iGEM 2025 — Team xjtlu-china

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{% block title %}Diversity and inclusivity{% endblock %}
{% block lead %}Describe all the safety issues of your project.{% endblock %}
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Diversity and inclusivity

## Project Design

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## June

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## July

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## August

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## *Ulva Prolifera* Diary

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{% block title %}Engineering{% endblock %}
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## Overview

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![engineering figure](https://static.igem.wiki/teams/5578/image/engineering/engineer.webp)

To address the root cause of the proliferation
of *Ulva prolifera*, we have undergone multiple iterations and launched "super carpet"
as a new solution. The super carpet we designed is divided into three major spells:First,
the 'Ennervate' survival spell ensures that our engineered bacteria can survive in
high-salinity seawater environments. Second, the 'Permanent Sticking' functional spell is
responsible for efficiently and specifically capturing Enteromorpha spores with high specificity.
Finally, the 'Avada Kedavra' biosafety spell guarantees the controllability and safety of
the project in open environments. Each module has been verified through multiple rounds of
design-Build-Test-Learn (DBTL) cycles, and the subsequent optimization direction has been clearly
defined.

#### Five Core Challenges with Resolution in Our Project

Expand/Collapse All

Bacterial survival in seawater
▼

* Strategy: Salt-tolerant gspM and echM genes
* Successes: Near-normal growth at 600mM NaCl
* Limitations: Slight growth inhibition remains

![Bacterial survival in seawater](https://static.igem.wiki/teams/5578/image/engineering/context-image1-1.webp)

Adhesive biofilms
▼

* Strategy: Ag43 and OmpA expression
* Successes: Enhanced biofilm formation verified by crystal violet
  staining
* Limitations: High expression burden on bacteria

![Bacterial survival in seawater](https://static.igem.wiki/teams/5578/image/engineering/context-image2-1.webp)

Alternative spore capture
▼

* Strategy: CsgA-Arginine oligopeptides fusion protein
* Successes: Successful design and structural simulation
* Failures: Failed to express target protein

![Bacterial survival in seawater](https://static.igem.wiki/teams/5578/image/engineering/context-image3-1.webp)

Programmable biofilm structure
▼

* Strategy: Ag-Nb orthogonal adhesion pairs
* Successes: Successful checkerboard pattern modeling and adhesion pair construction
* Limitations: Complete integration not finished

![Bacterial survival in seawater](https://static.igem.wiki/teams/5578/image/engineering/context-image4-1.webp)

Biosafety control
▼

* Strategy: Blue light-induced suicide switch (L.U.C.I.A.)
* Successes: TetR-LOVdeg fusion protein design and construction
* Limitations: Sensitivity testing incomplete

![Bacterial survival in seawater](https://static.igem.wiki/teams/5578/image/engineering/context-image5-1.webp)

### CLICK THE CHOSEN BOX TO EXPLORE OUR DBTL CYCLES:

[🛡️

##### Ennervate

Survival Spell](#Cycle1)
[🔗

##### Permanent Sticking

Functional Spell](#Cycle2)
[⚡

##### Avada Kedavra

Biosafety Spell](#Cycle3)

## Ennervate - survival spell

**Escherichia coli** is the core host of our design, but natural *Escherichia coli* is
extremely prone to death in the high-salt environment of seawater. In the communication with
Professor Xiaolei Wang from Ocean University of China, we were directly pointed out: "You must verify the growth
of the bacteria under a salinity close to that of seawater. If the survival problem of *Escherichia coli*
in seawater cannot be solved, no matter how creative the functional module is, it is out of the question."

As we recognized during our communication with Professor Wang, solving the survival problem of engineered
bacteria in seawater is a fundamental prerequisite for the establishment of our entire project. Therefore, the
first core task of our engineering cycle is to create a powerful 'survival module' for our chassis cell.

![engineering figure](https://static.igem.wiki/teams/5578/image/engineering/survival-5.webp)

#### Cycle 1: Construct salt-tolerant plasmids

![Design](https://static.igem.wiki/teams/5578/image/engineering/design-1.webp)

We aim to develop an engineered bacterium capable of normal growth and proliferation in a medium simulating
seawater salinity (approximately 600 mM NaCl). Based on the in-depth research by Raj Kishor
Kapardar[[1]](#fn1) et al., we confirmed from the
metagenome of the pond that the salt-tolerant genes gspM and
echM can enable bacteria to survive at 750mM sodium chloride concentration. They respectively express
salt stress tolerance proteins and proteins with enyl-coA hydratase activity. We plan to introduce them
into Escherichia coli to enable them to survive in seawater. For details, please refer to the [Design](https://2025.igem.wiki/xjtlu-china/design)
page.

---

![Build](https://static.igem.wiki/teams/5578/image/engineering/build-1.webp)

After confirming the sequence, we designed the
[echM-gspM]-pET28a(+) plasmid, which can be used to construct salt-tolerant survival systems in
Escherichia coli. The plasmid was successfully constructed,
and the following figure shows detailed plasmid information.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/1.webp)

Fig1. echM-gspM Genetic Circuit

---

![Test](https://static.igem.wiki/teams/5578/image/engineering/test-1.webp)

We aim to quantitatively compare the growth curve differences between the engineered bacteria and wild-type
strains introduced with the [echM-gspM]-pET28a(+) plasmid in high-salt (600mM NaCl) and salt-free
media to verify the function of the salt-tolerant gene. We measured the OD600 absorbance values of
each group of strains in different culture media at regular intervals within 0 to 27 hours and
conducted fitting analysis using the Logistic model.

Our design observation shows that the engineered
bacteria in 600 mM NaCl NaCl medium exhibited normal growth patterns comparable to those in NaCl-free LB
medium. In contrast, growth of negative control E. coli was significantly inhibited under high-salt
conditions, confirming that salt-tolerant genes enhance engineered bacteria's adaptability to
high-salt environments. However, further analysis revealed slightly NaCl-free LB medium supported
slightly better growth of engineered bacteria than 600 mM NaCl-containing LB medium, indicating residual
growth inhibition by high-salt stress.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/2.webp)
![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/3.webp)

Fig2&3. Growth Curves of E.coli Strains with Logistic Model

---

![Learn](https://static.igem.wiki/teams/5578/image/engineering/learn-1.webp)

This DBTL cycle verified that gspM and echM expression enables engineered bacteria to achieve near-normal
growth in 600 mM NaCl (high-salt) environments, comparable to salt-free conditions. However, the
K-value of their growth curve remained slightly lower than the salt-free control, indicating
potential metabolic burden from high-salt osmotic pressure or exogenous protein expression. This
"slight gap" discovery directly gave rise to the problems in our next round of engineering
cycle. In the future, we will conduct metabolic flux analysis at the modeling level to determine
whether the slight K-value reduction primarily stems from salinity-induced osmotic pressure or
protein expression burden.

---

![Design](https://static.igem.wiki/teams/5578/image/engineering/design-1.webp)

Although we already know that engineered bacteria can
survive normally in a high-salt environment. However, the seawater environment is very complex. It not
only contains sodium chloride but also a large amount of other minerals. To test the survival ability of
engineered bacteria in natural seawater environments, we redesigned the experiment for the future:

1. The engineered bacteria were inoculated in naturally sterilized seawater to test their response
   ability to environmental stress in the natural environment.

2. Explore the size of the dominant ecological niche of engineered bacteria in the natural seawater
   environment.

## Permanent Sticking -- functional spell

![engineering figure](https://static.igem.wiki/teams/5578/image/engineering/nbag.webp)

Fisherman Bingyang Wang mentioned, "One square meter of green algae
today, and 100 square meters tomorrow. Salvage is always a step behind." The
paper points out that during the physical salvage process, it will cause the tiny
reproduction of algae to newly enter the water, further promoting the growth of
green algae[[3]](#fn3). These pieces of evidence indicate
that physical salvage is far
from sufficient to solve this difficult problem!

According to the literature, the spores of the green algae in the shallow
beaches of northern Jiangsu will be buried in the sediment, serving as the
source of the green algae outbreak the following year [[2]](#fn2). This indicates that
spores are the source of the annual proliferation. If the total number of spores
decreases, the problem of green algae proliferation will also be alleviated or
even solved.

![engineering figure](https://static.igem.wiki/teams/5578/image/engineering/4.webp)

Therefore, we chose Ulva prolifera spores as the governance object of the
main functional module

The spores will be fixed on the raft racks for nori cultivation in the shallow beaches of northern Jiangsu.
When nori is harvested, it falls off in large quantities, thus changing from a fixed ecological state to a
floating state[[3]](#fn3). So we want to capture the spores
of green algae in advance to prevent them from settling in
the natural environment.

##### Our functional system consists of three major submodules:

1. Adhesion Submodule

We enhance the adhesion ability of the bacterial surface by introducing relevant plasmids
to form a stable and highly adhesive biofilm as the main body for capturing spores.

2. Sedimentation Submodule

We took advantage of the specific sedimentation feature of arginine short peptides on
green algae spores to express arginine short peptides on the hairs of Aspergillus escherichia coli. This can
cause the irreversible colonization process of spores to occur earlier, prevent spore escape, and enhance
specificity[[4]](#fn4).

3. Ag-Nb system

Ultimately, we used Ag-Nb adhesion pairs to connect the two major submodules in a
programmable manner.

### 1. Adhesion submodule:

In order to provide sufficient adhesion substrates for
zoospores and fully consider the adhesion effect of the hardware carrier and the stress resistance of
the seawater environment, we decided to complete the adhesion submodule by enhancing the biofilm.

#### Cycle 1: Predict the optimal induction concentration range of IPTG of ag43-ompA

![Design](https://static.igem.wiki/teams/5578/image/engineering/design-1.webp)

Our design specification is: to achieve
the controllable formation of engineered bacterial biofilms by regulating the expression of key
proteins. Literature analysis identified Ag43 and OmpA as core regulators of biofilm formation: Ag43
specifically mediates cell aggregation, adhesion, and biofilm development; OmpA, a key determinant of
bacterial colonization and biofilm establishment, significantly enhances biofilm formation on
hydrophobic surfaces[[6]](#fn6)[[7]](#fn7). The design employs lactose operon-mediated
regulation of these exogenous
genes. Optimization of IPTG induction concentration ensures sufficient target protein expression while
minimizing host cytotoxicity. To streamline experimental workflow, a predictive model adapted from
XJTLU-CHINA (2023) was utilized to estimate the optimal IPTG concentration range, facilitating
experimental design and improving efficiency[[5]](#fn5).

---

![Build](https://static.igem.wiki/teams/5578/image/engineering/build-1.webp)

We use ordinary differential equations (ODEs) to describe the mRNA expression processes of *ag43*
and
*ompA*. These ODEs are numerically solved using Python. Given that *ag43* and
*ompA*
are driven by the same operon and co-transcribed into a single polycistronic mRNA, their mRNA expression
levels are collectively represented by a single variable, mRNAAG43·ompA.

For details, please refer to the [Model](https://2025.igem.wiki/xjtlu-china/model) page.

