iGEM 2025 — Team nmu-china

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{% block title %}Notebook{% endblock %}
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day for your project.{% endblock %}
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### Notebook

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Experiments
▼

Human practices
▼

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{% extends "layout.html" %} {% block title %}engineering{% endblock %}
{% block lead %}Demonstrate product success in a technical aspect of your
project by going through at least one iteration of the product design cycle.
This achievement should be distinct from your Contribution for Bronze.{%
endblock %} {% block page\_content %} {# 初始化环形导航栏内容 #} {% set labels =
[ {"name": "Design", "x": 61, "y": 19, "r": 1}, {"name": "Build", "x": 63, "y":
68, "r": -1}, {"name": "Test", "x": 14, "y": 67, "r": 1}, {"name": "Learn", "x":
14, "y": 16, "r": -1} ] %} {# 定义生成单个组的宏，每次使用都依据全局计数器#}
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+ 1%} {% set offset\_item = (offset-1) \* 4 + 1%}

Click
the
cycle!

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![base](https://static.igem.wiki/teams/5291/images/engineering/no-activation.svg)

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### Engineering

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# Overview

![Gut-sweeper](https://static.igem.wiki/teams/5847/engineering/over1.webp)

"Can a Biologist Fix a Radio?" In the article, reflections on the methodology of biological research
are proposed, and the close integration of biology and engineering in the field of synthetic biology is
expounded. The concept of synthetic biology is gradually becoming clear. The engineering cycle is the
core framework for the development of synthetic biology projects, featuring innovative methods for
optimizing design, verifying hypotheses, and enhancing overall performance. Adhering to the above
philosophy, our team integrates engineering thinking into different project functional modules and sets
up engineering cycles for continuous iteration to ensure compatibility and feasibility among the
modules. We strictly follow each stage of design, construction, testing and learning, and continuously
improve project solutions from multiple aspects. During the design phase, we clarified the core
objective of the project and proposed an innovative design concept in combination with literature
research and expert suggestions. During the construction phase, we send the designed gene fragments to
the company for synthesis, integrating the synthesized genes into the Saccharomyces cerevisiae genome or
synthesizing the corresponding plasmids and transforming them into Saccharomyces cerevisiae.

We focus on the accuracy and reproducibility of construction and have developed detailed experimental
procedures to ensure consistency in experiments. Therefore, during the testing phase, we strictly
verified whether the module construction was feasible through experiments. In the learning phase, we
analyzed the test results and identified the shortcomings and improvement spaces in the design. Between
learning and the next round of design, the wet and dry teams ensured the feasibility and innovativeness
of each engineering progress through further investigation and imagination. After determining whether
the project should continue to be improved, the ideas were integrated into the next cycle. We
objectively view the successes and failures in each engineering cycle, do our best to optimize our
project, and build safe and effective engineering products to say no to the harm of microplastics.

Our treatment module includes the following parts - the degradation and adsorption part and the
anti-inflammatory and antioxidant part. In the engineering cycle of the degradation and adsorption part,
we finally obtained a safe and efficient strategy for degrading PET microplastics. In the engineering
cycle of the anti-inflammatory and antioxidant part, we started from the harm caused by microplastics in
the human intestinal tract and iteratively obtained the expected anti-inflammatory and antioxidant
circuit step by step.

Our safety module, from strain safety guarantee to personal safety guarantee, has undergone repeated
iterations, constantly exploring safety issues in all aspects of product application and providing our
unique solutions.

In addition, the dual-bacteria symbiotic system we designed is closely related to the two major modules
and plays a key role in the iteration. Here, we first present the functional overview diagram of our
symbiotic system.

![Fig XXX](https://static.igem.wiki/teams/5847/engineering/over2.webp)

Treatment Part 1

Treatment Part 2

Treatment Part 3
Safety Part

Engineering

Cycle 1: Optimization of wild-type degrading enzymes

Cycle 2: Methods for experiencing the intestinal environment

Cycle 3: Control continuous expression in the gut

Cycle 4: Control enzyme expression with ternary gates
pathways

Cycle 5: Group sensing signal

Cycle 6: Logic gate refactoring and system integration

References

Cycle 1：Optimization of wild-type degrading enzymes

(1) Research

Microplastics have become ubiquitous in nature, spreading continuously through
ecological cycles and food chains, becoming the "invisible killer" in our lives. In the
past, many iGEM teams also focused on microplastics and provided many industrial-level
treatment methods. However, nowadays the impact of microplastics is extremely
widespread, and different scenarios make the treatment methods limited. The
environmental impact caused by microplastics naturally needs to be considered, but in
recent years, research on the harm of microplastics to the human body has been
continuously deepened. Based on the fact that the diffusion range of microplastics is
large, we believe that while taking degradation measures in the external environment, we
also need to prevent the harm of microplastics to the human body. This is what we
consider to be a safety measure[1].Due to the wide variety of microplastics,
we
conducted a survey based on our project objectives. We found that in a study analyzing
various microplastics in the blood of healthy subjects, the average quantifiable total
concentration of plastic particles in the blood was 1.6 μg/ml. This was the first time
that the mass concentration of plastic polymer components in human blood was measured.
Among these microplastics, PET plastic was the most common. Therefore, we decided to use
this type of plastic as the subject of our project[2].

We recognize the role of degradation in the treatment module. Therefore, through
databases such as Scifinder and Pubchem, we identified the reactions related to PET
plastic degradation. We discovered the specific roles of the PETase and MHETase enzymes
in the degradation process[3][4].

![img](https://static.igem.wiki/teams/5847/engineering/part1-1.webp)

Fig 1. The degradation process of PET and related enzymes

(2) Imagine

Since PETase and MHETase are the wild-type enzymes, we aim to optimize these two enzymes
to initially enhance the degradation efficiency. The joint optimization test for these
two enzymes is a major innovation in the iGEM project.

(3) Design and Build

Our main task was to integrate the latest literature information and derive our design:
The machine learning-related literature predicted the mutation sites of a more efficient
PETase enzyme - the Fast-PEtase enzyme(Fig 2. B). Based on the degradation results of
the
literature chromatogram, it was reflected that we needed to construct an explanation for
a large amount of MHET substances. From the literature, we found the MHET conversion
rate of the highest MHETase mutant.(Fig 2. C)

![img](https://static.igem.wiki/teams/5847/engineering/part1-2.webp)

Fig 2. Design of degrading enzymes

In order to conduct experiments for verification, we constructed two plasmids,
Fast-PETase and MHETase-W397A, for yeast transformation experiments. (Fig.3. A, B) The
transformed
yeast cells could grow on the medium lacking His and Ura. Moreover, an alternative
solution for integrating the CRISPR/Cas9 technology into the genome was provided.

![img](https://static.igem.wiki/teams/5847/engineering/part1-3.webp)

Fig 3. Construction of degradation enzyme plasmids

(4) Test

We conducted double plasmid transformation but failed to achieve the desired
transformation effect. Therefore, we chose the "Fast-PETase plasmid transformation +
MHETase-W397A genomic integration" method and achieved good results(Fig.4. A).
We performed overlapping extension PCR for fusion and validated the final target strain
via PCR. (Fig 4. B, C, D)

![img](https://static.igem.wiki/teams/5847/engineering/part1-4.webp)

Fig 4. Graph of MHETase integrating the genome

After the transformation is successful, we will conduct a co-fermentation of different
control groups and experimental groups along with microplastics (films and particles),
and finally analyze through HPLC whether the microplastics have been degraded.

