*. However, the majority of the fungi we extracted were from the genera
*Penicillium* and *Aspergillus*. Therefore, it is necessary to verify the inhibitory effect of
Pn-AMP1 on these two types of fungi.

In order to enable our engineered bacteria to inhibit the growth of bacteria on the murals, we introduced
GRP-1 into our engineered bacteria. GRP1 is an antibacterial peptide extracted from *Limosilactobacillus
fermentum*. It shows a good inhibitory effect on *Staphylococcus aureus* and *Salmonella
Paratyphi B*. However, the inhibitory effect of this substance on the microorganisms on the murals
still requires further verification.

### Build

We entrusted Tsingke Biotechnology Co., Ltd. to synthesize the complete nucleotide sequence of
[BBa\_259HIEMB](https://registry.igem.org/parts/bba-259hiemb) and
[BBa\_25XI83ND](https://registry.igem.org/parts/bba-25xi83nd), and constructed pET-30a(+) -
BBa\_259HIEMB and pET-30a(+) - BBa\_25XI83ND for the subsequent
testing work.

### Test

Three species of fungi and six species of bacteria were isolated from the mural samples. Antimicrobial
susceptibility testing via the zone of inhibition method was conducted on each microbial strain, followed by
simulated conservation tests on mural specimens pre-colonized by these microorganisms.

![eng1](https://static.igem.wiki/teams/5550/pic/project/eng1-new.webp)

![eng1.2](https://static.igem.wiki/teams/5550/pic/project/eng1-2-new.webp)

Fig 1 Microorganisms isolated from mural paintings

The inhibition zone experiment demonstrated that GRP1 has a good inhibitory effect on bacteria, and PnAMP1
has a good inhibitory effect on fungi.

![eng2](https://static.igem.wiki/teams/5550/pic/project/eng2-new.webp)

Fig 2 The size of the inhibition zone produced by GRP1 against different bacteria

![eng3](https://static.igem.wiki/teams/5550/pic/project/eng3.webp)

Fig 3 The morphology of the inhibition zones of different bacteria
a: Bacteria
4.1, b: Bacteria 6.1, c: Bacteria 7.1, d: Bacteria 8.1
The white arrow indicates the experimental group.

![eng4](https://static.igem.wiki/teams/5550/pic/project/eng4-new.webp)

Fig 4 The size of the inhibition zone produced by PnAMP1 against different fungi

![eng5](https://static.igem.wiki/teams/5550/pic/project/eng5-new.webp)

Fig 5 The morphology of the inhibition zones of different fungi
a: Fungal 3.1, b:
Fungal 4.1, c: Fungal 5.1

In order to test the survival of our engineered bacteria on the mural, we obtained simulated mural samples from
Dr. Wu Fasi's laboratory.

After inoculation with BL21-BBa\_25XI83ND/BBa\_259HIEMB and mural microbes, no distinct microbial plaques were
observed on the simulated mural (Fig 6a). However, murals not inoculated with the antimicrobial engineered
bacteria displayed large plaques and exhibited significant damage (Fig 6b), demonstrating that the antimicrobial
engineered bacteria can prevent microbial growth on the murals. Murals with plaques were treated with the
engineered bacteria. Calcein-AM/PI staining revealed a significant number of viable bacteria on the untreated
murals (Fig 6c). However, 24 h after treatment with the engineered bacteria, widespread microbial cell death was
observed on the murals (Fig 6d), demonstrating that the antimicrobial engineered bacteria can effectively treat
microbial infestations in the murals. Figure 7 shows that the microbial mortality rate on the murals not treated
with the antimicrobial engineered bacteria was 30.03%, while that on the murals treated with the engineered
bacteria reached 68.23%.

![eng6](https://static.igem.wiki/teams/5550/pic/project/eng6-new.webp)

Fig 6 Verification of the inhibitory effect of the antimicrobial engineered bacteria
BL21-BBa\_25XI83ND/BBa\_259HIEMB on mural microorganisms.
(a) Simultaneous inoculation of antimicrobial engineered bacteria and mural microorganisms; (b) Inoculation of
mural microorganisms alone. (c) Calcein-AM/PI staining results after 5 d of inoculation with mural
microorganisms; (d) staining results after 5 d of incubation and 1 d of treatment with engineered bacteria.
Green represents viable cells, and red represents dead cells.