---

![Test](https://static.igem.wiki/teams/5578/image/engineering/test-1.webp)

We drew the prediction graphs of mRNA
synthesis amounts for ag43 and ompA under IPTG gradient induction by solving the ODE equations using
Python.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/5.webp)

Fig4. Effect of IPTG Concentration on mRNA\_AG43\_ompA Expression

---

![Learn](https://static.igem.wiki/teams/5578/image/engineering/learn-1.webp)

Our model
prediction shows that the mRNA expression levels of ag43 and ompA tend to saturate when the IPTG
concentration reaches approximately 400-600μM, with diminishing returns from further concentration
increases.

This round of modeling provides guidance for the
subsequent DBTL cycle: the IPTG concentration gradient in wet experiments will be focused on the 0.1–0.8
mM range for testing. This design avoids blind large-scale screening and conserves valuable time and
resources. While the model cannot fully replicate real-world conditions, it has effectively delineated
high-value regions for experimental exploration.

#### Cycle 2: Experimental verification of Ag43-OmpA expression

![Design](https://static.igem.wiki/teams/5578/image/engineering/design-1.webp)

To actually
construct a strong biofilm, we designed a plasmid that overexpresses Ag43-OmpA using a strong
promoter and strong ribosome binding site (RBS)and
used the data obtained from cycle 1 to induce expression at 400-600μM IPTG.

---

![Build](https://static.igem.wiki/teams/5578/image/engineering/build-1.webp)

We designed recombinant plasmids containing ag43 and
ompA genes to regulate gene overexpression by assembling strong promoters and strong RBS sequences.
The plasmid was successfully constructed, and the following figure shows detailed plasmid information.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/6.webp)

Fig5. ag43-ompA Genetic Circuit

---

![Test](https://static.igem.wiki/teams/5578/image/engineering/test-1.webp)

The wet experiment verified the expression of the
target protein by Western blot and detected the biofilm formation ability by crystal violet staining.
According to Fig6, it was found that the biofilm formation ability of the engineered bacteria in the
experimental group introduced with [ag43-ompA]-pET28a(+) plasmid was significantly enhanced compared
with that of the control group introduced with pET28a(+) empty plasmid. During the experiment of
inducing protein expression, we used the prediction graph of the optimal concentration of mRNA synthesis
induced by IPTG in cycle 1 and conducted gradient attempts near the 400μM IPTG concentration. The final
experiment found that the optimal induction concentration of IPTG was approximately 600 μM.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/1008-6.webp)

Fig6. The Western Blot(WB) result induced by different IPTG concentrations.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/7.webp)

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/8.webp)

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/9.webp)

Fig6 & 7 & 8. Crystal Violet Staining Results of 24-hour Growth Biofilm

---

![Learn](https://static.igem.wiki/teams/5578/image/engineering/learn-1.webp)

We successfully utilized strong promoters and strong RBS to regulate the overexpression of ag43 and ompA to
achieve the purpose of enhancing biofilms, and ensured that the engineered bacteria could survive normally
and function properly. However, Professor Quanfeng Liang from Shandong University mentioned that excessive
expression of membrane proteins can impose an excessive burden on bacteria, and overexpression of OmpA poses
a risk of bacterial self-lysis. Subsequently, we plan to seek better ways to reduce the expression burden of
engineered bacteria and ensure the enhancement capacity of biofilms. We find bcsQ is another key gene to
promote the formation of biofilms.

#### Cycle 3: Repair bcsQ gene

![Design](https://static.igem.wiki/teams/5578/image/engineering/design-1.webp)

Considering Excessive expression of membrane proteins can impose an excessive burden on bacteria, and
overexpression of OmpA poses a risk of bacterial self-lysis. We find bcsQ is another key gene to promote the
formation of biofilms.

Exopolysaccharide (EPS) is an ideal abiotic component in biofilm by exerting the following functions:

1. Enhancing **surface adhesion** through interactions among functional groups.

2. **Defensing** against abiotic stressors, granting the ecological biofilm greater survival
capabilities.

The method of enhancing E. coli biofilms by repairing the **bcsQ mutation**[[14]](#fn14) works based on
the following
mechanism: In E. coli K-12 strains, the 6th codon of bcsQ is a premature termination codon (TAG), which
truncates BcsQ translation early. This triggers "operon polarity," inhibiting the expression of downstream
genes in the bcs operon (e.g., bcsA, bcsB) and thus blocking cellulose biosynthesis.

By prime editing repairing **TAG** to **TTG** (encoding leucine), functional BcsQ
is produced, and the continuous
ribosome binding during translation prevents Rho-dependent transcriptional termination, restoring the
expression of downstream bcs genes.

**This dual restoration of the bcs pathway promotes cellulose synthesis, which, together with curli,
forms an
elastic, cohesive matrix.** This enhances biofilm structure (e.g., larger colonies with complex
folds) and
mechanical properties, thereby strengthening the biofilm. For a more comprehensive explanation,
refer to the [Design](https://2025.igem.wiki/xjtlu-china/design) section.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/a.webp)

Fig9. Prediction Effect of bcsQ Repair

Based on the guidance of senior researchers and professors as well as literature research, and in
accordance with the principle of guided editing, the highly efficient site-directed mutagenesis binary
plasmid system PE-[bcsQ] -PacyCDUet-1 plasmid was successfully constructed[[15]](#fn15). Compared with λ-Red and
traditional CRISPR, guided editing has significant advantages: it does not require double-strand breaks in
the genome, has no "scar" problem of λ-Red editing, and has higher targeting accuracy and editing
efficiency, which can solve the problems of low efficiency of λ-Red and potential off-target effects of
traditional CRISPR.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/b.webp)

Fig10. Mechanism of Prime Editing

---

![Build](https://static.igem.wiki/teams/5578/image/engineering/build-1.webp)

Under the guidance of senior researchers and professors, and through reviewing relevant literature, we
successfully constructed two-in-one plasmid prime editing system for efficient site-directed mutagenesis
based on the principle of prime editing. The evopreq1-1 trimmed motif was added to protect pegRNA from
degradation[[16]](#fn16).

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/c.webp)

Fig11. The Synthesis Process of PE-[bcsQ]-pACYCDuet-1 Plasmid

![Learn](https://static.igem.wiki/teams/5578/image/engineering/learn-1.webp)

We have successfully made the purchase, but due to time constraints, the plasmid has not arrived yet. We
look forward to achieving successful results before jamboree.

### Settlement submodule

Merely enhancing the adhesion effect of the substrate
cannot prevent the active escape of spores. We focus on the important physiological process ——
sedimentation during spore settlement. To enhance the specific sedimentation ability of biofilms
for *Ulva prolifera* spores, we introduce a sedimentation submodule to assist the adhesion
submodule.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/1008-1.webp)

Fig12. Sketch map of spores sedimentation

#### Cycle1: AHL Diffusion Model

![Design](https://static.igem.wiki/teams/5578/image/engineering/design-1.webp)

Our design specification is: to construct a
"signal capture - spore anchoring system", enabling the engineered bacteria to effectively
settle and anchor spores by diffusing and releasing specific functional molecules in the water body and
forming a stable concentration gradient, thus preventing them from detaching from the biofilm.
Acyl-Homoserine Lactone (AHL) is a signaling molecule used by bacteria for quorum sensing and is widely
present near biological membranes. We chose it as our first functional molecule. According to reference
[[8]](#fn8), to induce irreversible spore deposition,
the concentration of AHL must be at least 25μmol · L⁻¹.
Based on this, the functional molecule release and gradient formation mechanism of the engineered
bacteria is designed to achieve spore deposition through molecular action and anchor on the biofilm.

---

![Build](https://static.igem.wiki/teams/5578/image/engineering/build-1.webp)

**During the model-building process, we assume:**

1. The diffusion coefficient of AHL can be calculated by the Einstein–Smolukhovsky equation.
   $$D = \frac{\mu\_q k\_B T}{q} \approx 4.65 \times
   10^{-10}
   \text{ m}^2/\text{s}$$
2. Only consider two environments:
   1. Still water environment: Only consider diffusion.
   2. Marine environment: Consider convection and diffusion.

Diffusion models are mainly divided into the following two types:

1. In a still water environment, Fick's Second Law was used to simulate the variation of AHL concentration
   with time and space.
2. In the marine environment, the Convection–diffusion Equation was adopted and integrated with the
   seawater
   harmonic diffusion model to simulate the ocean flow velocity in both the horizontal and vertical
   directions.

For details, please refer to the [Model](https://2025.igem.wiki/xjtlu-china/model) page.

---

![Test](https://static.igem.wiki/teams/5578/image/engineering/test-1.webp)

1. Simulation results of still water environment

Although AHL molecules theoretically continue to diffuse, due to their extremely low diffusion coefficient
\( \sim 10^{-10} \, \text{m}^2/\text{s} \), their effective diffusion range is very limited.
Even if the initial concentration of AHL is as high as 600 μM, it is difficult for AHL to spread
beyond 5 cm with the injection point as the center within 24 hours. For details, please refer to the Fig13
below.

Fig13. AHL Simulation Results of Still Water Environment

2. Marine environment simulation results:

In the presence of convection, the spatial diffusion rate of AHL increases, but it is still limited by an
extremely low generation rate.

The calculation results show that the diffusion rate of AHL is much higher than the physiological reasonable
production rate of bacteria
\( \sim 10^{-7} \, \text{m}^2/\text{s} \), making it difficult for its concentration to accumulate
effectively. For details, please refer to the Fig14 below.

Fig14. AHL Simulation Results in the Presence of Convection

---

![Learn](https://static.igem.wiki/teams/5578/image/engineering/learn-1.webp)

Even under convective conditions, the diffusion rate
of AHL is far higher than its generation rate, failing to reach the minimum concentration required for
spore sedimentation. This indicates that the AHL-based spore capture strategy is not feasible in real
aquatic environments.

Thus, the scheme of using AHL for spore sedimentation
was abandoned. Notably, an alternative substance capable of inducing *Ulva prolifera* spore
sedimentation—**arginine short peptide**—was identified, which can accelerate the natural
sedimentation process of spores[[4]](#fn4). To avoid
the recurrence of diffusion-related issues, the design is
adjusted to immobilize the arginine short peptide onto *Escherichia coli*.

#### Cycle 2: Arginine fusion short peptide

##### Cycle 2.1: Design arginine fusion short peptides

![Design](https://static.igem.wiki/teams/5578/image/engineering/design-1.webp)

Under the modeling guidance of Cycle 1, we aimed to
identify a substance that can be immobilized on engineered bacteria to induce premature spore
sedimentation. Notably, T. Ederth demonstrated that arginine oligopeptides induce *Enteromorpha
margin* spores to prematurely release adherent vesicle contents, shortening spore formation time
from 2 hours to 5 minutes[[4]](#fn4). Accordingly, we
designed to fuse arginine oligopeptides with the
*csgA* gene (derived from *Aspergillus niger* fimbriae) in *Escherichia coli*,
enabling spores to anchor more firmly to the biofilm matrix.