(5) Learn

The degradation of PET films and particles has been achieved. By comparing the peak area
results with the standard curve, the concentration of degradation products can be
obtained. We found that the degradation effect of particles was significantly greater
than that of films. Thus, we obtained a yeast strain that can significantly degrade PET.
In this cycle, we also noticed that when MHETase and PETase were connected through a
flexible linker to form a chimeric fusion protein, the degradation effect was better
than simply mixing the two enzymes together. This improved the degradation of PET
plastic and the conversion of MHET. This provided a clear direction for our subsequent
optimization. And since our application environment is in the intestinal tract, it is
very important to control the specific spatial expression of the degradation part.

![img](https://static.igem.wiki/teams/5847/02/11.avif)

Fig 5. HPLC Result Graph(PET Particles)

Cycle 2：Methods for experiencing the intestinal environment

(1) Research

In cycle 1, a yeast strain capable of more efficiently degrading PET was obtained.
Therefore, it was hoped that the engineered bacteria could recognize the surrounding
environment and precisely secrete. Since microplastics are mainly ingested in the
intestinal tract, we conducted in-depth research on recognizing the intestinal
environment. We discovered that the "location" of the intestinal tract can be identified
from four aspects: physical signals, conventional biochemical signals, microbial group
metabolite signals, and host-related signals. Through this in-depth exploration in this
stage, we obtained a multi-dimensional signal candidate library.

(2) Imagine

We aim to integrate signals from different dimensions that are mutually orthogonal and
simple yet straightforward, and that are already mature. Thus, we can accurately and
stably identify the intestinal environment. Therefore, we compared various signals in
the research one by one and found that combining a macroscopic physical signal -
hypoxia, and a microscopic biochemical signal - copper ions, can construct a highly
precise "location" perception system.

(3) Design and Build

In order to verify the functions and characterize the performance of the two "position
sensors" we envisioned, we chose green fluorescent protein as the reporter gene and
designed the copper ion detection sequence ‘Ⅺ6up-CUP1p-GFP-ADH1t-Ⅺ6dw’ and the hypoxia
detection sequence ‘Ⅻ5up-KlPDC1p-GFP-FBA1t-Ⅻ5dw’ respectively.

![img](https://static.igem.wiki/teams/5847/engineering/part1-6.webp)
![img](https://static.igem.wiki/teams/5847/engineering/part1-7.webp)

Fig 6. Verification of promoter function construction(Cu2+ and Oxygen Sensor)

During the construction phase, we used synthetic primers to introduce the sequences into
yeast cells through overlapping extension PCR, homologous recombination, and CRISPR/Cas
technology. Then, we screened out the positive strains for functional testing.

(4) Test

We conducted targeted induction tests on the strains. To verify that the engineered
bacteria can sense copper ions, we added different concentrations of copper sulfate
(CuSO4) to the culture medium for induction and quantitatively detected the GFP
fluorescence intensity using an enzyme detector. For the low-oxygen promoter, we
cultivated the strains separately in a regular shaker (with oxygen conditions) and an
anaerobic incubator (without oxygen conditions), and measured the GFP fluorescence
intensity to verify their response to the low-oxygen environment.

![img](https://static.igem.wiki/teams/5847/02/07.avif)

Fig 7. Workflow for Promoter Validation using Genome Editing

(5) Learn

The test results successfully verified that both the P\_CUP1 and P\_KIPDC1 promoters can
precisely and reliably respond to copper ions and low-oxygen environments, proving that
our concept of using "physical + biochemical" dual signals to locate the intestinal
environment is completely feasible. However, merely identifying the intestinal
environment is not comprehensive enough. We hope to be able to further control its
expression in the time dimension after it enters the intestine.

Cycle 3: Control continuous expression in the gut

(1) Research

In Cycle 2, we successfully identified the physical and biochemical signals that can
recognize the intestinal tract, solving the problem of "where" expression. Further, we
hope to achieve more precise regulation of its expression - solving the problem of
"when" expression, so as to maximize the degradation efficiency and reduce the metabolic
burden on the strain. To this end, we investigated and learned that the possible
available signals include direct recognition of the PET plastic itself, degradation
intermediates, direct signals of plastic additives, as well as intermediate signals such
as sugars, amino acids, lipids, and osmotic pressure associated with feeding behavior.。

(2) Imagine

Initially, we aimed to directly detect the signals of microplastics. However, after
conducting in-depth research, we discovered that in the currently known biological
systems, there are almost no natural sensors that can specifically recognize PET.
Therefore, we turned to search for indirect "feeding signals" and screened to find that
the intensity and universality of the glucose signal were the highest. Based on this, we
successfully obtained the high-affinity glucose transporter promoter HXT1. It can be
activated in a high-sugar environment and is in a closed state in a low-sugar
environment, perfectly meeting our design requirements.

(3) Design and Built

To verify the effectiveness of the P\_HXT1 promoter as an "eating signal sensor", we
continued to use green fluorescent protein as the reporter gene and designed a reporting
circuit that responds to changes in glucose concentration: Ⅺ6up-P\_HXT1-GFP-ADH1t-Ⅺ6dw.

![img](https://static.igem.wiki/teams/5847/engineering/part1-8.webp)

Fig 8. Verification of promoter function construction(Temperature Sensor)

And the sequences were constructed using the same method, yeast was introduced, and the
positive strains were screened out.

(4) Test

We conducted experiments to simulate the "fed" and "fasted" states, respectively
culturing the engineered bacteria in media containing 2% and 0.1% glucose for a certain
period of time. We quantitatively detected the GFP fluorescence intensity of the two
groups of strains using an enzyme detector and conducted a comparative analysis to test
the effect of the P\_HXT1 promoter.

![img](https://static.igem.wiki/teams/5847/engineering/engf9.avif)

Fig 9. Construction of a Fluorescent Strain for Promoter Validation

(5) Learn

At this point, we have successfully identified "locations" in Cycle 2 and "times" in
this current cycle. All the key input components are now in place. Next, we hope to
connect these three independent signals and build a complete core control system.

Cycle 4：Control enzyme expression with ternary gates

(1) Research

In Cycle 2 and Cycle 3, we successfully verified two "location" signals of low oxygen
and copper ions, as well as a "time" signal of glucose. Now, we aim to integrate these
three independent signals while triggering the downstream output at the right time. To
achieve this, we reviewed a large number of literature in the field of synthetic biology
and found the following circuit design strategies:
a. Promoter concatenation/cascading: Link multiple regulatory
elements in series to form a transcriptional unit that requires multiple activations
to be turned on.
b. Recombinase logic gates: Use site-specific recombinases
(such as Cre/LoxP, Flp/FRT) to irreversibly flip or remove DNA sequences to achieve
logical judgments.
c. Transcription factor logic gates: Design complex protein
interaction networks where multiple transcription factors need to act together to
activate downstream genes.
d. CRISPR/dCas9 system: Utilize the modular characteristics of
dCas9 protein and gRNA to separately control the expression of dCas9 and gRNA,
achieving coordinated regulation of the target gene.