![eng6](https://static.igem.wiki/teams/5550/pic/project/eng-new.webp)

Fig 7 Mortality of microorganisms on simulated murals before and after treatment with
antibacterial engineered bacteria.

### Learn

The results indicate that our engineered bacteria largely fulfilled the intended objectives. In subsequent
work, we will explore adjustments to the types and expression levels of antimicrobial peptides to achieve
enhanced efficacy.

## Pigment Degradation Module

### Cycle 1

#### Design

In order to enable our engineered bacteria to degrade melanin and carotenoids, we introduced the laccase
CueO and dye decolorizing peroxidase DyP into our engineered bacteria. CueO can degrade a variety of
substrates, including melanin. DyP can utilize hydrogen peroxide to degrade various substrates including
carotenoids.

#### Build

We entrusted Beijing Tsingke Biotech Co., Ltd. to synthesize the complete sequence and construct pET-30a(+) -
[BBa\_256VH953](https://registry.igem.org/parts/bba-256vh953) and pET-30a(+) -
[BBa\_25OTZVL1](https://registry.igem.org/parts/bba-25otzvl1)
for subsequent experiments.

#### Test

As shown in Fig 8, the degradation rate of melanin by the engineered bacteria without CueO protein expression
was 10.08%. However, the degradation rate of melanin by the engineered bacteria expressing CueO increased to
29.67% (Fig 8b)..

![eng7](https://static.igem.wiki/teams/5550/pic/project/eng8-new.webp)

Fig 8 Melanin degradation by CueO-expressing bacteria.
(a) Melanin standard curve; (b) Melanin degradation rate by the engineered bacteria expressing CueO.
CueO: engineered bacteria without induced expression; E-CueO: engineered bacteria with induced expression.

As shown in Fig 9, the degradation rate of β-carotene by the engineered bacteria without DyP protein was
11.99%, while that by the engineered bacteria expressing DyP increased to 35.32% (Fig 9b).

![eng8](https://static.igem.wiki/teams/5550/pic/project/eng9-new.webp)

Fig 9 β-carotene degradation by DyP-expressing bacteria.
(a) β-carotene standard curve; (b) β-carotene degradation rate by the engineered bacteria expressing DyP.
DyP: engineered bacteria without induced expression; E-DyP: engineered bacteria with induced expression.

#### Learn

Our constructed pET-30a-CueO and pET-30a-Dyp enzymes were effective at degrading melanin and β-carotene,
respectively, but the degradation efficiency was low, potentially limiting their practical application. Based on
literature analysis, this may be due to the low efficiency of the functional enzymes in capturing the pigments,
necessitating the addition of a pigment-capturing protein.

### Cycle 2

#### Design

Because CueO and DyP alone have limited effectiveness in pigment degradation, we introduced a modified
scaffoldin (BBa\_25TLZ7K5) into the engineered bacteria. This scaffoldin binds to CueO and DyP via Dockerin,
enhancing the degradation of melanin and β-carotene.

![eng8](https://static.igem.wiki/teams/5550/pic/project/eng10-new.webp)

Fig 10 Principle of Functional Complex Composition

#### Test

As shown in Fig 11, after incubation with melanin, the absorbance of melanin decreased (Fig 11a), and the color
of melanin changed from dark blue to lighter (Fig 11c). The melanin degradation efficiency increased to 59.07%,
significantly higher than that of E-CueO (Fig 11b). The CueO-scaffoldin engineered bacteria further enhanced the
melanin degradation efficiency of CueO.

![eng8](https://static.igem.wiki/teams/5550/pic/project/eng11-new.webp)

Fig 11 Melanin degradation by CueO-scaffoldin-expressing bacteria.
(a) UV-visible spectrum of melanin after degradation by CueO-scaffoldin-expressing bacteria; (b) Melanin
degradation efficiency by the bacteria; (c) Visual representation of melanin decolorization by
CueO-scaffoldin-expressing bacteria.

As shown in Fig 12, after incubation with β-carotene, the absorbance of β-carotene decreased (Fig 12a), and the
color of β-carotene changed from dark yellow to light (Fig 12c). The degradation efficiency of β-carotene
increased to 49.57%, significantly higher than that of E-DyP (Fig 12b). The DyP-scaffoldin-expressing bacteria
further enhanced the degradation efficiency of DyP for β-carotene.