---

![Build](https://static.igem.wiki/teams/5578/image/engineering/build-1.webp)

Literature
indicates that in the experimental system of Ederth et al., peptides with similar length and
terminal exposure to Arg can significantly increase the adhesion rate of Ulva swimming spores
(pseudo-colonization ratio >70%).

We fused
short peptide sequences containing arginine into the outer membrane protein CsgA of Escherichia coli
to construct a functional module capable of extracellular display and mediating spore deposition.
AlphaFold3 simulations predicted the three-dimensional structure of the fusion protein, revealing
stable extracellular exposure of the short peptide—this confirms the module's potential to
endow host cells with spore deposition-inducing functionality. For the specific 3D structure, please refer
to Fig15.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/15.webp)

Fig15. CsgA-Arginine Oligopeptide 3D Structure

---

![Test](https://static.igem.wiki/teams/5578/image/engineering/test-1.webp)

AlphaFold3 predictions show high confidence in the
overall structure of our designed CsgA-Arg fusion protein. The β-sheet region of the CsgA backbone
remains highly reliable, confirming the fusion peptide does not disrupt the core scaffold. However, as
amino acid count increases, the arginine short peptide terminus exhibits lower prediction confidence
(evidenced by lighter areas in the
Fig16), indicating it is an intrinsically disordered region (IDR).
This design avoids stable folding domains, allowing the short peptide to act as an "oscillating
pendulum" to maximize contact between terminal arginines and external targets, such as
*Ulva* spore membranes.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/16.webp)

Fig16. CsgA-Arginine Oligopeptide PAE Picture

---

![Learn](https://static.igem.wiki/teams/5578/image/engineering/learn-1.webp)

The results demonstrate that short peptides can be
stably exposed outside the membrane, promising to endow host cells with a novel function of inducing
spore deposition. With this structural basis verified, we proceeded to the next phase of Cycle 2.2,
concentrating on the practical construction of arginine fusion short peptides.

##### Cycle 2.2: Construction of arginine fusion short peptides

![Design](https://static.igem.wiki/teams/5578/image/engineering/design-1.webp)

To further
enhance the targeting and sedimentation efficiency of the short peptide system and better achieve
the core goal of inducing Ulva prolifera spore deposition, we planned to construct arginine fusion
short peptides. Based on the need for high sedimentation activity, we selected the arginine
oligopeptide coding gene combination GSGGSG-RYRYRYR, which was reported in the literature to have
the highest sedimentation efficiency among similar arginine oligopeptide sequences—this choice
ensures the designed fusion short peptide can effectively exert spore sedimentation
function[[4]](#fn4).

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/e.webp)

Fig17. The sketch map of CsgA-Arginine oligopeptide

---

![Build](https://static.igem.wiki/teams/5578/image/engineering/build-1.webp)

We fused and expressed this type of small molecule
oligopeptide with the common extracellular display platform protein CsgA of Escherichia coli to address
the issue of rapid diffusion of small molecules. We constructed a recombinant plasmid map containing the
CsgA-arginine oligopeptide fusion gene. The plasmid was
successfully constructed, and the following figure shows detailed plasmid information.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/17.webp)

Fig18. [CsgA-GSGGSG-RYRYRYR]-pET28a(+) Plasmid

---

![Test](https://static.igem.wiki/teams/5578/image/engineering/test-1.webp)

We plan to introduce the recombinant plasmid of
CsgA-arginine oligopeptide fusion gene into Escherichia coli. The experiment verified the expression of
the target protein by Western blot and compared the effectiveness of sedimentation function through
spore release assay. We attempted gradient induction at 0.6 to 1.2mM and different temperature induction
at 16,30, and 37°C, but failed to obtain the target protein from the supernatant and precipitate of cell
lysis.

Experiments were designed to verify target protein
expression via Western blot and compare spore sedimentation efficacy through spore release assays. We
introduced the recombinant plasmid harboring the CsgA-arginine oligopeptide fusion gene
into *Escherichia coli*. Gradient IPTG induction (0.6–1.2 mM) and temperature-specific induction
(16, 30, and 37°C) were attempted; however, target protein was not detected in either the supernatant or
pellet of cell lysates. For specific WB images, please refer to Fig19.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/f.webp)

Fig19. WB Results of CsgA-arginine Oligopeptide at Different Gradient Induction
and Temperature Induction

---

![Learn](https://static.igem.wiki/teams/5578/image/engineering/learn-1.webp)

Despite the
correct construction of the plasmid verified by restriction enzyme digestion, the failure of protein
expression poses a significant challenge. This outcome revealed that even if in silico design and
plasmid construction are successful, the expression, folding, and stability of the target protein
remain highly uncertain. Based on this failure, we have formulated the following subsequent
plans:

1. Sequence the plasmid to rule out the possibility of
   unexpected mutations in the coding region;

2. Attempt nickel column affinity chromatography for
   purification to capture the target protein that may exist in inclusion bodies or be expressed at
   extremely low levels;

3. If the above methods fail, we will redesign the fusion
   protein to address potential issues such as protein toxicity, misfolding, or expression
   inhibition.

Although
this experimental cycle yielded negative results, it is crucial for us to identify the key
bottlenecks of the project and resolve problems through a systematic engineering approach.

##### Cycle 2.3: Sanger sequencing of the [CsgA-GSGGSG-RYRYRYR]-pET28a(+) plasmid

![Design](https://static.igem.wiki/teams/5578/image/engineering/design-1.webp)

To identify the cause of failed protein expression, we performed Ni-NTA column purification in parallel with
Sanger sequencing of the original recombinant plasmid and the mini-prep plasmid from the engineered E. coli
strain. This approach enabled us to distinguish between expression-related issues and genetic problems such as
mutations or rearrangements in the fusion gene.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/g.webp)

Fig20. The result of the Sanger sequencing

---

![Build](https://static.igem.wiki/teams/5578/image/engineering/learn-1.webp)

Based on the Sanger sequencing results, we identified the presence of a premature stop codon within the
coding sequence of the gene in JM109(DE3), which truncates the polypeptide chain and prevents full-length
protein synthesis. This finding directly explains the lack of detectable protein expression in our previous
experiments. To address this issue, we plan to retransformation the plasmid, ensuring the integrity of the
open reading frame and restoring the intended protein expression.

---

### Ag-Nb adhesion system

During the process of constructing engineered bacteria, we found that multiple modules rely on the overexpression of
specific proteins. To reduce the burden of protein expression and enable efficient coupling of different functional
zones, we expressed the adhesion and sediment modules separately and connected them using Ag-Nb orthogonal adhesion.
Building on this strategy, we also innovatively proposed the concept of 4-bit encoded biofilms, which achieves the
programmability of biofilm structures through up to four adhesion pairs[[12]](#fn12) and ultimately forms programmable biofilms
with specific functional zones.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/h.webp)

Fig21. Nb-Ag interactions between cells can mediate production of microscopic patterns (spatial organization of cell
types, denoted in color) and morphologies (overall spatial structure of all cells, denoted by gray background).

#### Cycle 1: Design programmable biofilms

![Design](https://static.igem.wiki/teams/5578/image/engineering/design-1.webp)

To ensure the dense and stable biofilm, we adjust the
length of the interface between different colonies and the connectivity of the intersection points, and
combine geometric modeling analysis to select the most suitable biofilm shape from multiple patterns.

---

![Build](https://static.igem.wiki/teams/5578/image/engineering/build-1.webp)

We have developed a function-oriented biofilm scoring
system that allows users to assign different weights based on their actual needs. The system will
automatically rate different patterns and output recommended ones. Meanwhile, we also offer a
non-weighted radar chart visualization interface for intuitively comparing the performance of each
pattern under different indicators to assist in decision-making.

We take stability as the primary consideration. After
several rounds of scoring, we finally selected the checkerboard pattern as the target biofilm structure.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/20.webp)

Fig22. Function-oriented Biofilm Scoring System

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/21.webp)

Fig23. A Radar Chart Visualization Interface without Weight

After the pattern was determined, we further provided
the adhesive allocation scheme that matched the pattern and the corresponding plasmid design strategy. For
details, please refer to the [Model](https://2025.igem.wiki/xjtlu-china/model) page.

---

![Test](https://static.igem.wiki/teams/5578/image/engineering/test-1.webp)

We simulated the designed colony pattern through
models, including:

1. Two-strain system: Observe how the two strains form a stable interface under an ideal initial
   distribution.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/22-1.webp)

Fig24. The Stable Interface Formed by the Two Strains under the Ideal Initial
Distribution

2. Multi-strain system: Explore how different adhesion pairs work together to form complex structures
   [[10]](#fn10).

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/23.webp)

Fig25. Different Adhesion Pairs Work Together to Form Complex Structures

---

![Learn](https://static.igem.wiki/teams/5578/image/engineering/learn-1.webp)

The simulation results show that the colonies can
indeed form clear and regular interfaces through the set rules, preliminarily verifying the feasibility
of the programmable biofilm scheme and providing a reliable basis for subsequent experiments.

#### Cycle 2: Constructing programmable biofilms

![Design](https://static.igem.wiki/teams/5578/image/engineering/design-1.webp)

Our design specification is: To develop a programmable
strategy for constructing biofilms with controllable inter-colony interfaces, leveraging Ag-Nb
orthogonal adhesion pairs. The outer membrane-anchored nanoantibody-antigen (Nb-Ag) programmable
adhesion cassette displays antigen-nanoantibody pairs on *Escherichia coli* surface, enabling
rapid assembly of stable biofilms[[9]](#fn9). Guided by
Cycle 1 modeling results, the target biofilm
configuration is determined as the "checkerboard pattern", which is achieved via the above
strategy.

---

![Build](https://static.igem.wiki/teams/5578/image/engineering/build-1.webp)

We designed two plasmids by referring to the BBa\_K4765105 and BBa\_K4765106[[17]](#fn17) components designed by Fudan
University in 2023 since Ag3-Nb3 adhesive pair has got the best aggregation result[[11]](#fn11). To make the
experimental
results more intuitive, we respectively used mCherry and sfGFP to characterize the expressions of Ag and Nb.

1. Insert the intimin-Ag3 fusion protein and characterize it with mCherry.

2. Insert the intimin-Nb3 fusion protein and characterize it with sfGFP.

The plasmid
was successfully constructed, and the following figures show detailed plasmid information.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/24.webp)

Fig26. Ag3-mCherry Genetic Circuit

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/25.webp)

Fig27. Nb3-sfGFP Genetic Circuit

---

![Test](https://static.igem.wiki/teams/5578/image/engineering/test-1.webp)

We conducted a mixed culture of the two engineered Escherichia coli strains constructed above and compared
the functional efficiency of the two surface display systems through self-aggregation determination
experiments. As can be seen from the following figure, the bacterial density of the supernatant in the mixed
strain group has significantly decreased over time compared with that in the single strain group.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/26.webp)

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/27.webp)

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/28.webp)

Fig28& 29 &30. Aggregation Assay of *E. coli* at Different Time Points

Meanwhile, we observed the luminescence of the fluorescent proteins mCherry and sfGFP with the aid of a
fluorescence microscope to verify the display effect and localization characteristics of the fusion proteins
on the bacterial outer membrane. In the culture medium, the alternating distribution of red- and green-labeled
*Escherichia coli* is clearly observable. Additionally, large bacterial clusters are present, within
which
individual red and green bacterial cells are difficult to distinguish.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/i.webp)

Fig31. Characterization of Ag-Nb in fluorescence quantification system(30X)(Red signals depict Ag
expression; green signals depict Nb expression.)