(2) Imagine

In order to achieve the goals in Research, the circuit should be able to integrate three
signals - glucose, copper ions, and low oxygen - and generate a unified output,
following a strict "AND gate" logic. Additionally, this design must possess excellent
engineering characteristics: high modularity for easy iteration, low metabolic burden to
ensure the health of the host, and system orthogonality to avoid interference with the
cell's own functions. Based on these criteria, we compared various designs found in the
Research stage and ultimately decided to adopt the CRISPR/dCas9-based system to
construct our logic gate.

(3) Design and Build

According to the project requirements, we further referred to the paper[7] ,
designed
and constructed the following circuit:
We connected the dcas9 binding protein gene downstream of the glucose-sensitive promoter
HXT1, the scRNA sequence downstream of the copper-ion-sensitive promoter CUP1, and the
MCP-VP64 gene downstream of the hypoxia-sensitive promoter. When the three downstream
elements are expressed simultaneously, they will assemble to promote the expression of
the degradation protein.

![img](https://static.igem.wiki/teams/5847/engineering/part1-9.webp)

Fig 10. Three-input AND gate regulation

(4) Test

In this round of iteration, we mainly referred to Hofmann et al.'s 2019 paper to test
our circuit. Compared to the original circuit, we specifically expressed scRNA and
replaced the required three promoters[7]. While they successfully constructed
a
dual-input "AND gate" based on CRISPR/dCas9; and through experimental data and ROC curve
analysis, they proved the rigor of the system; and also demonstrated the modular design.
Therefore, the research results of this paper provided a solid "proof of concept" for
our "ternary AND gate", proved the feasibility of the circuit design, and met our
expectations for the rigor and low leakage of the system in the Imagine stage.

(5) Learn

After this round of iteration, we successfully designed a highly specific three-input
"AND gate" that can precisely respond to "time" and "location" signals. However, how to
further optimize and upgrade the circuitry towards the ultimate goal - the expression of
the degrading enzyme - still requires further research.

Cycle 5: Group sensing signal

(1) Research

In Cycle 4, we successfully designed the "ternary gate" and achieved the expression of
degradation enzymes to be activated at specific times and locations. Further, we aimed
to focus on "how many cells are working" to improve the secretion efficiency of the
enzymes and amplify the upstream signal. Therefore, we conducted a new round of
literature research and discovered the following possible approaches:

* Metabolic load sensor: A line that can sense the internal
  metabolic state of the cell (such as ATP level) and regulate protein expression
  through feedback.
* Stage-dependent promoters: Promoters that are activated
  only at specific stages of cell growth (such as the stationary phase).
* Quorum sensing system: A "communication" system used by
  microorganisms to sense the density of their own population and coordinate group
  behavior. In Saccharomyces cerevisiae, this system based on pheromones (α-factor)
  has been studied very thoroughly.

(2) Imagine

We hope that the optimization strategy we adopt can achieve the goal of reducing the
early metabolic burden and regulate expression based on the population density.
Therefore, we compared various optimization approaches during the Research stage: the
metabolic load sensor can only passively protect itself; while the growth
stage-dependent promoters are interfered by the complex intestinal environment. However,
population sensing directly focuses on the "size and density" of the population, which
can meet the requirements of improving efficiency and amplifying signals. Thus, we
decided to use the population sensing signal for regulation.

(3) Design and Build

Based on the ideas in Imagine, we reviewed the relevant papers on group sensing and
discovered a group sensing circuit based on positive feedback[9]. Therefore, we modified
it based on this and connected the degrading enzymes Fastpetase and MHETase\_W397A to the
downstream of the core promoter FUS1p of the group sensing pathway, achieving group
sensing regulation.

![img](https://static.igem.wiki/teams/5847/engineering/part1-10.webp)

Fig 11. Quorum sensing in Saccharomyces cerevisiae[8]

(4) Test

To demonstrate the effectiveness of the group communication signal pathway, we
constructed and verified it based on the establishment of a series of chemical reaction
equations and differential equations. Firstly, we investigated how the extracellular α
pheromone binds to the Ste2 receptor. We also used the Autodock software to dock the α
factor and the receptor of the Ste2 signaling pathway, and selected the optimal model
for visualization in PyMOL, as shown in the figure. The binding energy is -6.7 kcal/mol.

![img](https://static.igem.wiki/teams/5847/engineering/part1-11.webp)

Fig 12. Molecular docking results between Ste2 and α factor

Furthermore, we demonstrated that the Ste5 scaffold protein activates a series of
cascade reactions (MAPK kinase chain) within the cell. The final product of this process
activates the transcription factor Ste12, thereby prompting the cell to produce more α
pheromones, forming a positive feedback loop that amplifies the signal. Through
mathematical modeling, it was shown that the quorum sensing system can effectively
function, enabling efficient expression of enzymes and signal amplification.

(5) learn

After this round of iteration, we obtained the "group effect" module, which further
optimized the expression of the enzyme. This naturally led to the next engineering
challenge: How to ingeniously connect these two modules to achieve complete expression
regulation?

Cycle 6: Logic gate refactoring and system integration

(1) Research

We intend to use the "active" AND gate of Cycle 4 to control the "group sensing" switch
of Cycle 5. Therefore, we reviewed the literature and noticed a key gene: BAR1. This
gene encodes an enzyme that degrades and removes the α-factor pheromone, rendering the
group sensing signal ineffective. In the lines we referred to, the BAR1 gene also needs
to be knocked out to ensure the effective accumulation of the signal.

(2) Imagine

Taking the BAR1 gene as the breakthrough point, we discovered that: since its expression
would render the group communication signal ineffective, then by inhibiting its
expression, we could activate the group communication system. Based on this, if we
replace the activation signal of the AND gate with an inhibitory signal and connect it
to BAR1, we can control the downstream group communication system.

(3) Design and Build

Based on the concept in Imagine, we first replaced the activating MCP-VP64 expression
box in the original circuit with a repressive MCP-MXI1 expression box; then we precisely
designed the scRNA target on the promoter BAR1p of the BAR1 gene. Thus, we achieved
control of the quorum sensing system through a ternary AND gate and formed the final
circuit.

![img](https://static.igem.wiki/teams/5847/engineering/part1-12.webp)

Fig 13. The tri-functional AND gate targeting the BAR1 gene

At the construction level, we completed the design and optimization of the final circuit
through in silico methods.

(4) Test

Firstly, through motif scanning on MXI1, we identified potential SID sites that might
recruit SIN3. Subsequently, using protein interaction networks, we linked
MXI1-SIN3B-HDAC1 into a known deacetylation inhibition axis. Genome-wide occupancy
analysis revealed that this complex was highly enriched in the vicinity of the promoter
and was accompanied by a significant enrichment of active acetylation marks (H3K27ac),
suggesting that MXI1-SIN3B-HDAC1 is not located at silent gene loci but is targeted to
active promoters to negatively fine-tune/transcribe output, thereby supporting the
inhibitory effect of MXI1. (Fig.16.)