![eng8](https://static.igem.wiki/teams/5550/pic/project/eng12-new.webp)

Fig 12 β-carotene degradation by DyP-scaffoldin-expressing bacteria.
(a) UV-visible spectrum of β-carotene after degradation by DyP-scaffoldin-expressing bacteria; (b) β-carotene
degradation efficiency by the bacteria; (c) Visualization of β-carotene decolorization by
DyP-scaffoldin-expressing bacteria.

## pH-Responsive Switch

### Design

To enable our engineered bacteria to regulate the synthesis of products according to pH changes, we need to
design a regulatory element. We used the Pasr promoter, which can significantly enhance expression strength
in acidic environments. We combined the Pasr promoter (BBa\_K4716000), TetR (BBa\_K5237005), and PtetO3
promoter (BBa\_K3606007) to enable the transcription of some genes to be turned off while others are turned
on at different pH levels.

### Build

We constructed BBa\_25WCSXND and introduced fluorescent proteins downstream of the two promoters as reporter
proteins. We tested the function of the regulatory element by detecting changes in fluorescence intensity.

![eng12](https://static.igem.wiki/teams/5550/pic/project/eng12.webp)

Fig 13 Design of test plan for BBa\_25WCSXND

### Test

We cultivated our engineered bacteria at different pH levels and used an enzyme detector to measure the
trend of fluorescence protein fluorescence intensity over time. The increase and decrease in fluorescence
intensity reflect the activation and deactivation of fluorescent protein synthesis.

![eng13](https://static.igem.wiki/teams/5550/pic/project/eng13.webp)

Fig 14 Changes in fluorescence intensity of EGFP at different pH values.
Eight
gradients were set up within the pH range of 4 to 7 for the experiment. The fluorescence intensity of EGFP
showed a downward trend over time at pH = 4.25 and pH = 5, and an upward trend at pH = 5.5. The trends were
not obvious at other pH values.

![eng14](https://static.igem.wiki/teams/5550/pic/project/eng14.webp)

Fig 15 Changes in fluorescence intensity of mCherry at different pH values.
Eight
gradients were set up within the pH range of 4 to 7 for the experiment. The fluorescence intensity of
mCherry showed an upward trend over time at pH values ranging from 4 to 4.75, and a downward trend at pH 5.
The trend was not obvious at other pH values.

### Learn

Our part met the expected results under certain conditions when the pH was less than 5.5. However, the
performance of Part under other conditions is confusing. The fluorescence intensities of the two fluorescent
proteins did not show any significant increase or decrease trend when the pH value was greater than 5.5. The
fluorescence intensity of mCHerry decreased perplexingly within the pH range of 4.5 to 5. We believe that it
is possible that the transcriptional activation strength of PtetO3 at pH > 5.5 is not optimal, and the
decrease of mCherry might have other causes. These require further experimentation for verification and
improvement.

## Suicide Module

### Cycle 1

#### Design

To prevent genetic leakage beyond the repair materials, we designed a suicide module. When the engineered
*E. coli* carrying this plasmid detach from the repair materials, the suicide system can be activated
to prevent genetic leakage.

We employed the extracellular nuclease gene *nuc* from *Serratia* as the suicide gene. The
signal peptide sequence was removed to enable the nuclease to degrade DNA within the chassis *E.
coli*, thereby inducing bacterial suicide. The suicide gene is driven by a moderately weak *E.
coli* constitutive promoter J23105 (BBa\_ J23105), coupled with the RBS sequence J61100 (BBa\_ J61100).
Downstream of the promoter, we incorporated the operator sites O1, O2, and O3 from the *E. coli* λ
phage. These operator sites can bind to the λ phage CI protein（BBa\_C0051）to repress gene expression. The CI
protein is expressed under the control of the arabinose-inducible promoter pBAD（BBa\_I13453）. In the absence
of arabinose, this promoter forms a specific structure that inhibits transcription. When arabinose is
present, the araC protein binds to arabinose and promotes transcription of the downstream gene.

In the presence of arabinose, the PBAD promoter successfully binds RNA polymerase and initiates
transcription, expressing CI protein to suppress the expression of the nuclease *nuc*. In the absence
of arabinose, the expression of *nuc* is no longer repressed by CI protein, ultimately leading to
genomic DNA degradation and bacterial suicide. This design allows us to add arabinose to the repair
materials, enabling normal bacterial function. Upon removal of the repair materials from the mural, the
engineered bacteria activate the suicide mechanism, effectively preventing genetic leakage.