---

![Learn](https://static.igem.wiki/teams/5578/image/engineering/learn-1.webp)

To date, the
validation of our basic principle confirms the theoretical feasibility of this project. The Ag3-Nb3
adhesion pair of BL21 (DE3) demonstrated a stronger orthogonal adhesion ability than JM109 (DE3) in
self-aggregation determination. Moving forward, we will attempt the integration step: Incorporate
the Ag3 adhesion system with the "adhesion subunit module" into strain A, and integrate
the Nb3 adhesion system with the "sedimentation subunit module" into strain B. By
co-culturing these two engineered strains, we anticipate constructing the final multifunctional
biofilm with spatial functional partitioning.

## Avada Kedavra - Biosafety spell

In the iGEM competition, safety is not only a
compliance criterion but also the core cornerstone of synthetic biology innovation. We are well aware of
the potential risks in bioengineering and have integrated safety throughout the
entire Design-Build-Test-Learn (DBTL) cycle since the project started.

We would like to divide the process of synthetic
biology entering the market into the several nodes. At present, the footprint of synthetic biology in
the world mostly remains at factory production increase, which is a "50%" value that the
general public can accept. However, we aim to create a transferable component to further open up the
market for synthetic biology.

![engineering figure](https://static.igem.wiki/teams/5578/image/engineering/bio-safety.webp)

![engineering figure](https://static.igem.wiki/teams/5578/image/engineering/29.webp)

In this year's project, we applied modified
Escherichia coli and placed it in the sea to capture green algae spores. Escherichia coli is an
environmental pollutant, and organisms edited by synthetic biology are not allowed to be placed in the
natural environment either. So in an effort to achieve a transformation in synthetic biology from
"50%" to "75%", we designed a new type of blue light-induced suicide switch - **L.U.C.I.A.(Light Unlocks
Cytotoxic Inducible Adaptor)**.

#### Cycle 1: Design L.U.C.I.A. fusion proteins

![Design](https://static.igem.wiki/teams/5578/image/engineering/design-1.webp)

We select the tetracycline operon as the main body of the
suicide switch. We fused the LOVdeg tag into the C-terminal of the galactose repressor TetR, enabling
TetR to be rapidly degraded under blue light irradiation and regulating the expression of the downstream
suicide gene mazF. According to the literature[[13]](#fn13), we know that the C-terminal of this tag will expand and
expose the degradation signal under the irradiation of blue light with a wavelength of 465nm, and then
be recognized by the ClpXP protease system of Escherichia coli, thereby achieving light-dependent
protein degradation[[11]](#fn11). This new type of
blue light-controlled suicide system will be more sensitive
and controllable, and have stronger mobility. For details, please refer to the [Model](https://2025.igem.wiki/xjtlu-china/model) page.

1. No light

When light (about 465 nm light irradiation) is absent, the tetR repressor binds tightly to the operator,
preventing transcription by RNA polymerase.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/j.webp)

Fig32. The gene circuit has no transcription without light.

2. With light

When cells are under about 465 nm light irradiation, the LOVdeg tag promotes the degradation of the tetR
repressor protein. This allows transcription by RNA polymerase.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/k.webp)

Fig33. TetR-LOVdeg is degraded irradiated by blue light.

This initiates the downstream expression of the mazF toxin gene, triggering programmed cell death.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/l.webp)

Fig34. The gene circuit finally initiates because of the removement of the repressor.

---

![Build](https://static.igem.wiki/teams/5578/image/engineering/build-1.webp)

According to the content of the paper[[11]](#fn11), we adopted
eight dark state stability mutations in the iLoD system to enhance the effect of the light-controlled
switch. We used AlphaFold3 to predict and verify the structural rationality of the fusion protein to
ensure that the binding of TetR and LOVdeg does not affect the core function. For details, please refer
to the following figure.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/33.webp)

Fig35. TetR-LOV 3D Structure

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/34.webp)

Fig36. TetR-LOV PAE Picture

---

![Test](https://static.igem.wiki/teams/5578/image/engineering/test-1.webp)

We used molecular docking and molecular dynamics
simulations to verify the binding ability of fusion proteins and DNA operon regions. The following
figures show part of the analysis results. For details, please refer to the [Model](https://2025.igem.wiki/xjtlu-china/model) page.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/model/md/docking-fusiondnafinal2.webp)
![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/model/md/docking-fmn-lovfinal.webp)

Fig37 &38. Partial Images of Molecular Docking and Molecular Dynamics
Simulation

---

![Learn](https://static.igem.wiki/teams/5578/image/engineering/learn-1.webp)

After comparative analysis of docking and simulation
results for multiple protein-DNA groups, the Lucia fusion protein exhibited remarkably high confidence.
This confirms that the LOVdeg can bind to the TetR protein effectively and stably, thereby providing
theoretical basis and support for subsequent experiments.

#### Cycle 2: Construct Lucia fusion proteins

![Design](https://static.igem.wiki/teams/5578/image/engineering/design-1.webp)

Through
literature research and functional screening, the core components required for constructing the blue
light-inducible suicide system were identified, with specific confirmation details as
follows:

1. Genes: ① Genes related to the optogenetic degradation tag—LOVdeg; ②
   Regulatory gene *tetR*; ③ Suicide effector gene
   *mazF*, which can induce cell death by degrading RNA;

2. **Plasmids**: Recombinant plasmids containing the complete set of
   components for the blue light-inducible suicide system, which need to include a promoter, the
   Tet operon, and a resistance marker gene for positive clone screening;

3. **Strains**: Host *E. coli* strains,
   which need to exclude the interference of
   endogenous LacI expression on the regulatory system to ensure the specificity of the Tet operon
   regulatory pathway.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/m.webp)

Fig39. tetR\*-sfGFP Genetic Circuit (The \* represents LOVdeg tag)

---

![Build](https://static.igem.wiki/teams/5578/image/engineering/build-1.webp)

We inserted the LOVdeg fusion protein gene fragment
obtained from cycle 1 based on the Tet operon to regulate the expression of the downstream mazF gene.

We have designed two plasmids in total:

1. Insert a green fluorescent protein, aiming to visually verify the degradation efficiency of the TetR
   repressor protein and potential leakage of the genetic circuit under light-induced conditions through
   fluorescent signals, thereby providing
   direct visual evidence for subsequent functional studies.

2. Insert the suicide gene mazF, which is used to execute the actual cell-killing function.

This design strategy helps to conduct step-by-step
troubleshooting when abnormalities occur in the experiment, thereby ensuring that the location and
resolution of experimental problems are more precise and efficient.

The plasmid
was successfully constructed, and the following figures show detailed genetic circuit information.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/37.webp)

Fig40. tetR\*-sfGFP Genetic Circuit (The \* represents LOVdeg tag)

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/38.webp)

Fig41. tetR\*-mazF Genetic Circuit (The \* represents LOVdeg tag)

---

![Test](https://static.igem.wiki/teams/5578/image/engineering/test-1.webp)

1. tetR\*-sfGFP genetic circuit test

Given that the sfGFP gene circuit can directly present experimental results through visualization, it has
the advantages of intuitive result observation and easy interpretation. Therefore, we give priority to using
this gene circuit for subsequent experimental verification.

To verify the successful expression of TetR and sfGFP modified by LOVdeg tags, we lysed the bacteria under
dark and light conditions respectively, extracted the proteins and detected them through Western blot (WB)
experiments. Experiments have proved that both of these two proteins are expressed normally.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/n.webp)

Fig42. The WB results induced by different light conditions showed that sfGFP and TetR-LOVdeg monomers
were detected to be normally expressed in the supernatant. After SDS treatment, the LOVdeg portion of the
TetR-LOVdeg fusion protein was also successfully detected. In addition to the target protein band,
"non-specific bands" were also observed. It is speculated that this is due to the successful unfolding of
the Jα domain of LOVdeg, exposing the SsrA tag containing EAA, which in turn recruited endogenous ClpX and
ClpA enzymes, triggering N-terminal degradation.

In order to measure the rate of gene expression leakage, we diluted bacterial suspension and uniformly
spread it on LK plates; following a certain period of light-protected incubation, color-based colony
screening was performed using ImageJ software, enabling the extraction and counting of green colonies.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/o.webp)

Fig43. Expected Results of the Experiment

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/p.webp)

Fig44. The Leakage Rate Results Produced by Image J

In this experiment, the leakage rate of the fluorescence quantification system was determined to be 84.92%
for the BL21(DE3) strain and 82.51% for the JM109(DE3) strain. It was indeed observed that the system
exhibits severe leakage.

Yet we remain undeterred, as **a high leakage rate is not equivalent to a high leakage
magnitude** and does not
necessarily imply constitutive overexpression of the toxic MazF protein in the light-induced suicide system,
nor does it indicate a failure to exert the intended light-induced lethal function.

2. tetR\*-mazF genetic circuit

After verifying the green fluorescent protein system, we tested the target light-induced suicide module
(suicide system) : qualitatively detecting the correct characterization of the TetR-LOVdeg protein in the
gene circuit and the normal expression of the MazF protein; At the same time, the suicide rate was
qualitatively reflected by diluting and spreading the bacterial liquid, and the growth curve was combined to
determine and evaluate whether the strain did not grow as expected under light.

![Light-off circuit diagram](https://static.igem.wiki/teams/5578/image/engineering/q.webp)

Fig45. The overview of the fluorescence quantification system genetic circuit

To verify the successful expression of TetR and MazF modified by LOVdeg tags, we lysed the bacteria under
dark and light conditions respectively, extracted the proteins and detected them through Western blot (WB)
experiments. Experiments have proved that both of these two proteins are expressed normally.

![这里没有图](https://static.igem.wiki/teams/5578/image/engineering/s.webp)

Fig46. The Western Blot result induced by different lighting conditions

Normal expressions of sfGFP and TetR-LOVdeg were detected in the supernatant. Light exposure combined to
form a large amount of toxic protein MazF, confirming the correctness of the gene pathway. However, a small
amount of MazF was also detected in JM109 (DE3) (Lane 5) under dark conditions, indicating a leak in the
genetic circuit. At the same time, it was observed that the content of the target protein in the precipitate
was significantly higher than that in the supernatant. It is speculated that the circuit leakage may be
related to the abnormal folding of the TetR-LOVdeg fusion protein.

A distinctive non-viable colony morphology was observed, which we interpret as colonies in which cells had
undergone MazF-mediated killing and thus failed to survive. In contrast, colonies that remained viable were
defined as “survivors.” The suicide efficiency of the system in this experiment was determined to be 44.19%.

To more intuitively verify the inhibitory effect of MazF toxic proteins on the growth of strains induced by
light, it is necessary to measure the mazF genotype of E.coli JM109(DE3) growth curve under lighting
conditions.