![img](https://static.igem.wiki/teams/5847/engineering/part1-13.webp)

Fig 14. Modeling results of MXI inhibitory effect

(5) Learn

This round of the cycle achieved a crucial "logic gate reconfiguration", which not only
solved the problem of module connection but also realized the coordinated regulation of
three dimensions: time, location, and population density. The successive engineering
cycles continuously optimized based on the degradation of microplastics, ultimately
providing a solution that balances precision, efficiency, and robustness.
In the future, we will further test and optimize the degradation of microplastics,
including increasing the linker to enhance the activity of the degradation enzyme,
conducting wet experiments to further verify the circuit design, and verifying its
comprehensive performance in a simulated intestinal environment, etc.

References

* 1. de Souza Machado AA, Kloas W, Zarfl C, Hempel S, Rillig MC. Microplastics as an
  emerging threat to terrestrial ecosystems. Glob Chang Biol. 2018;24(4):1405-1416.
  doi:10.1111/gcb.14020
* 2. Leslie HA, van Velzen MJM, Brandsma SH, Vethaak AD, Garcia-Vallejo JJ, Lamoree MH.
  Discovery and quantification of plastic particle pollution in human blood. Environ Int.
  2022;163:107199. doi:10.1016/j.envint.2022.107199
* 3. Burgin T, Pollard BC, Knott BC, et al. The reaction mechanism of the Ideonella
  sakaiensis PETase enzyme. Commun Chem. 2024;7(1):65. Published 2024 Mar 27.
  doi:10.1038/s42004-024-01154-x
* 4. Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate
* 5. Lu H, Diaz DJ, Czarnecki NJ, et al. Machine learning-aided engineering of hydrolases
  for PET depolymerization. Nature. 2022;604(7907):662-667. doi:10.1038/s41586-022-04599-z
* 6. Yoshida S, Hiraga K, Takehana T, et al. A bacterium that degrades and assimilates
  poly(ethylene terephthalate). Science. 2016;351(6278):1196-1199.
  doi:10.1126/science.aad6359
* 7. Hofmann A, Falk J, Prangemeier T, et al. A tightly regulated and adjustable
  CRISPR-dCas9 based AND gate in yeast. Nucleic Acids Res. 2019;47(1):509-520.
  doi:10.1093/nar/gky1191
* 8. Williams TC, Nielsen LK, Vickers CE. Engineered quorum sensing using
  pheromone-mediated cell-to-cell communication in Saccharomyces cerevisiae. ACS Synth
  Biol. 2013;2(3):136-149. doi:10.1021/sb300110b
* 9. Chen Z, Duan R, Xiao Y, et al. Biodegradation of highly crystallized poly(ethylene
  terephthalate) through cell surface codisplay of bacterial PETase and hydrophobin. Nat
  Commun. 2022;13(1):7138. Published 2022 Nov 21. doi:10.1038/s41467-022-34908-z

Engineering

Cycle 1: A new perspective for enhanced degradation-adsorption

Cycle 2: Exploration of the Orthogonality of Degradation Adsorption

Cycle 3: The optimization direction of the surface display system

Cycle 4: The adsorption effect of symbiotic system colonization

References

Cycle 1: A new perspective for enhanced degradation-adsorption

(1) Research

We continued our literature search and found that adsorbing PET has the potential to
further enhance degradation. Current research has utilized hydrophobins to enhance the
adsorption effect. The methods described in the literature all involve the co-secretion
or co-display on the surface with degradation enzymes[1].

(2) Imagine

We believe that by using surface display technology combined with excreted degrading
enzymes, we can also achieve an enhanced degradation effect. This is our unique
innovation compared to other methods!

(3) Design and Build

We employed the traditional yeast surface display technology - using the α-agglutinin of
Saccharomyces cerevisiae - to construct a fusion protein that can adsorb PET and has the
function of surface display technology. Saccharomyces cerevisiae constitutively
expresses Aga1, which can bind to the Aga2 functional domain in the fusion protein and
secrete it as cell display. Initially, we controlled this process through the high
expression of the constitutive promoter PTEF. Additionally, we selected a flexible
linker (GGGGS)n to connect the key protein domains, and we tested how to determine the
length of the linker.

![img](https://static.igem.wiki/teams/5847/engineering/part2-1.webp)

Fig 1. HFBI fusion protein gene map

![img](https://static.igem.wiki/teams/5847/engineering/part2-2.webp)

Fig 2. HFBI fusion protein expressing process

(4) Test

We conducted a preliminary prediction on the Alphafold Server. We found that the
length of Linker2 had little impact on the prediction of V5 epitope, while the
length of Linker1 had a significant effect on the structure of the HFBI and Aga2
functional domains. Therefore, we chose to test with different Linker1 lengths:
(GGGGS)3, (GGGGS)5, and (GGGGS)8. They were numbered as 3\_1, 5\_1, and 8\_1
respectively.

![img](https://static.igem.wiki/teams/5847/engineering/part2-3.webp)

Fig 3. HFBI fusion proteins with different linker lengths

We conducted transmembrane domain and hydrophobicity score analyses on the three
fusion protein structures. The results initially demonstrated that the fusion
proteins could be successfully displayed on the surface and that the hydrophobicity
of the HFBI functional domain was good.

![img](https://static.igem.wiki/teams/5847/engineering/part2-4.webp)

Fig 4. HFBI fusion protein transmembrane and hydrophobicity analysis

Based on this, we compared the structures of the active domains to preliminarily
verify the structural fidelity. We used two key monomeric proteins and fusion
proteins for docking. The smaller the RMSD result obtained, the better.

![img](https://static.igem.wiki/teams/5847/engineering/part2-5.webp)

Fig 5. The structure of key functional domains of fusion proteins

![img](https://static.igem.wiki/teams/5847/engineering/part2-6.webp)

Fig 6. Verification of the structural fidelity of fusion proteins

Next, we conducted a docking between the fusion protein and PET. The binding
energies obtained were all within the expected range. The larger the absolute value
of the binding energy, the better the affinity.

![img](https://static.igem.wiki/teams/5847/engineering/part2-7.webp)

Fig 7. Fusion protein and PET docking site and binding energy

(5) Learn

After an initial analysis, we concluded that all three designs were feasible. To
highlight the differences, we chose 3\_1 and 8\_1 for constructing plasmids for the
experiments.

Cycle 2: Exploration of the Orthogonality of Degradation
Adsorption

(1) Research

Before conducting the route design, we had already taken into account the impact of
the metabolic toxicity of the chassis bacteria. We plan to reduce the metabolic
toxicity by adjusting different control conditions of the engineering route and
minimizing the constitutive expression.

(2) Imagine

Is there a dynamic regulatory system that can control the expression of the
adsorption module?

(3) Design and Build

In the experiment, we learned about the "Pgal1+Δgal80" strategy. Under high glucose
conditions, the expression of downstream genes controlled by this dynamic regulatory
system is inhibited, and at this time, yeast energy is used for growth. While under
low glucose conditions, the downstream genes are induced to express, and this
process has a temporal sequence[2].Furthermore, we discovered that this
dynamic
regulatory system forms an orthogonal relationship with the PHXT-1 promoter of our
degradation module. PHXT-1 responds to feeding signals and induces the expression of
downstream genes under high glucose conditions.

![img](https://static.igem.wiki/teams/5847/engineering/part2-8.webp)

Fig 8. Pgal1/Δgal80 dynamic regulation system

(4) Test

So, based on our imagination, we constructed the following plasmid transformation
yeast, and conducted an adsorption verification experiment after co-fermentation
with microplastics. In both immunofluorescence and transmission electron microscopy,
the desired results were observed.