#### Build

We obtained the suicide module flanked by biobrick prefix and suffix through full-sequence synthesis and
transformed it into *E. coli* strains DH5α and BL21. The functionality of the suicide plasmid DNA was
then assessed.

#### Test

The pSB1A3 plasmid containing the suicide module was transformed into *E. coli* strains DH5α and
BL21, respectively. The bacteria were plated on LB solid medium with varying concentrations. However, no
suicide phenomenon was observed in the *E. coli*.

![eng15](https://static.igem.wiki/teams/5550/pic/project/eng15.webp)

Fig 16 Growth of *E. coli* carrying the suicide plasmid gene under culture
media with different arabinose concentrations
Five different arabinose concentration gradients were set
(0, 0.01%, 0.1%, 0.5%, and 1%). After transforming 1 μL of the suicide plasmid, 100 μL of bacterial
suspension was plated per dish. All plates contained ampicillin. The number of *E. coli* colonies
grown in media with different concentrations showed no significant concentration-dependent differences.

To investigate the specific reasons for the failure, we designed a forward primer with a PstI restriction
site at the terminus and a reverse primer with an EcoRI restriction site. The nuclease gene carrying the
J61100 RBS was cloned individually and ligated into the pUC19 vector using restriction enzyme digestion and
ligation. Upon inducing protein expression with IPTG, no significant bactericidal activity was observed.

![eng16](https://static.igem.wiki/teams/5550/pic/project/eng16.webp)

Fig 17 Growth of *E. coli* with the cloned nuclease gene under conditions
with and without IPTG induction
*E. coli* transformed with the pUC19 vector plasmid carrying the
nuclease gene was induced using 50 mM IPTG. No significant difference was observed between the two groups.

#### Learn

Our suicide plasmid failed to meet the requirements, as it did not induce bacterial suicide under growth
conditions lacking arabinose. Additionally, the isolated expression of the nuclease gene alone did not
result in engineered bacterial suicide. This necessitates further refinement of the suicide plasmid.

### Cycle 2

#### Design

Since the initial nuclease did not achieve the desired bactericidal effect, we sought an alternative
suicide gene and identified the protein MazF. MazF is a toxin protein in the *E. coli*
toxin-antitoxin system, functioning as a sequence-specific endoribonuclease that cleaves single-stranded RNA
at ACA sequences to induce bacterial suicide. We plan to use the method of restriction and ligation to link our
regulatory elements with the MazF suicide gene, incorporating biobrick interfaces at both ends for convenient
vector integration.

#### Build

We performed full gene synthesis of the MazF gene carrying an RBS and ligated it into the pET-30a vector.
The functionality was tested via IPTG induction. Subsequently, fusion PCR was employed to connect it with
the regulatory elements, constructing a suicide module carrying the MazF toxin gene.

#### Test

Induction with 50 mM IPTG to express the MazF protein demonstrated effective bactericidal activity under
induction conditions.

![eng17](https://static.igem.wiki/teams/5550/pic/project/eng17.webp)

Fig 18 Growth of *E. coli* carrying the MazF gene under conditions with
and without IPTG induction
*E. coli* transformed with the pET-30a vector plasmid carrying the MazF
gene was induced using 50 mM IPTG. No *E. coli* growth was observed on the IPTG-containing plates.

To integrate MazF into the suicide module, we employed restriction enzyme digestion and ligation for
modification. We separately cloned the upstream regulatory sequence of the suicide module and the MazF gene
sequence. The upstream regulatory sequence was first ligated into the pSB1A3 vector, followed by restriction
enzyme digestion and ligation to incorporate the MazF toxin protein gene at the end of the suicide
regulatory sequence. This process successfully yielded the modified vector.In the presence of 1% arabinose,
the engineered bacteria exhibited normal growth. However, in the absence of arabinose, the cell death
program was successfully executed, and no bacterial colonies grew on the culture medium.