![这里没有图](https://static.igem.wiki/teams/5578/image/engineering/r.webp)

Fig47. Growth curves of the mazF genotype of E.coli JM109(DE3) with logistic model under lighting
conditions

It was observed that the growth of the JM109(DE3) strain was indeed severely affected by the toxic
expression of MazF: growth was temporarily arrested at one stage, yet it eventually resumed normal growth
and failed to achieve the desired permanent growth arrest.

---

![Learn](https://static.igem.wiki/teams/5578/image/engineering/learn-1.webp)

The experimental results show that the construction scheme of the blue-induced Escherichia coli suicide
system is feasible. This system can serve as a potential migratory biosafety module in synthetic biology -
if the engineered bacteria are accidentally released into the environment, the blue light in natural light
can initiate the suicide program, effectively preventing the leakage of the engineered bacteria. However,
this system has problems such as severe leakage, being able to only inhibit the growth of strains but not
achieving permanent stagnation. Due to time constraints, the related exploration is still ongoing and we
look forward to presenting it at jamboree.

## **References**

[1] Kapardar, K. *et al.* (2010)
‘Identification and characterization of genes conferring salt tolerance to Escherichia coli from pond
water metagenome’, *Bioresource Technology*, 101(11), pp. 3917–3924.
doi:10.1016/j.biortech.2010.01.017.[↩︎](#fnref1)

[2] Li, A.*et al.* (2024) ‘Overwintering and
summer survival of Ulva prolifera in sediments: Indoor simulation of temperature impacts’, *Marine
Pollution Bulletin*, 201. doi:10.1016/j.marpolbul.2024.116233.[↩︎](#fnref2)

[3] Xia, Z.*et al.* (2022) ‘A review of
physical, chemical, and biological green tide prevention methods in the Southern Yellow Sea’, *Marine
Pollution Bulletin*, 180. doi:10.1016/j.marpolbul.2022.113772.[↩︎](#fnref3)

[4] Ederth, T.*et al.* (2009) ‘*Interactions
of Zoospores of Ulva linza with Arginine-Rich Oligopeptide Monolayers*’. Available at:
https://search.ebscohost.com/login.aspx?direct=true&db=edsoai&AN=edsoai.on1234364910&site=eds-live&scope=site
(Accessed: 11 September 2025).[↩︎](#fnref4)

[5] <https://2023.igem.wiki/xjtlu-china/index.html>[↩︎](#fnref5)

[6] Ma, Q. and Wood, T.K. (2009) ‘OmpA influences
Escherichia coli biofilm formation by repressing cellulose production through the CpxRA two-component
system’,*Environmental Microbiology*, 11(10), pp. 2735-2746–2746.
doi:10.1111/j.1462-2920.2009.02000.x.[↩︎](#fnref6)

[7] Serra, D. O., Richter, A. M., & Hengge, R.
(2013). Cellulose as an architectural element in spatially structured Escherichia coli
biofilms. *Journal of Bacteriology*, *195*(24). <https://doi.org/10.1128/jb.00946-13>[↩︎](#fnref7)

[8] Tong, Y., Jørgensen, T. S., Whitford, C. M.,
Weber, T., & Lee, S. Y. (2021). A versatile genetic engineering toolkit for E. coli based on
CRISPR-prime editing. *Nature Communications*, *12*(1), 5206. <https://doi.org/10.1038/s41467-021-25541-3>[↩︎](#fnref8)

[9] Ageorges, V.*et al.* (2019) ‘Differential
homotypic and heterotypic interactions of antigen 43 (Ag43) variants in autotransporter-mediated
bacterial autoaggregation’, *Scientific Reports*, 9(1). doi:10.1038/s41598-019-47608-4.[↩︎](#fnref9)

[10] Joint, I., Tait, K. and Wheeler, G. (2007)
‘Cross-Kingdom Signalling: Exploitation of Bacterial Quorum Sensing Molecules by the Green Seaweed
Ulva’, *Philosophical Transactions: Biological Sciences*, 362(1483), pp. 1223–1233.
doi:10.1098/rstb.2007.2047.[↩︎](#fnref10)

[11] Glass, D.S. and Riedel-Kruse, I.H. (2018) ‘A
Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and
Patterns’,*Cell*, 174(3), pp. 649–658. doi:10.1016/j.cell.2018.06.041.[↩︎](#fnref11)

[12] Kim, H.*et al.* (2022) ‘4-bit adhesion
logic enables universal multicellular interface patterning’, *Nature: International weekly journal of
science*, 608(7922), pp. 324–329. doi:10.1038/s41586-022-04944-2.[↩︎](#fnref12)

[13] Nathan Tague*et al.* (2024)
‘Light-inducible protein degradation in E. coli with the LOVdeg tag’, *eLife*, 12.
doi:10.7554/eLife.87303.[↩︎](#fnref13)

[14] Serra, D.O., Richter, A.M. and Hengge, R. (2013) ‘Cellulose as an architectural element in spatially
structured *Escherichia coli* biofilms’, *Journal of Bacteriology*, 195(24), pp. 5540–5554.
doi:10.1128/JB.00946-13.[↩︎](#fnref14)

[15] Tong, Y. *et al.* (2021) ‘A versatile genetic engineering toolkit for *E. coli* based
on CRISPR-prime editing’, *Nature Communications*, 12(1), 5206 [Preprint]. Available at:
https://search.ebscohost.com/login.aspx?direct=true&db=edsoai&AN=edsoai.on1280591207&site=eds-live&scope=site
(Accessed: 5 October 2025).[↩︎](#fnref15)

[16] Nelson, J.W. *et al.* (2022) ‘Engineered pegRNAs improve prime editing efficiency’,
*Nature Biotechnology: The Science and Business of Biotechnology*, 40(3), pp. 402–410. doi:
10.1038/s41587-021-01039-7.[↩︎](#fnref16)

[17] [2023 iGEM Fudan Team
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2025).[↩︎](#fnref17)

{% endblock %}

{% extends "layout\_page.html" %}
{% block title %}Experiments{% endblock %}
{% block banner\_image %}https://static.igem.wiki/teams/5578/image/experiment/experiment.webp{% endblock %}
{% block page\_content %}

## **Bacterial Transformation**

---

1. Add 2.0 μL of DNA solution (purified) to Competent cells (Top10/BL21(DE3)) 50 μL (Please use from
   the “used” cells in our deep freezer after melting.)

2. Stand on ice 30 min

3. Heat 42℃for 45 s

4. Stand on ice for 2 min

5. Add 250 μL of SOC/LB medium

6. Incubate tubes at 37℃at 200 rpm for 1 hour

7. Plate50 μL/200 μL onto LK plates

8. Put the plate upside down and cultivate overnight at 37 °C

## **Restriction Enzyme Digestion**

---

**Preparation of linearized vector
fragments**

1. Prepare reaction mixture as follows.

   |  |  |
   | --- | --- |
   | Plasmid (about 0.1-1 μg/μL) | 5 μL |
   | 10X Cut Buffer | 5 μL |
   | Restriction enzyme | 0.4 μL |
   | dd H2O | 39.6 μL |
   | Total reaction volume | 50 μL |

   **\*Note:** Confirm the
   restriction enzyme units are enough to digest all plasmids. All operations should be done on
   ice. Don't forget to bring enzymes back to -25 °C after using. Please use the water bath to
   incubate the mixture overnight, but not heat block.

2. Please incubate the mixture at 37 °C overnight.

## **Plasmid DNA Mini Preparation**

---

1. Column Equilibrium Step: Add 500 μLof equilibration buffer BL to the adsorption column CP4 (place
   the column in a collection tube). Centrifuge at 12,000 rpm (~13,400×g) for 1 min, discard the waste
   liquid in the collection tube, and return the adsorption column to the collection tube. *(Please
   use columns processed on the same day.)*

2. Transfer 5–15 mLof overnight bacterial culture into a centrifuge tube and centrifuge at 5,000×g for
   10 min. Invert the tube on paper for 5 min to remove as much supernatant as possible.

   **\*Note**:
   If the bacterial
   culture volume is large, pellet the cells through multiple centrifugations and combine them into
   one tube. The amount of bacterial cells should be optimal for complete lysis. Excessive
   bacterial cells may lead to insufficient lysis and reduce plasmid extraction efficiency.

3. Add 500 μLof Solution P1 (*ensure RNase A has been added*) to the centrifuge tube containing
   the cell pellet. Re-suspend the bacterial cell pellet thoroughly using a pipette or vortex mixer.
   *(1.5 ml EP tube)*

   **\*Note**: Ensure complete
   re-suspension of the bacterial pellet. Incomplete mixing may affect lysis, leading to lower
   yield and purity.

4. Add 500 μLof Solution P2 to the centrifuge tube and gently invert 6–8 times to ensure complete
   lysis.

   **\*Note**: Mix gently—avoid
   vigorous shaking to prevent genomic DNA contamination. The lysate should become clear and
   viscous within no more than 5 min to avoid plasmid damage. If the solution remains cloudy, the
   bacterial load may be too high; reduce the amount for complete lysis.

5. Add 500 μLof Solution P4 to the centrifuge tube and immediately invert gently 6–8 times to mix
   thoroughly. A white flocculent precipitate will form. Let it stand at room temperature for ~10 min,
   then centrifuge at 12,000 rpm (~13,400×g) for 10 min. A pellet will form at the bottom of the tube.

   **\*Note**: After adding P4, mix
   immediately to avoid localized precipitation. If small white precipitates remain in the
   supernatant, centrifuge again and collect the supernatant.

6. Transfer the supernatant from the previous step in batches to the filtration column CS (*placed
   in a collection tube*). Centrifuge at 12,000 rpm (~13,400×g) for 2 min, and collect the
   filtrate in a clean 2 mLcentrifuge tube (*user-provided*).

7. Add isopropanol (0.3× the filtrate volume, 400–500μL) to the filtrate. (*Excessive isopropanol
   may cause RNA contamination.*) Mix by inverting, then transfer the mixture to the adsorption
   column CP4 (*placed in a collection tube*).

   **\*Note**: Due to potential filtrate loss,
   adjust the isopropanol volume
   accordingly. The CP4 column has a maximum capacity of 700 μL, so load the sample in batches.

8. Centrifuge at 12,000 rpm (~13,400×g) for 1 min at room temperature. Discard the flow-through and
   return the adsorption column to the collection tube.

   **\*Note**: If the solution from Step 7 is
   loaded in batches, repeat this
   step for each aliquot.

9. Add 500 μLof protein removal buffer PD to the adsorption column CP4. Centrifuge at 12,000 rpm
   (~13,400×g) for 1 min, discard the flow-through, and return the column to the collection tube.

10. Add 600μL of wash buffer PW (*ensure absolute ethanol has been added*) to the adsorption
    column CP4. Centrifuge at 12,000 rpm (~13,400×g) for 1 min, discard the flow-through, and return the
    column to the collection tube.