(5) Learn

![img](https://static.igem.wiki/teams/5847/engineering/part2-9.webp)

Fig 9. Pgal1/Δgal80 dynamic regulation system

Since the Saccharomyces cerevisiae itself can express Aga1, but its basal expression
level may not be high, resulting in poor surface display effect, we decided to
enhance the expression by co-integrating Aga1 into a plasmid to improve the expected
surface display effect. We used the Pgal1,10 bidirectional promoter to control Aga1
and the fusion protein part separately. Moreover, in the laboratory, we successfully
obtained the Aga1 fragment in the genome through PCR, linearized the plasmid
containing the HFBI-Aga2 fusion protein,and will proceed with a further cloning step
to enhance the expression of Aga1.

Cycle 3: The optimization direction of the surface display
system

(1) Research

In addition to the optimization strategy of enhancing Aga1 expression by using the
Pgal1,10 bidirectional promoter, we also learned that the surface display system
applied to Saccharomyces cerevisiae is very diverse. Although only α-agglutinin has
been applied to HFBI, we still want to enrich the application of the surface display
system in the future.

(2) Imagine

Although we have achieved good surface display results using α-agglutinin, can we
achieve even better results through other surface display systems, and can the
process be presented more concisely?

(3) Design and Build

Our Aga1 is not the entire protein that is used for attaching to the surface of the
yeast cell membrane, as shown in our schematic diagram. The amino acids from 150 to
725 act as the anchor protein. We hypothesize that directly displaying HFBI through
the anchor protein might be a better approach. Therefore, we designed and
constructed a new surface display system.

![img](https://static.igem.wiki/teams/5847/engineering/part2-10.webp)

Fig 10. The surface display system of utilizing GPI

(4) Test

We conducted a literature search to preliminarily explore the feasibility. We found
that this surface display method was more effective than α-agglutinin for other
target proteins. The article also compared various types of GPI and demonstrated the
great potential of this display method.

![img](https://static.igem.wiki/teams/5847/engineering/f10.avif)

Fig 11. The surface display system of utilizing GPI[3]

(5) Learn

The results in the literature have greatly boosted our confidence for future
experiments. Additionally, the literature also proposed a method applicable to the
surface display system. We plan to conduct preliminary dry experiments to narrow
down the range of GPI types first. At the same time, we will use online software
such as Signalp to screen different signal peptides. Finally, we will construct
plasmids for experiments, thus forming another engineering cycle.

![img](https://static.igem.wiki/teams/5847/engineering/part2-13.webp)

Fig 12. Surface display of the system's signal peptide selection[4]

Cycle 4: The adsorption effect of symbiotic system colonization

(1) Research

After optimizing the adsorption capacity of the engineered yeast, we encountered a
core challenge: how to ensure that it can function stably and persistently in the
complex intestinal environment. Through literature research, we discovered that
microorganisms often form multi-species biofilms through co-aggregation in nature to
enhance their survival advantages. This strategy not only enhances their resistance
to environmental stress but also facilitates colonization in specific ecological
niches (such as intestinal mucosa).

(2) Imagine

We envision that an ideal symbiotic partner for engineered yeast can be identified.
This strain would be able to aggregate with the yeast to enhance intestinal
colonization, and also work in tandem with us to accomplish the task of adsorbing
microplastics.

(3) Design and Build

By reviewing the literature, we focused our attention on Lactobacillus bacteria. A
large number of studies have confirmed that specific Lactobacillus strains can form
co-aggregation with Saccharomyces cerevisiae. Among them, the mannose-specific
adhesin (Msa) on the surface of Lactiplantibacillus plantarum can specifically bind
to the mannoprotein on the yeast cell wall, forming a stable physical connection.

![img](https://static.igem.wiki/teams/5847/engineering/part2-14.webp)

Fig 13. Synergistic adsorption in a symbiotic system

The adhesive ability of Lactobacillus plantarum to intestinal mucosal cells is a key
aspect in exerting its probiotic effects. Probiotics attach to the intestinal
mucosal cells, thereby preventing the penetration of harmful microorganisms and
toxins, and play an important role in enhancing the intestinal barrier function.
Moreover, they can compete with potential pathogens for nutrients and living space,
disrupt the biofilm of pathogenic bacteria, and hinder the colonization of harmful
bacteria[5].Researchers identified a collagen-binding protein from
Lactobacillus
plantarum. This protein not only promotes adhesion but also inhibits the adhesion of
Escherichia coli O157:H7 to extracellular matrix components[6]. Furthermore,
through adhesion and interaction with intestinal mucosal cells, probiotics help
maintain the immune balance within the intestinal tract[7]. The elongation
factor
Tu is a cell surface-related protein that has been shown to interact with
fibronectin on intestinal epithelial cells. It may play a role in intestinal
immunity and homeostasis by inhibiting cell apoptosis[8].Lactobacillus
plantarum
can also enhance its adhesion ability to the intestinal mucosa by sensing unknown
intestinal signals and relieving the inhibition of transcription factor MbpR on the
adhesion protein genes.

![img](https://static.igem.wiki/teams/5847/engineering/part2-15.webp)

Fig 14. The key regulatory mechanism for achieving intestinal adhesion

More importantly, we discovered a strain named Lactobacillus plantarum DT88. This
strain not only has the potential to aggregate with yeast, but also demonstrates a
strong ability to adsorb microplastics on its own[9]. Therefore, we
constructed a
dual-bacterial symbiotic system consisting of engineered Saccharomyces cerevisiae
and Lactobacillus plantarum DT88, aiming to leverage the synergistic effects of the
two organisms to achieve a "1 + 1 > 2" colonization and adsorption outcome.

(4) Test

After envisioning the dual-bacteria symbiotic system, we conducted further
literature research to assess the potential shortcomings of this strategy. We found
that although co-culture theoretically has advantages, it faces challenges in
practical applications:
Internal stability and competitive relationship: Under ideal laboratory conditions,
yeast and lactobacillus can exhibit mutualistic symbiosis. However, in the dynamic
and complex intestinal microenvironment, the relationship between the two may become
unstable and even turn into a competitive relationship, causing one of the organisms
to be eliminated, thereby rendering the entire system ineffective.

(5) Learn

In response to these challenges, we once again turned to the literature to seek
innovative solutions to optimize our symbiotic system:
Utilize "postbiotics": To avoid the instability risk of live bacteria in the body, a
more advanced strategy is to use "postbiotics" or "paraprobiotics", which involve
the use of inactivated probiotic cells or their metabolites. Studies have shown that
even inactivated bacteria or their cellular components retain most of their
biological activity, such as immune regulation and adsorption[10].This method
not
only ensures the stability and safety of the product, but also avoids the
uncertainty of viable bacteria colonization. It provides another highly promising
optimization path for our project.