![eng18](https://static.igem.wiki/teams/5550/pic/project/eng18.webp)

Fig 19 Growth of *E. coli* carrying the suicide module on LB solid medium
with 1% arabinose and without arabinose

Subsequently, to test the survival of the engineered bacteria on the mural surface, we obtained mural samples
from Dr. Wu Fasi's laboratory. E. coli carrying EGFP was sprayed onto the mural surface, and the number of
surviving cells was observed under a microscope. The experiment demonstrated that the majority of the engineered
bacteria failed to survive on the mural surface (Fig 20), aligning with our expectations.

![eng19](https://static.igem.wiki/teams/5550/pic/project/eng20-new.webp)

Fig 20 Survival status of the engineered bacteria on the mural at days 0, 5, and
10

#### Learn

The MazF gene effectively achieves the intended suicide effect and, after being incorporated into the
suicide module, fully meets our requirements by activating the death program in the absence of arabinose.
Furthermore, it cannot survive independently on the mural.

{% endblock %}


{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}**Protecting Ancient Murals from Microbial Destruction** - Team LZU 2025{% endblock %}
{% block styles %}
{% endblock %}
{% block page\_content %}

[Antibacterial Module](#sec-antibacterial) [Pigment Degradation](#sec-pigment) [pH-Responsive Switch](#sec-ph) [Suicide Module](#sec-suicide)

# Results

## Antibacterial Module

### The microorganisms isolated from the murals

Three species of fungi and six species of bacteria were isolated from the mural samples. Antimicrobial
susceptibility testing via the zone of inhibition method was conducted on each microbial strain, followed by
simulated conservation tests on mural specimens pre-colonized by these microorganisms.

![result1](https://static.igem.wiki/teams/5550/pic/wetlab/result1-new3.webp)

![result2](https://static.igem.wiki/teams/5550/pic/wetlab/result2-new3.webp)

Fig 1 Microorganisms isolated from mural paintings

### Bacteriostatic zone experiment

After testing, GRP1 showed varying degrees of inhibitory effects on Bacillus 4-1, Priestia 6-1, Priestia 7-1,
Priestia 8-1

![result3](https://static.igem.wiki/teams/5550/pic/wetlab/result3-new.webp)

Fig 2 The size of the inhibition zone produced by GRP1 against
different bacteria

![result4](https://static.igem.wiki/teams/5550/pic/wetlab/result4.webp)

Fig 3 The morphology of the inhibition zones of different bacteria
a: Bacillus 4-1, b: Priestia 6-1, c: Priestia 7-1, d: Priestia 8-1
The white arrow indicates the experimental group.

After testing, Pn-AMP1 showed varying degrees of inhibitory effects on
Scopulariopsis 4-1, Scopulariopsis5-1, Aspergillus 3-1.

![result5](https://static.igem.wiki/teams/5550/pic/wetlab/result5-new2.webp)

Fig 4 The size of the inhibition zone produced by PnAMP1 against
different fungi

![result6](https://static.igem.wiki/teams/5550/pic/wetlab/result6.webp)

Fig 5 The morphology of the inhibition zones of different fungi
a: Aspergillus 3-1, b: Scopulariopsis 4-1, c: Scopulariopsis5-1

### Test on mural briquette

After inoculation with BL21-
[BBa\_25XI83ND](https://registry.igem.org/parts/bba-25xi83nd)/
[BBa\_259HIEMB](https://registry.igem.org/parts/bba-259hiemb) and mural microbes, no distinct
microbial plaques were
observed on the simulated mural (Fig 6a). However, murals not inoculated with the antimicrobial engineered
bacteria displayed large plaques and exhibited significant damage (Fig 6b), demonstrating that the
antimicrobial engineered bacteria can prevent microbial growth on the murals. Murals with plaques were treated
with the engineered bacteria. Calcein-AM/PI staining revealed a significant number of viable bacteria on the
untreated murals (Fig 6c). However, 24 h after treatment with the engineered bacteria, widespread microbial
cell death was observed on the murals (Fig 6d), demonstrating that the antimicrobial engineered bacteria can
effectively treat microbial infestations in the murals. Figure 7 shows that the microbial mortality rate on the
murals not treated with the antimicrobial engineered bacteria was 30.03%, while that on
the murals treated with the engineered bacteria reached 68.23%.