    **\*Note**: Letting the column sit for 2–5 min at room temperature after adding PW
    may improve impurity removal.

11. Repeat Step 10 with another 600μL of wash buffer PW. Centrifuge and discard the flow-through.

12. Reinsert the adsorption column CP4 into the collection tube and centrifuge at 12,000 rpm (~13,400×g)
    for 2 min to remove residual wash buffer.

    **\*Note**: Ethanol residue may interfere with downstream enzymatic reactions (e.g.,
    digestion, PCR). To ensure complete removal, air-dry the column at room temperature for a few
    minutes with the cap open.

13. Place the adsorption column CP4 in a clean centrifuge tube. Apply 50–300 μLof preheated 60 °C ddH₂O
    or elution buffer TB directly to the center of the membrane. Incubate at room temperature for 5 min,
    then centrifuge at 12,000 rpm (~13,400×g) for 1 min to collect the plasmid solution.

    **\*Note**: For
    higher yield,
    reload the eluate onto the column and repeat Step 13. Elution efficiency depends on pH: Use
    elution buffers with pH 7.0–8.5 (pH <7.0 reduces yield). Minimum elution volume: 100
    μL (lower volumes decrease recovery). Store DNA at –20℃ to prevent degradation. (*Perform two
    elutions totaling 100 μL.*)

## **PCR**

---

1. Please make two tubes of PCR mixtures as follows.

   |  |  |
   | --- | --- |
   | **Component** | **Volume** |
   | ddH2O | (18.25 - x) μL |
   | 10X PCR Buffer (with Mg²⁺) | 2.5 μL |
   | dNTP (2.5 mM each) | 2 μL |
   | Template DNA (final 50 ng/μL) | x μL |
   | Primer 1 (10 μM each) | 1 μL |
   | Primer 2 (10 μM each) | 1 μL |
   | Taq DNA Polymerase (2.5 U/μL) | 0.25 μL |
   | **Total** | **25 μL** |

2. PCR by following program PCR program

   1) 94 ℃
   3 min --- denature

   2) 94 ℃ 30 sec --- denature

   3) 55 ℃ 30 sec --- annealing

   4) 72 ℃ t min (min/kb) --- extension

   5) GOTO step 2), 30 times

   6) 72 ℃ 10 min

   7) 12 ℃ Forever

3. Check the PCR products by 1.0% agarose electrophoresis

## **Gel Electrophoresis**

1. 1% or 1.5% Agarose gel electrophoresis, 100 V for checking (apply 12 μL/lane)

2. Use 5 μL/lane for Beyotime DNA Ladder (0.1-10 kb, 21 bands, figure below)

3. Stain the gel for 30 min with about 200 mL of GelRed (1/3300 diluted for bubble dyed)

4. Check the stained bands by Imaging system. Put the stained agarose gel on the UV tray.

5. Please recycle the GelRed solution in the light-protected boxes.

![1](https://static.igem.wiki/teams/5578/image/experiment/image.webp)

Figure 1. DNA
ladder (0.1-10 kb) used as a molecular size marker.

## **IPTG induction**

**Materials and Source**

* Overnight culture
* LB broth with 50 μg/mL Kanamycin
* Refrigeration Centrifuge
* 100 mM IPTG

**\*Note**: To prepare a 100 mM IPTG solution, 2.383 g of IPTG powder should be weighed and dissolved
in 100 mL of deionized water. After filtration and sterilization, it should be packaged and stored at -20 ℃.

**Procedure**

1. The overnight cultures are diluted 100-fold in fresh LB medium with 50 μg/mL Kanamycin,
   and cultured at 37 ℃ until they reached an optical density (OD) at 600 nm of 0.6.
2. Cells could be pelleted at 4000 g and gently resuspended in an induction medium containing
   0.6 mM, 0.8 mM, 1.2 mM IPTG and 50 μg/mL antibiotics.
   Protein expression is allowed to occur at 37 ℃ for 4 hours or 16 ℃ overnight.

**\*Note**: 0.4 mM IPTG is not necessarily the optimal concentration.
It is proper to make the concentration gradient, such as from 0.2 mM to 1.2 mM IPTG (0, 0.5, 1 mM first).

## **Nanodrop**

---

**Materials used in the Nanodrop process**

* Nanodrop instrument
* ddH₂O

* Liquid to be tested
* Solvent
* Delicate task wiper

**Procedure**

1. Before measuring the concentration of the substrate, first use 2 μL of ddH₂O in the cuvette of the instrument,
   and gently close and open the cover. Then use a delicate task wiper to wipe the cuvette.

2. Repeat the first step 2 times.
3. Use a pipette to drop 2 μL of the solvent, turn on Nano 2000c, click the blank button, and measure.
   Perform the zeroing procedure.

   **\*Note**: If using ddH₂O (EB buffer) to elute the column which contains DNA,
   use ddH₂O (EB buffer) to measure the standard curve.

4. Use a pipette to drop 2 μL of the liquid to be tested on the cuvette,
   close the cover gently, and click the start button.
5. Write down the statistics of concentration, A260/280, and A260/230 on the screen
   and compare them with the expected data.
6. After finishing the Nanodrop process, use ddH₂O to wash the cuvette.

## **Streaking**

---

**Procedure**

1. Pick a well-separated colony on a plate with relatively few colonies. Using a heat-sterilised inoculating loop,
   streak the colony out onto a separate agar plate.
2. Aseptically remove a loopful of bacteria from the sample and transfer it to section 1 of the agar. Evenly
   distribute the inoculum over the surface of section 1 in a back-and-forth motion.
3. Flame sterilize the loop and let it cool.
4. Turn the plate and spread the bacteria from section 1 to section 2 as diagrammed.
5. Flame sterilize the loop again and repeat spreading the bacteria from section 2 to section 3.
6. Flame sterilize the loop again and repeat going into section 4 of the plate.

## **Mini-scale liquid culture in tubes**

---

1. Check the streaked colonies.
2. Bring LK medium from 4 ºC to room temperature.
3. Put 10 mL of LK medium into 50 mL tubes.
4. Pick up a colony with a pipette tip or inoculating loop and inoculate into the LK medium.
5. Please don’t forget to open the cap and seal with tape!
6. Shake at 37 ºC at 150–200 rpm for more than 12 hrs or overnight.
7. Transfer bacterial solution from 50 mL tubes to 2 mL Eppendorf tubes.

8. Centrifuge at 3000–5000 ×g for 10 min in SIGMA 1-14K centrifuge at room temperature.
9. Discard the supernatants to the bacteria drain.

**Aim:** various culture for mini-prep & growth curve

## **Preparation of LB medium for small culture**

---

**Materials**

1. 500 mL Bottle
2. LB premix powder (20 g for 1 L)

**Procedure**

1. Take 400 mL of MilliQ water into a cylinder.
2. Pour the water into the 500 mL bottle.
3. Weigh 8 g of premix LB powder and add it into the bottle.
4. Cover with aluminum foil and fix with sterilization tape or seal, then put into the autoclave.
5. Never forget to open the cap!
6. Autoclave for 20 min, at 121 ℃, under 2 atm pressure.
7. After cooling to 60 ℃, add 1/1000 volume of kanamycin (1 g/mL) before use.
8. Store at 4 ℃.

## **Preparation of LK plates**

---

**Materials**

1. 200–300 mL Flask
2. LB premix powder (LB nutrient agar)

**Procedure**

1. Measure 100 mL of MilliQ water using a cylinder.
2. Pour the water into a 200–300 mL flask.
3. Weigh LB nutrient agar for 100 mL and add into the flask.
4. Cover with aluminum foil and fix with sterilization tape or seal, then put into the autoclave.
5. If there is a cap, never forget to open it!

6. Autoclave for 20 min at 121 ℃, under 2 atm pressure.
7. After around 90 min, check the autoclave temperature. If below 60 ℃, move the bottles to the bench and add
   1/1000 volume of kanamycin (1 g/mL).
   *If there are bottles with agar, move them to a 60 ℃ dry heater. Ensure the kanamycin concentration is 50
   μg/mL. Mix gently!*
8. Pour 20 mL medium into each 9 cm dish.
9. Let the dishes stand until solidified.
   *If space and time allow, keep lids open during solidification to prevent condensation on the lid.*
10. Store the plates upside down at 4 ℃.

## **RIPA lysis**

---

**Procedure**

1. For 1 mL bacterial solution, centrifuge to remove the supernatant.
2. Wash the pellet once with PBS. Centrifuge (12,000 rpm, 5 min), remove the supernatant, and gently tap the tube
   bottom to disperse the pellet.
3. Add 200 μL lysis buffer (protease inhibitor optional). Vortex gently or tap to mix, then lyse on ice for 20 min,
   vortexing every 4 min.
4. *Optional:* For better lysis, treat bacteria with lysozyme before using this buffer.

## **Ultrasonication**

---

**Materials**

* Fisherbrand FB505 Ultrasonic Crusher
* Processing capacity: 0.2–1000 mL
* 500 W, 20 kHz
* Programmable, 1 s to 10 h
* Fisherbrand FB50 Ultrasonic Crusher
* Processing capacity: 0.2–50 mL
* 50 W, 20 kHz
* Manual operation

**Procedure**

1. Centrifuge the induced sample (3000 ×g, 5 min) and discard the supernatant.
2. Wash the cells 1–2 times with equal volume of PBS buffer, centrifuging each time.
3. Resuspend the pellet in 10 mL PBS buffer.
4. Insert the ultrasonic probe into the suspension (kept in an ice-water bath).

5. Ultrasonic conditions: 150 W, work for 1 s with a 3 s interval, total 20 min.
6. After sonication, centrifuge at 4 ℃, 12,000 ×g for 20 min.
7. The supernatant is the crude enzyme solution (take 40 μL as the supernatant sample).
8. Resuspend the pellet in 10 mL PBS buffer (take 40 μL as the pellet sample).
9. Store both the supernatant and pellet suspension at –20 ℃.

## **Bicinchoninic Acid Assay**

---

**Equipment**

* Microplate reader (capable of measuring absorbance at 562 nm)
* 96-well plate
* 37 ℃ incubator
* Adjustable pipettes and tips
* Deionized water
* EP tubes

**Reagent Preparation**

**\*Note:** If crystals appear in BCA Reagent A, gently heat until dissolved.

1. 1× PBS: Dilute 5× PBS with deionized water to 1× before use. Store at 4 ℃ for up to 12 months.
2. BSA Standard (2 mg/mL): Dissolve 2 mg BSA in 1 mL 1× PBS. Aliquot and store at –20 ℃ to avoid freeze–thaw
   cycles.
3. BCA working solution: Mix BCA Reagent A and Reagent B at 50:1 immediately before use. Store at 4 ℃ for up to 24
   h.

**\*Note**: If crystals form in BCA
Reagent A, gently heat at 37°C until fully dissolved. If flocculent precipitates appear, mix well before
use.