References

* 1. Chen Z, Duan R, Xiao Y, et al. Biodegradation of highly crystallized poly(ethylene
  terephthalate) through cell surface codisplay of bacterial PETase and hydrophobin. Nat
  Commun. 2022;13(1):7138. Published 2022 Nov 21. doi:10.1038/s41467-022-34908-z
* 2. Deng J, Wu Y, Zheng Z, et al. A synthetic promoter system for well-controlled protein
  expression with different carbon sources in Saccharomyces cerevisiae. Microb Cell Fact.
  2021;20(1):202. Published 2021 Oct 18. doi:10.1186/s12934-021-01691-3
* 3. Yang X, Tang H, Song M, Shen Y, Hou J, Bao X. Development of novel surface display
  platforms for anchoring heterologous proteins in Saccharomyces cerevisiae. Microb Cell
  Fact.
  2019;18(1):85. Published 2019 May 18. doi:10.1186/s12934-019-1133-x
* 4. O'Riordan NM, Jurić V, O'Neill SK, Roche AP, Young PW. A Yeast Modular Cloning (MoClo)
  Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display
  in Saccharomyces cerevisiae. ACS Synth Biol. 2024;13(4):1246-1258.
  doi:10.1021/acssynbio.3c00743
* 5. Wang, J., Y. Zeng, S. Wang, H. Liu, D. Zhang, W. Zhang, Y. Wang, and H. Ji. 2018.
  Swine-derived probiotic Lactobacillus plantarum inhibits growth and adhesion of
  enterotoxigenic Escherichia Coli and mediates host defense. Frontiers in Microbiology
  9:1364. doi: 10.3389/fmicb.2018.01364.
* 6. Yadav, A. K., A. Tyagi, J. K. Kaushik, A. C. Saklani, S. Grover, and V. K. Batish.
  2013.
  Role of surface layer collagen binding protein from indigenous Lactobacillus plantarum
  91 in
  adhesion and its anti-adhesion potential against gut pathogen. Microbiological Research
  168
  (10):639–45. doi: 10.1016/j.micres.2013.05.003.
* 7. Santarmaki, V., Y. Kourkoutas, G. Zoumpopoulou, E. Mavrogonatou, M. Kiourtzidis, N.
  Chorianopoulos, C. Tassou, E. Tsakalidou, C. Simopoulos, and P. Ypsilantis. 2017.
  Survival,
  intestinal mucosa adhesion, and immunomodulatory potential of Lactobacillus plantarum
  strains. Current Microbiology 74 (9):1061–7. doi: 10.1007/s00284-017-1285-z.
* 8. Du, Y., H. Li, W. Xu, X. Hu, T. Wu, and J. Chen. 2023. Cell surface-associated protein
  elongation factor tu interacts with fibronectin mediating the adhesion of Lactobacillus
  plantarum HC-2 to penaeus vannamei intestinal epithelium and inhibiting the apoptosis
  induced by LPS and pathogen in Caco-2 cells. International Journal of Biological
  Macromolecules 224 (January):32–47. doi: 10.1016/j.ijbiomac.2022.11.252.
* 9. O'Riordan NM, Jurić V, O'Neill SK, Roche AP, Young PW. A Yeast Modular Cloning (MoClo)
  Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display
  in
  Saccharomyces cerevisiae. ACS Synth Biol. 2024;13(4):1246-1258.
  doi:10.1021/acssynbio.3c00743
* 10. Batista VL, da Silva TF, de Jesus LCL, et al. Probiotics, Prebiotics, Synbiotics, and
  Paraprobiotics as a Therapeutic Alternative for Intestinal Mucositis.Front Microbiol.
  2020;11:544490. Published 2020 Sep 17. doi:10.3389/fmicb.2020.544490

Engineering

Cycle 1: Investigate the main hazards of microplastics to the human body

Cycle 2: Search for therapeutic elements - Antioxidants

Cycle 3: Search for therapeutic elements - anti-inflammatory

Cycle 4: ROS-Responsive Promoter Design

Cycle 5: How can the joint control of anti-inflammation and anti-oxidation be achieved

Cycle 6: The auxiliary role of symbiotic systems in treatment

References

Cycle 1: Investigate the main hazards of microplastics to the human
body

(1) Research

Initially, we assumed that microplastics mainly caused physical damage. However, through
research and preliminary experiments, it was discovered that oxidative stress and
inflammatory responses were the key pathogenic mechanisms. This refuted our initial
assumption of only considering physical damage. Therefore, we turned to intervention at
the molecular mechanism level. We investigated the most common harmful diseases caused
by microplastics in the human intestinal tract. Currently, PET has not found any
diseases that are significantly related to specific diseases. Among them, inflammatory
bowel disease (IBD) has the most active research on the association with microplastic
exposure and has been proven to have a certain correlation. Additionally, microplastics
can also cause dysregulation of the intestinal microbiota and damage to the intestinal
barrier function[1-3].

(2) Imagine

In addition to literature review, we aim to identify the targets of microplastics
causing intestinal diseases through bioinformatics methods for the purpose of designing
treatments. We plan to start with IBD, a common disease.

(3) Design and Build

The students in the modeling group came up with specific plans for target
identification:
a. Data collection and intersection with targets.
b. Construction of protein networks and screening of core nodes.
c. Multi-dimensional functional enrichment and verification.
d. Final determination through machine learning algorithms.
The specific methods are presented in the Test section.

(4) Test

[For details, see the Model
section.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)

(5) Data collection and intersection with the target

Firstly, the researchers established two independent gene databases. One was for PET
microplastics, where potential targets that might interact with it were comprehensively
collected from multiple databases such as PubChem, CHEMBL, and STITCH based on the
SMILES chemical encoding of its main degradation products. The other was for
inflammatory bowel disease (IBD), where a group of genes with the highest association
with IBD were selected from disease databases such as GeneCards and OMIM. By taking the
intersection of these two large databases, the "common targets" that were related to
both PET microplastics and IBD were finally identified. This step ensured that the
targets for the subsequent analysis had dual correlations.

![img](https://static.igem.wiki/teams/5847/engineering/part3-1.webp)

Fig 1. The search for cross-targets between PET and IBD

(6) Protein network construction and core node selection

Just having a list of genes is not enough, because genes usually work together through
complex networks. Researchers used the STRING database to construct a protein-protein
interaction (PPI) network based on the common targets from the previous step.
Subsequently, this network was imported into the Cytoscape software for visual analysis.

The researchers employed two complementary algorithms to identify the key genes in the
network:
a. CytoHubba\_MCC algorithm: This is used to find the nodes with the most connections
and located in the "central hub" position. This method can directly identify the
proteins that play a key role in information transmission.
b. MCODE plugin: This is used to discover the most densely connected "functional
modules" or protein clusters in the network. This helps to identify the protein groups
that collaborate to perform specific functions in the cell.

![img](https://static.igem.wiki/teams/5847/engineering/part3-2.webp)

Fig 2. Protein interaction network results

(7) Multidimensional functional enrichment and validation

To understand the specific biological functions of these core genes, the researchers
conducted Gene Ontology (GO) and KEGG pathway enrichment analyses. The results showed
that these genes were highly enriched in biological processes such as "inflammatory
response" and "immune response", which are highly relevant to the pathology of IBD, and
were closely related to important signaling pathways such as PI3K-Akt and Jak-STAT.

![img](https://static.igem.wiki/teams/5847/engineering/part3-3.webp)

Fig 3. Metabolic pathway analysis

To verify whether PET can directly act on these targets at the physical level, we
conducted molecular docking and molecular dynamics simulation tests. The results showed
that the binding energies of PET to proteins such as JAK2, IL2, and TGFBR2 were
negative, which proved in physics that there is a stable and spontaneous binding between
them.