![result7](https://static.igem.wiki/teams/5550/pic/wetlab/result7-new.webp)

Fig 6 Verification of the inhibitory effect of the antimicrobial engineered bacteria
BL21-BBa\_25XI83ND/BBa\_259HIEMB on mural microorganisms.
(a) Simultaneous inoculation of antimicrobial engineered bacteria and mural microorganisms; (b) Inoculation of
mural microorganisms alone. (c) Calcein-AM/PI staining results after 5 d of inoculation with mural
microorganisms; (d) staining results after 5 d of incubation and 1 d of treatment with engineered bacteria.
Green represents viable cells, and red represents dead cells.

![result7.1](https://static.igem.wiki/teams/5550/pic/wetlab/result7-1.webp)

Fig 7 Mortality of microorganisms on simulated murals before and after treatment with antibacterial engineered
bacteria.

## Pigment Degradation Module

As shown in Fig 8, after incubation with melanin, the absorbance of melanin decreased (Figure 8a), and the
color of melanin changed from dark blue to lighter (Fig 8c). The melanin degradation efficiency increased to
59.07%, significantly higher than that of E-CueO (Fig 8b). The CueO-scaffoldin engineered bacteria further
enhanced the melanin degradation efficiency of CueO.

![result11](https://static.igem.wiki/teams/5550/pic/wetlab/result8-new2.webp)

Fig 8 Melanin degradation by CueO-scaffoldin-expressing bacteria.
(a) UV-visible spectrum of melanin after degradation by CueO-scaffoldin-expressing bacteria; (b) Melanin
degradation efficiency by the bacteria; (c) Visual representation of melanin decolorization by
CueO-scaffoldin-expressing bacteria.

As shown in Fig 9, after incubation with β-carotene, the absorbance of β-carotene decreased (Fig 9a), and the
color of β-carotene changed from dark yellow to light (Fig 9c). The degradation efficiency of β-carotene
increased to 49.57%, significantly higher than that of E-DyP (Fig 9b). The DyP-scaffoldin-expressing bacteria
further enhanced the degradation efficiency of DyP for β-carotene.

![result12](https://static.igem.wiki/teams/5550/pic/wetlab/result9-new2.webp)

Fig 9 β-carotene degradation by DyP-scaffoldin-expressing bacteria.
(a) UV-visible spectrum of β-carotene after degradation by DyP-scaffoldin-expressing bacteria; (b) β-carotene
degradation efficiency by the bacteria; (c) Visualization of β-carotene decolorization by
DyP-scaffoldin-expressing bacteria.

## pH-Responsive Switch

To enable our Escherichia coli strain to regulate product synthesis in
response to pH variations, we engineered a genetic regulatory module.
The Pasr promoter, which exhibits significantly enhanced expression
under acidic conditions, was employed. By integrating Pasr with the TetR
gene and PtetO3 operator, we constructed a system capable of selectively
activating or repressing transcription of specific genes at different pH
levels. To evaluate the regulatory performance of this module,
fluorescent proteins were incorporated at both termini as
transcriptional reporters, with fluorescence intensity serving as the
metric for transcriptional control. The engineered bacterial strains
were cultured under varying pH conditions, and time-dependent changes in
fluorescence intensity of distinct fluorescent proteins were quantified
using a microplate reader. The observed increase and decrease in
fluorescence intensity directly indicated the activation and repression
of fluorescent protein synthesis, respectively.

![result15](https://static.igem.wiki/teams/5550/pic/wetlab/result15.webp)

Fig 10 Changes in fluorescence intensity of EGFP at different pH
values.
Eight gradients were set up within the pH range of 4 to 7 for the
experiment. The fluorescence intensity of EGFP showed a downward trend
over time at pH = 4.25 and pH = 5, and an upward trend at pH = 5.5. The
trends were not obvious at other pH values.

![result16](https://static.igem.wiki/teams/5550/pic/wetlab/result16.webp)

Fig 11 Changes in fluorescence intensity of mCherry at different pH
values.
Eight gradients were set up within the pH range of 4 to 7 for the
experiment. The fluorescence intensity of mCherry showed an upward trend
over time at pH values ranging from 4 to 4.75, and a downward trend at
pH 5. The trend was not obvious at other pH values.

As shown in Fig 10 and Fig 11, our regulatory elements meet our design
goals within the pH range of 4 to 5.5, which can be concluded from the
changes in fluorescence intensity.