**Procedure**

1. Preheat the microplate reader for ≥30 min and set the wavelength to 562 nm.

2. 96-well plate

   ![1](https://static.igem.wiki/teams/5578/image/experiment/image-1.webp)

{% raw %}3. Prepare 20 μL standard solutions, then add 20 μL samples to the 96-well plate. Add 200 μL BCA
   working solution to each well and mix thoroughly. Incubate at 37°C for 30 min, then measure the OD
   values at 562 nm.
   * Blank well (0 µg/mL): Record as $A\_{empty}$
   * Standard wells: Record as $A\_{standard}$
   * Sample wells: Record as $A\_{sample}$
{% endraw %}

4. Calculate:
   * ΔAstandard = Astandard − Aempty
   * ΔAsample = Asample − Aempty

5. Data Analyse
   * Standard Curve: Plot ΔA(y-axis) vs. standard concentration
     (µg/mL, x-axis).

     standard
   * Sample Concentration: Substitute ΔAinto the standard curve
     equation to obtain x (µg/mL).

     sample

   \*Tip: If samples were further diluted,
   multiply the result by the dilution factor (n).

## **Western Blotting**

---

**SDS-PAGE Electrophoresis**

1. Run the SDS-PAGE by constant 15 mA for stacking gel and 30 mA for separating, or constant 200Vfor
   whole gels.

2. When dye reached 5 mm in distance from the bottom of gel, stop running the PAGE.

**Procedure**

1. Immerse a new PVDF membrane in 100% methanol for 30 sec (you can recycle this methanol.)

2. Add the Wet transfer buffer to the PVDF membrane, and shake it for >15 min.

3. During shaking, immerse 2 filter papers in the transfer buffer together with the membrane.

4. Immediately after finishing gel, apart the gel plates from the device, open the glasses, and remove
   the stacking gel carefully and cut the bottom corner at the 1st lane.

5. Immerse the separating gel in transfer buffer and shake it for 15 min.

6. Wear transparent PE gloves and put a filter paper onto the black sponge on the black plastic side,
   and put the gel on the filter papers. Note, please construct the gel holder cassette in the large
   box with sufficient transfer buffers. Check bubbles, please remove them.

7. Put the PVDF on the gel, and put another filter paper on it.

8. Please push the gel from the surface of papers to remove bubbles.

9. Close the gel holder cassette and insert it to the electrode module.

10. Please put 2 frozen ice boxes in the buffer tank, and apply constant 100V for 60 min. Due to your
    results, you should refine suitable time for transferring (40-90 min).

11. Please prepare the blocking solution (2% BSA or 5% skim milk in PBS-T, and so on) during
    transferring.

12. Immediately after transferring, open the lid and mark the cutting corner of gel and the transferred
    side of PVDF membrane by a water-resistant ball pen.

13. Immerse the membrane into the blocking solution and shake it for 30-60 min.

14. Wash the membrane 1-3 times of 3-5 min shaking in the PBS-T.

15. Please dilute the 1st antibody by PBS-T (or blocking solution) to make 1-1.4 mL. In case of
    anti-FLAG, recommended dilution is 1/2,000 for the first trial. Note, please bring antibody on ice
    and after using, please bring antibody back to -25 ºC immediately!

16. Sand the membrane between parafilms or clear pockets to start 1st antibody reaction.

17. After 45-60 min, wash the membrane with the PBS-T, 3-5 times by 3-5 min shaking.

18. Please dilute the 2nd antibody by PBS-T to make 1-1.4 mL. In case of anti-mouse IgG, 20,000 times
    dilution should be recommended. Note, please bring antibody on ice and after using, please bring
    antibody back to -25 ºC immediately!

19. Add the diluted 2nd antibody (1-1.4 mL) to the PVDF membrane between parafilms.

20. After 45-60 min, wash the membrane with the PBS-T, 3-5 times by 3-5 min shaking.

21. Before finishing reaction, bring ECL solution A and B (Biosharp) and take each 0.5-0.7 mL and warm
    them at room temperature, and turn on the BioRad ChemiDoc MP.

22. Mix ECL A with B and incubate the mixture (total 1-1.4 mL) with the PVDF membrane on a parafilm or
    in a clear pocket for 1 min.

23. Take images of chemiluminescence from the membrane using BioRad ChemiDoc MP. Choose
    Application-Blot-Chemiluminescence. Put the membrane on the Blot/UV/StainFree tray.

24. To merge molecular markers, please choose Application-Blot-Colorimetric after obtaining the
    chemiluminescence image.

## **Crystal Violet Staining**

---

**Procedure**

The experiments were performed on a 96 well plate.

1. The initial *E. coli* stock culture was prepared by adding 1mLinoculum into 9mL standard LB broth and
   allowing the *E. coli* to grow overnight (16-18 hrs) at 37℃ (induced by IPTG whenunder orbital shaker
   at 200 rpm.

2. Next day, the optical density (OD) at 600nm was checked. Value at 600nm must be 2.0 or dilute it.

3. Few wells of plate are filled with 200μL of fresh LB as blank.

4. The culture was diluted in the ratio of 1:100 with 195μL of fresh LB broth and 5μL of culture in the
   well.

5. The plates were then transferred to thermostatic static incubators maintained at 37℃.

6. To avoid evaporation, the external area of the plate was filled with less amount of distilled water.

7. After 24 hrs, the 96 well plate was taken from the incubator and the planktonic cells were removed.

8. The wells were then washed using sterile PBS.

9. Each well was then added with 200 μLof 0.1% crystal violet and was kept for 15 min for staining.

10. The crystal violet was then removed and washed with sterile PBS and kept the plate for air drying
    for 5 min.

11. The biofilm was quantified in duplicate, after adding 200 μL of 95% ethanol, by an enzyme-linked
    immunosorbent assay plate reader at 570 nm.

{% endblock %}

{% extends "layout\_page.html" %}
{% block title %}Project Description{% endblock %}
{% block lead %}Describe how and why you chose your iGEM project.{% endblock %}
{% block banner\_image %}https://static.igem.wiki/teams/5578/image/result/2025-10-07-20-00-48.webp{% endblock %}
{% block page\_content %}

Two high-expression *Escherichia coli* (*E. coli*) strains, **BL21(DE3)** and **JM109(DE3)**, were employed throughout the wet experiment. T
his approach aimed to provide dual guarantees for the reliable acquisition of experimental
data and the proper characterization of proteins; Additionally, we compared the activity
and functionality of the two strains and selected the superior one as the final engineered
strain to better perform the task of *Ulva* spore clearance.

![magician](https://static.igem.wiki/teams/5578/image/result/magician.webp)

## Survival Module

In the Survival module, we introduced the **salt-tolerant** **genes,*****gspM*** **and** ***echM***
to arm our biofilm with the necessary adaptability against inhospitable conditions.

#### Bacterial growth curve analysis

![](https://static.igem.wiki/teams/5578/image/result/sketch-map.webp)

Fig1. The sketch map of bacterial growth curve analysis.

We aim to quantitatively compare the growth curves of the engineered bacteria and
wild-type strains carrying the [echM-gspM]-pET28a(+) plasmid under high-salt (600 mM NaCl)
and salt-free conditions, in order to validate the function of the salt-tolerance gene. The
OD600 values of each strain group in different media were measured at regular intervals
from 0 to 27 hours, and a Logistic model was applied for curve fitting and analysis.

![](https://static.igem.wiki/teams/5578/image/result/growth-curves.webp)

Fig2 & 3 & 4 & 5 & 6. Growth curves of E.coli strains with logistic model.

We found that the engineered bacteria in 600mM NaCl medium showed normal growth
conditions similar to those in LB medium without NaCl, while the growth of
*Escherichia coli* in the negative control group was significantly inhibited
in a high-salt environment. These results proves that salt-tolerant genes do indeed make
engineered bacteria more adaptable to high-salt environments. However, further analysis
reveals that the salt-free LB medium is slightly more suitable for the growth of
engineered bacteria compared to the 600mM NaCl salt-containing LB medium.
This indicates that the engineered bacteria are still to some extent inhibited
by the stress of a high-salt environment.

## Light suicide module

By fusing the **LOVdeg** tag to **TetR**, the engineered
*E. coli* gains the ability to sense blue light (450–490 nm) and thereby regulate
activation of the suicide mechanism downstream of the Tet operon. Consequently, upon
entering open marine environments and encountering blue light, the engineered bacteria
will autonomously initiate their suicide program, thereby mitigating the risk of
engineered bacteria contamination.

#### Fluorescence quantification system

The fluorescence quantification system was established to simulate experimental
conditions and qualitatively detect three key indicators: the proper characterization
of the TetR-LOVdeg protein, the normal inducible expression of sfGFP of gene circuits,
and the leakage of the light-induced suicide system. Additionally,
**we used** this system to semi-quantitatively determine the
leakage level of gene circuits via fluorescence quantification.

![](https://static.igem.wiki/teams/5578/image/result/tetr-gfp.webp)

Fig7. The overview of the fluorescence quantification system genetic circuit.

##### Protein expression of sfGFP and TetR-LOVdeg

Bacteria were lysed under both dark and light conditions, followed by protein extraction
and subsequent Western blot (WB) assay.

![](https://static.igem.wiki/teams/5578/image/result/wb1.webp)

Fig8. The Western Blot(WB) result induced by different lighting conditions.

Normal expression of sfGFP and TetR-LOVdeg monomer (Dimer would be degraded by SDS
treatment due to non-covalent bond connection.) was detected in the supernatant.
Furthermore, following SDS treatment, the LOVdeg moiety of the TetR-LOVdeg fusion
protein—harboring a 6×His-tag—was successfully detected. Notably, besides the band
corresponding to the target protein, we always observed additional "non-specific bands"
even after optimizing the blocking step to its optimal state. We propose that this
phenomenon arises from the successful unfolding of the Jα domain in LOVdeg: this unfolding
exposes the EAA-containing SsrA tag, which subsequently mediates the recruitment of
endogenous ClpX and ClpA enzymes, thereby triggering N-terminal degradation.

##### Leakage rate measurement

Bacterial suspension was diluted and uniformly spread on LK plates; following
a certain period of light-protected incubation, color-based colony screening was
performed using ImageJ software, enabling the extraction and counting of green
colonies.

![](https://static.igem.wiki/teams/5578/image/result/fluorescence-quantification-experiment.webp)

Fig9. Expected results of the experiment.

![](https://static.igem.wiki/teams/5578/image/result/example.webp)

Fig10 & 11 & 12. The example of BL21(DE3) leakage rate results produced by Image J.

In this
experiment, the leakage rate of the fluorescence quantification system was determined
to be 84.92% for the BL21(DE3) strain and 82.51% for the JM109(DE3) strain. It was
indeed observed that the system exhibits severe leakage; yet we remain undeterred,
as a high leakage rate is not equivalent to a high leakage magnitude and does not
necessarily imply constitutive overexpression of the toxic MazF protein in the
light-induced suicide system, nor does it indicate a failure to exert the intended
light-induced lethal function.

#### Suicide system (L.U.C.I.A.)

A suicide system was established, which serves as the light-induced suicide module
we aim to ultimately implement. Additionally, we qualitatively detected the proper
characterization of the TetR-LOVdeg protein in the gene circuit and the normal inducible
expression of the toxic MazF protein in the same circuit. Meanwhile, the suicide rate
was qualitatively reflected via bacterial liquid dilution and spreading, and growth
curve determination was used to assess whether the strains failed to grow as expected
under inducing conditions.