![img](https://static.igem.wiki/teams/5847/engineering/part3-4.webp)

Fig 4. PET and three target molecules docking

(8) The machine learning algorithm is finally locked

After going through the above multiple rounds of screening and verification, a series of
machine learning algorithms (including LASSO regression, SVM-RFE, boruta and XGBoost)
were finally employed for the final and most rigorous screening. These algorithms
further evaluated the importance of each gene from a statistical perspective, and
ultimately precisely identified the three core targets: JAK2, IL2 and TGFBR2.

![img](https://static.igem.wiki/teams/5847/engineering/part3-5.webp)

Fig 5. Machine learning screening

(9) Localization of pathogenic function of the target

JAK2: It belongs to non-receptor tyrosine kinases. The accumulation of ROS can directly
activate JAK2, making it an important upstream trigger point in the inflammatory
cascade.
IL2: T-cell growth factor, over-activation is associated with immune dysregulation and
abnormal T-cell proliferation in IBD.
TGFBR2: A key receptor of the TGF-β signaling pathway, regulating cell proliferation,
differentiation, and tissue repair. Its abnormality may lead to failure of intestinal
epithelial repair or a tendency towards fibrosis.

(1) Learn

We have identified three therapeutic targets for the harm caused by microplastics to the
human body. In the future, when we are searching for therapeutic components, we will pay
particular attention to their relationship with the target sites.

Cycle 2: Search for therapeutic elements - Antioxidants

(2) Research

Firstly, we address the issue from the first aspect of microplastics causing disease -
oxidative stress. Research indicates that reactive oxygen species (ROS) play a
significant role in the pathogenesis of inflammatory bowel disease. Superoxide anion
(O₂⁻) and hydrogen peroxide (H₂O₂) are two core types of ROS. The accumulation of ROS
can directly activate the JAK2 pathway and trigger an inflammatory cascade reaction.
Therefore, eliminating ROS is a key strategy for inhibiting inflammation induced by
microplastics.

(3) Imagine

Can we establish a "cohesive" antioxidant regulatory system to rapidly eliminate ROS at
the source and achieve antioxidant stress response? Does the molecular mechanism layer
have any effect on the targets we are exploring?

(4) Design and Build

There are two key enzymes that play a dominant role in the removal of ROS:
a. SOD1: Converts O₂⁻ into H₂O₂;
b. CTT1: Further decomposes H₂O₂ into water and oxygen.
Theoretically, the cascade reaction formed by these two enzymes can efficiently remove
ROS.

![img](https://static.igem.wiki/teams/5847/engineering/part3-6.webp)

Fig 6. The function of the antioxidant module

(5) Test

After reviewing the literature, SOD1 and CTT1 were found to be capable of performing
their intended functions in our chassis - Saccharomyces cerevisiae. Therefore, we
selected these two genes as the effect modules and designed a dual-enzyme expression
pathway to enable the engineered bacteria to possess systematic antioxidant
capabilities.
In terms of the molecular mechanism, this antioxidant component directly eliminates
reactive oxygen species (ROS) to block the activation of JAK2 at the source, achieving
direct intervention in the downstream inflammation[22][23].

(6) Learn

The team realized that the antioxidant module can precisely address the "downstream
trigger point" of ROS → JAK2, but the inflammatory network is complex, and relying on a
single module is not sufficient to completely inhibit the pathological process.
Oxidative stress is merely a "trigger", not the sole effector. Once the inflammatory
pathway is activated, merely removing the trigger factor is not enough to stop the
pathological progression. This discovery prompted the project to shift to a more
comprehensive treatment approach - in addition to the antioxidant module, functional
modules such as immune regulation and tissue repair need to be added.
Furthermore, model analysis and literature comparison indicate that this system can
indeed significantly clear ROS and directly block the activation of JAK2 in theory.
However, there is a fundamental defect: it can only eliminate the initial oxidative
signal, but cannot inhibit the downstream inflammatory cascade reaction triggered by
ROS. This deserves our team to continue to think about optimization strategies.

Cycle 3: Search for therapeutic elements - anti-inflammatory

(1) Research

The team first realized that direct intervention in the inflammation itself was
necessary to solve the problem. Thus, they embarked on in-depth research on the core
signaling pathways of the host immune system (such as
**JAK2**, **IL-2**, and **TGFBR2**). By analyzing the mechanisms of these
pathways, the team realized that they were key nodes regulating the immune system, and
theoretically, the inflammation could be controlled through external factors.

(2) Imagine

Based on this understanding, the team proposed an ambitious idea:
Utilizing the synthetic biology pathways of engineered bacteria, to secrete inhibitors
or agonists, to "remote control" the functions of the host immune cells from the
outside, directly acting on the JAK2, IL-2 and TGFBR2 pathways, in order to achieve
precise control of inflammation.

(3) Design and Build

During the specific design and analysis phase, the team decisively rejected this
approach. The reason was as follows: These host immune pathways are **extremely
complex** and mainly function within the host immune cells; engineered
microorganisms (such as yeast) are difficult to stably, safely and precisely directly
regulate them; directly interfering with key kinases such as JAK2 might cause
unforeseeable off-target effects on systemic functions such as **hematopoiesis and
immune surveillance**; and there are significant risks in terms of **biological
safety**.
Therefore, the team shifted its focus from "Is it technically possible?" to "Is it
functionally safe and controllable?". This strategic retreat avoided the high-risk dead
end. Subsequently, the team shifted to designing new solutions, seeking molecules that
could **the organization's repair and stabilization recovery.** Eventually,
**Trefoil Factor 3 (TFF3)** was identified as the ideal effector factor.

(4) Test

The team further analyzed the mechanism of action of TFF3:
Promoting epithelial repair: Activating the JAK2/STAT3 pathway in intestinal epithelial
cells in a local manner, promoting mucosal healing and recovery of barrier function.

Actively inhibiting inflammation: Reducing key pro-inflammatory cytokines (such as
TNF-α, IL-1β) in the inflammatory microenvironment, and weakening the excessive
activation of IL-2 and TGFBR2-related signals.
This dual function ensures both local and controllable efficacy, meeting safety
requirements[6].

(5) Learn

Through this cycle, the team learned that:
Direct intervention in the core host immune pathways is an unbridgeable red line because
it is unpredictable and high-risk;
Strategically giving up high-risk solutions and instead adopting more indirect and safer
mechanisms reflects engineering wisdom and a high sense of responsibility for biological
safety;
Ultimately, the team established the "antioxidation + mucosal repair" strategy to
replace the direct blocking of inflammation, thereby achieving the anti-inflammatory
goal.

Cycle 4: ROS-Responsive Promoter Design

(1) Research

In the inflammatory environment of the human intestinal tract, a large amount of
reactive oxygen species (ROS) accumulate, driving epithelial damage and inflammatory
cascade reactions. We need a regulatory element that can precisely sense ROS signals as
a molecular switch for the therapeutic pathway, so as to achieve the expression of
conditionally induced anti-inflammatory factors.