## Suicide Module

To prevent genetic leakage outside the repair materials, we designed a
suicide module. In the presence of arabinose, this module expresses the
CI protein, which binds to the O1, O2, and O3 sites upstream of the
nuclease gene's RBS, inhibiting nuclease expression. In the absence of
arabinose, the CI protein cannot activate transcription, allowing the
nuclease gene to be expressed and kill the engineered E. coli. We
synthesized the suicide module using full gene synthesis, inserted it
into the pSB1A3 vector, and transformed it into E. coli (Fig 12). We
tested different arabinose concentration gradients, but the number of E.
coli colonies did not decrease with reduced arabinose concentrations,
nor was there a significant difference compared to the no-arabinose
control group. These results indicate that the initially designed
suicide module did not meet our requirements.

![result42](https://static.igem.wiki/teams/5550/pic/wetlab/result42.webp)

Fig 12 Growth of E. coli carrying the suicide plasmid gene under
culture media with different arabinose concentrations
Five different arabinose concentration gradients were set (0, 0.01%,
0.1%, 0.5%, and 1%). After transforming 1 μL of the suicide plasmid, 100
μL of bacterial suspension was plated per dish. All plates contained
ampicillin. The number of E. coli colonies grown in media with different
concentrations showed no significant concentration-dependent
differences.

To investigate the specific reasons for the
failure, we designed a forward primer with a PstI restriction site at
the terminus and a reverse primer with an EcoRI restriction site. The
nuclease gene carrying the J61100 RBS was cloned individually and
ligated into the pUC19 vector using restriction enzyme digestion and
ligation. Upon inducing protein expression with IPTG, no significant
bactericidal activity was observed (Fig 13).

![result43](https://static.igem.wiki/teams/5550/pic/wetlab/result43.webp)

Fig 13 Growth of E. coli with the cloned nuclease gene under
conditions with and without IPTG induction
E. coli transformed with the pUC19 vector plasmid carrying the nuclease
gene was induced using 50 mM IPTG. No significant difference was
observed between the two groups.

Since the initial nuclease did not achieve
the desired bactericidal effect, we sought an alternative suicide gene
and identified the protein MazF. MazF is a toxin protein in the E. coli
toxin-antitoxin system, functioning as a sequence-specific
endoribonuclease that cleaves single-stranded RNA at ACA sequences to
induce bacterial suicide. We synthesized the MazF toxin gene carrying an
RBS sequence and ligated it into the pET-30a vector. Induction with 50
mM IPTG demonstrated effective bactericidal activity under induction
conditions (Fig 14).

![result50](https://static.igem.wiki/teams/5550/pic/wetlab/result50.webp)

Fig 14 Growth of E. coli carrying the MazF gene under conditions with
and without IPTG induction
E. coli transformed with the pET-30a vector plasmid carrying the MazF
gene was induced using 50 mM IPTG. No E. coli growth was observed on the
IPTG-containing plates.

After confirming that the MazF toxin protein could successfully kill E.
coli, we proceeded to integrate it into the suicide module. We employed
restriction enzyme digestion and ligation for modification. The upstream
regulatory sequence of the suicide module and the mazF gene were
separately cloned, with biobrick prefix and suffix interfaces added to
both ends. The upstream regulatory sequence was first ligated into the
pSB1A3 vector, followed by restriction enzyme digestion with PstI and
EcoRI and ligation of the vector and fragment, ultimately yielding the
successfully modified vector. Experimental validation showed that the
engineered bacteria exhibited normal growth in the presence of 1%
arabinose. However, in the absence of arabinose, the cell death program
was successfully executed, and no bacterial colonies grew on the culture
medium (Fig 15), indicating that the modified suicide module met our
requirements.

![result51](https://static.igem.wiki/teams/5550/pic/wetlab/result51.webp)

Fig 15 Growth of E. coli carrying the suicide module on LB solid
medium with 1% arabinose and without arabinose

Subsequently, to test the survival of the engineered bacteria on the
mural surface, we obtained mural samples from Dr. Wu Fasi's laboratory.
E. coli carrying EGFP was sprayed onto the mural surface, and the number
of surviving cells was observed under a microscope. The experiment
demonstrated that the majority of the engineered bacteria failed to
survive on the mural surface (Fig 16), aligning with our expectations.

![result52](https://static.igem.wiki/teams/5550/pic/wetlab/result52-new.webp)

Fig 16 Survival status of the engineered bacteria on the mural at day 0(a), day 5(b),
and day 10(c)

{% endblock %}