![](https://static.igem.wiki/teams/5578/image/result/tetr-mazf.webp)

Fig13. The overview of the fluorescence quantification system genetic circuit.

##### Protein expression of MazF and TetR-LOVdeg

Bacteria were lysed under both dark and light conditions, followed by protein extraction
and subsequent Western blot (WB) assay.

![](https://static.igem.wiki/teams/5578/image/result/suicide-experiment.webp)

Fig14. Expected results of the experiment.

![](https://static.igem.wiki/teams/5578/image/result/wb2.webp)

Fig15. The Western Blot(WB) result induced by different different lighting conditionst.

We detected sfGFP and TetR-LOVdeg at normal levels in the supernatant. Furthermore,
after SDS treatment, the LOVdeg moiety of the TetR-LOVdeg fusion protein—carrying
a 6×His-tag—was successfully detected. In the light-treated experimental group, a
significant amount of the toxic protein MazF was synthesized, confirming the
correctness of the gene pathway. However, in lane 5 (JM109(DE3) under dark conditions),
a small amount of MazF was also observed, indicating that the gene circuit still
exhibits leakage. Meanwhile, we clearly observed that the target protein content
in the pellet fraction was significantly higher than in the supernatant fraction.
We therefore speculate that the leakage of the gene circuit may be related to the
improper folding of the TetR-LOVdeg fusion protein.

##### Suicide strain growth curve measurement

We measured the mazF genotype of *E.coli* JM109(DE3) growth curve under
lighting conditions

![](https://static.igem.wiki/teams/5578/image/result/data-8.webp)

Fig16. Growth curves of the mazF genotype of E.coli JM109(DE3) with logistic model under lighting conditions.

It was observed that the growth
of the JM109(DE3) strain was indeed severely affected by the toxic expression
of MazF: growth was temporarily arrested at one stage, yet it eventually resumed
normal growth and failed to achieve the desired permanent growth arrest.

## Programmable biofilm

#### **Ag-Nb System**

To enable functional cooperation, we engineered *E. coli* to express outer
membrane-anchored nanobody–antigen (Nb–Ag) pairs. This modification not only facilitates
spontaneous cell adhesion but also allows the generation of programmable assembly patterns
within the living biofilm.

To verify that biofilm formation is mediated by the
intimin–antigen/nanobody (intimin–Ag3/Nb3) interaction, we performed both aggregation
assays and fluorescence microscopy imaging to confirm its capacity to induce biofilm
formation.

#### Escherichia coli self aggregation assay

We measured the
OD600 of the supernatant at 0, 2,4, 6 hours to reflect the bacteria quantity
remaining in the supernatant.

![](https://static.igem.wiki/teams/5578/image/result/sb.webp)

Fig17. Aggregation assay using microplate reader (OD600) measurements.

![](https://static.igem.wiki/teams/5578/image/result/bacteria-percentage1.webp)
![](https://static.igem.wiki/teams/5578/image/result/bacteria-percentage2.webp)
![](https://static.igem.wiki/teams/5578/image/result/bacteria-percentage3.webp)

Fig18 & 19 & 20. Bacteria percentage remaining in the supernatant.

We observed that at 6 hours, in the IPTG-induced E. coli samples,
bacteria percentage remaining in the supernatant was significantly lower
compared to the uninduced samples. This indicates that the intimin-Ag/Nb pairs can
effectively promote the binding between E. coli.

#### Fluorescence microscopy imaging

To make the experimental results more intuitive, we respectively used mCherry and
sfGFP to characterize the expressions of Ag and Nb.

1. Insert the intimin-Ag3 fusion protein and characterize it with mCherr
2. Insert the intimin-Nb3 fusion protein and characterize it with sfGFP

![](https://static.igem.wiki/teams/5578/image/result/13-1.webp)

Fig21 & 22 & 23 &24. Characterization of Ag-Nb in fluorescence quantification system(60X).

![](https://static.igem.wiki/teams/5578/image/result/merged-30x-ag-nb-copy-1.webp)

Fig25. Characterization of Ag-Nb in fluorescence quantification system(30X)(Red signals depict Ag expression;
green signals depict Nb expression.).

In the culture medium, the alternating distribution of red- and green-labeled *Escherichia coli* is clearly observable. Additionally, large bacterial clusters
are present, within which individual red and green bacterial cells are difficult to
distinguish.

## **Biofilm Enhancement**

#### **Verification of Ag43-OmpA expression**

T
hrough literature research, we identified the key proteins Ag43 and OmpA that
influence biofilm formation. Ag43 specifically mediates cell aggregation, adhesion,
and biofilm development; OmpA, as a key protein for bacterial colonization and biofilm
formation, significantly promotes biofilm formation on hydrophobic surfaces.

The wet experiment verified the expression of the target protein by Western blot
and detected the biofilm formation ability by crystal violet staining.

##### Exploration of the optimal induction concentration of IPTG

We utilize lactose operons to regulate the expression of these two exogenous genes.
Determining the optimal IPTG induction concentration ensures sufficient expression
of the target proteins while minimizing toxicity to host cells. To reduce experimental
workload, we referred to the study by XJTLU-CHINA (2023) and employed a predictive model
to estimate the optimal IPTG induction concentration range, thereby guiding experimental
design and improving efficiency.

![](https://static.igem.wiki/teams/5578/image/result/iptg.webp)

Fig26. Effect of IPTG concentration on mRNA\_ag43\_ompA expression.

![](https://static.igem.wiki/teams/5578/image/result/wb3.webp)

Fig27. The Western Blot(WB) result induced by different IPTG concentration.

During the experiment of inducing protein expression, we used the prediction graph
of the optimal concentration of mRNA synthesis induced by IPTG in cycle 1 and
conducted gradient attempts near the 400μM IPTG concentration. The final
experiment found that the optimal induction concentration of IPTG was approximately
600μM.

##### Crystal violet staining of biofilm

Crystal violet
staining is a foundational, widely used method for visualizing and semi-quantifying
microbial biofilms. Its principle relies on electrostatic binding between the
cationic crystal violet dye and anionic components of biofilms (e.g., extracellular
polysaccharides, peptidoglycan) in the extracellular polymeric substance (EPS) matrix,
resulting in visible purple staining; bound dye can also be solubilized with acetic
acid for absorbance-based biomass quantification. This method is favored for its
simplicity, cost-effectiveness, and utility as a preliminary screening tool for
biofilm research.

![](https://static.igem.wiki/teams/5578/image/result/crystal-violet-staining.webp)

Fig28. Visualization and semi-quantification of microbial biofilms.

![](https://static.igem.wiki/teams/5578/image/result/crystal-violet.webp)

Fig29 & 30 & 31. Crystal violet staining results of 24-Hour biofilm formation.

It was found that the biofilm formation ability of the engineered bacteria
in the experimental group introduced with [ag43-ompA]-pET28a(+) plasmid was
significantly enhanced compared with that of the control group introduced with
pET28a(+) empty plasmid.

## **Arginine-Inducible Sedimentation**

We aim to fuse arginine-rich oligopeptides to the common
*E. coli* extracellular platform CsgA, thereby inducing
diverse modes of spore settlement and enhancing the efficiency and
stability of spore adhesion through a biologically mediated mechanism.

##### Protein expression of CsgA-GSGGSG-RYRYRYR

We introduced a recombinant plasmid encoding the CsgA–arginine oligopeptide
fusion gene into *Escherichia coli*.

![](https://static.igem.wiki/teams/5578/image/result/wb4.webp)

Fig32 & 33 & 34. The Western Blot(WB) result of CsgA-GSGGSG-RYRYRYR induced by different
IPTG concentration.

Western blot analysis was performed to verify expression
of the target protein, while a spore release assay was used to compare the efficacy
of the sedimentation function. We tested gradient induction with IPTG concentrations
ranging from 0.6 to 1.2 mM and varied induction temperatures (16 °C, 30 °C, and 37 °C).
However, the target protein was not detected in either the supernatant or the pellet
following cell lysis.

##### Sanger sequencing of the [CsgA-GSGGSG-RYRYRYR]-pET28a(+) plasmid

To identify the cause of failed protein expression, we performed Ni-NTA column
purification in parallel with Sanger sequencing of the original recombinant plasmid
and the mini-prep plasmid from the engineered *E. coli* strain. This approach
enabled us to distinguish between expression-related issues and genetic problems such
as mutations or rearrangements in the fusion gene.

![](https://static.igem.wiki/teams/5578/image/result/snapgene.webp)

Fig35. The result of the Sanger sequencing.

Based on the Sanger sequencing results, we identified the presence of a
premature stop codon within the coding sequence of the gene in JM109(DE3),
which truncates the polypeptide chain and prevents full-length protein synthesis.
This finding directly explains the lack of detectable protein expression in our
previous experiments. To address this issue, we plan to retransformation the plasmid,
ensuring the integrity of the open reading frame and restoring the intended protein
expression.

## Proof of the concept

#### *Ulva* spores diffusion

Spore release and cultivation of *Ulva prolifera*  is also a critical
component of the wet experiments. Using a constant-temperature light incubator,
we successfully cultured *Ulva prolifera* from nutrient-supplemented spore
solution, obtaining both its juvenile and adult stages. Furthermore, via stimulation
with fresh seawater, a large number of motile spores were successfully released from
sporophyte-derived *Ulva prolifera* fragments, which were then used for the
spore attachment rate assay.

![](https://static.igem.wiki/teams/5578/image/result/image-3.webp)

Fig36. Juvenile and adult stage of *Ulva prolifera* were successfully
cultured from nutrient-enriched spore solution in a constant-temperature light
incubator.

![](https://static.igem.wiki/teams/5578/image/result/spore-ezgif-com-video-to-webp-converter.webp)

![](https://static.igem.wiki/teams/5578/image/result/4604-1759671035-ezgif-com-video-to-webp-converter.webp)

Video 1 & 2. Large amount of jumping swimming spores of *Ulva* genus.

#### Determination of adsorption rate of *Ulva* spores

To verify the ability of engineered bacteria to adsorb spores, we conducted
spore adsorption experiments. Currently, we selected BL21(DE3) engineered bacteria
for experimental verification. After spore release, the spore solution was diluted
to 10⁷ spores/ml. Subsequently, 15 ml of the diluted spore solution was added to
Petri dishes containing engineered bacterial biofilm in LK (LB + kanamycin) medium,
dishes containing only LK medium, and blank dishes without any medium. In the experiment,
we periodically (0 min, 5 min, 30 min,60 min, 120 min, 240 min, 480 min) used a
hemocytometer to observe the spore concentration in the supernatant, taking the
supernatant concentration at 0 min as the total concentration, and calculated
the adsorption rate using the formula (total concentration - supernatant concentration) /
total concentration. The results are shown in the figure below, indicating that the
engineered bacteria have a significant adsorption effect on spores.

![](https://static.igem.wiki/teams/5578/image/result/image-4.webp)

Figure 37: Results of spore adsorption experiments using BL21(DE3) engineered bacteria.

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