(2) Imagine

The literature indicates that the transcription factors Yap1 and Skn7 in Saccharomyces
cerevisiae are key regulatory elements for sensing ROS:
Yap1: Highly sensitive to ROS such as hydrogen peroxide, and can serve as the main
sensing module;
Skn7: Provides an auxiliary function, and can enhance the stability and intensity of the
response;Synergistic effect: When Yap1 and Skn7 synergistically bind to the promoter,
they can significantly increase the gene expression level and accelerate the response
speed.
Therefore, we hypothesize that the natural ROS-responsive promoter regulated by the
synergistic control of Yap1 and Skn7 can be used as a candidate regulatory tool.

(3) Design and Build

Based on the regulatory mechanism of Yap1/Skn7, we have identified three types of
naturally existing promoters in yeast:
pGSH1 (glutathione synthetase promoter): contains multiple Yap1 binding sites;
pTRX2 (thioredoxin reductase promoter): contains Yap1 sites and Skn7 elements;
pGPX2 (glutathione peroxidase promoter): contains Yap1 sites and Skn7 elements, and can
simultaneously sense ROS and inflammation-related oxidative signals.
These candidate promoters can all respond to oxidative stress signals, but there are
differences in their activation intensity, response speed, and background leakage.

We conducted a literature review and comparative analysis of the three types of
promoters[25]：

![img](https://static.igem.wiki/teams/5847/engineering/part3-7.webp)

Table 1. Oxidative stress promoter selection

After comprehensive analysis, pGPX2 stood out:
Low background expression → Avoid energy waste and side effects during non-inflammatory
states;
High induction intensity → Capable of rapidly producing sufficient therapeutic proteins
during inflammatory outbreaks;
Fast response → Exhibits a faster response speed to ROS and inflammatory signals
compared to other promoters;
Broad response spectrum → Covers ROS and a wider range of inflammatory-related oxidative
signals.
Therefore, the pGPX2 promoter has been confirmed as the optimal ROS-responsive
regulatory tool, constituting the core component of our treatment line design.
Based on the literature, we found the pGPX2 promoter from the yeast genome, located
upstream of the GPX2 CDS fragment (-709/+7).

![img](https://static.igem.wiki/teams/5847/engineering/part3-8.webp)

Fig 7. GPX2 promoter gene map

(4) Learn

We confirmed that pGPX2 is the optimal ROS-responsive promoter, capable of achieving low
background, high intensity, and rapid response characteristics in an inflammatory
environment, providing a reliable "molecular switch" for the therapeutic pathway.
However, in the subsequent design process, when we attempted to place SOD1, CTT1, and
TFF3 under three separate pGPX2 promoters respectively, new challenges emerged:
Metabolic burden: Multiple parallel pathways would significantly increase energy
consumption;
Expression incoordination: Even relying on the same promoters, the dynamics of the three
pathways are difficult to be completely consistent;
Uncontrollable dose ratio: The production of the three proteins is difficult to maintain
a stable stoichiometric ratio, thereby weakening the synergistic effect.
Therefore, the team realized that the selection of a single high-quality promoter is
only the first step. The more crucial issue lies in how to achieve safe, efficient, and
coordinated expression of multiple proteins within the same pathway. This thinking
directly led to the next round of iteration (Cycle 5): how to achieve the joint control
of the anti-inflammatory and antioxidant modules.

Cycle 5: How can the joint control of anti-inflammation and
anti-oxidation be achieved

(1) Research

After identifying the final three effect proteins, SOD1, CTT1 and TFF3, the team needed
to address a key issue:
How to rationally express these proteins in the engineered bacteria to maximize their
synergistic effect?
The research found that if each protein was driven by an independent expression pathway,
it would cause excessive metabolic burden; if the expression was not synchronized, it
would be difficult to ensure the precise stoichiometric ratio; and this would further
weaken the synergistic therapeutic effect of the three proteins.

(2) Imagine

Based on this, the team envisioned giving up the "parallel scheme" of multiple
independent lines and instead attempting a more efficient and integrated approach:
Integrate multiple effector proteins into a single line to ensure their synchronous
expression, thereby enhancing the robustness and safety of the system.

(3) Design and Build

The team finally constructed a single-line multi-module design: GPX2 → NCW2-SOD1 → P2A →
NCW2-TFF3 → T2A → NCW2-CTT1.
The key design points include:
Promoter (GPX2): Only drives expression when inflammatory signals are present, ensuring
the synchrony and spatiotemporal consistency of the three proteins.
2A self-cleavage peptide (P2A, T2A): Initially, an attempt was made to fuse the three
proteins through a flexible linker, but analysis indicated that it would disrupt their
three-dimensional structure and lead to functional loss. The team conducted literature
research and adopted 2A self-cleavage peptides, enabling a single mRNA to generate three
functionally independent proteins in a 1:1:1 ratio during translation, maintaining
structural integrity while reducing metabolic burden.

![img](https://static.igem.wiki/teams/5847/engineering/part3-9.webp)

Fig 8. The mechanism of action of 2A peptide[8]

Secretion signal peptide (NCW2): Add the NCW2 sequence before each protein, directing
the product to be secreted outside the cell and ensuring its release as needed in the
intestinal lumen[9].
By combining various elements, we have developed our treatment plan.

![img](https://static.igem.wiki/teams/5847/engineering/part3-10.webp)

Fig 9. Treatment Module Diagram

(4) Test

In the scheme simulation and literature comparison, the team verified that the single
promoter + 2A peptide strategy is feasible and has been widely applied in multi-protein
systems; the 2A peptide can ensure a stable stoichiometric ratio and avoid functional
loss caused by the fusion protein; the application of the secretion signal peptide NCW2
has been well reported in eukaryotic expression systems, and can reliably guide protein
secretion.

![img](https://static.igem.wiki/teams/5847/engineering/part3-11.webp)

Fig 10. Comparison of signal peptide efficiency[9]

Influences on heterologous protein secretion by accessory proteins linked to SPs with
different properties. a) α-amylase secretion titer in the ref, △SPC1 and ΔSEC72 strains
when using different SPs. The α-factor SP was used as a reference to compared with other
11 super-secreted SPs. b) Hydrophobicity of SPs determined α-amylase secretion change
tendency (increase or decrease) in the ΔSEC72 strain. The secretion change was
calculated as follow: (amylase secretion by the ΔSEC72 strain – amylase secretion by the
ref strain)/(amylase secretion by the ref strain) × 100%. was used as the control
strain. Data shown are mean values ±SDs of duplicates[27-28].

After the information was confirmed, we began the wet experiment work, which was divided
into two parts: the construction of ΔSEC72 Saccharomyces cerevisiae and the construction
and introduction of therapeutic genes. Preliminary laboratory results have been obtained
at present.

![img](https://static.igem.wiki/teams/5847/engineering/part3-12.webp)

Fig 11. Treatment module-related gene map

(5) Learn

Through this round of cycle, the team realized that a single-line multi-module design
can significantly reduce genetic complexity and metabolic burden; using 2A self-cleaving
peptides is the most elegant way to solve the problem of multi-protein co-expression,
which is both efficient and reliable; in the iGEM project, engineering wisdom lies not
only in "getting the proteins expressed", but also in how to express them safely,
efficiently and coordinately to maximize the therapeutic synergy.

Cycle 6: The auxiliary role of symbiotic systems in treatment

(1) Research

After modeling and testing the engineered yeast with the integrated SOD1–TFF3–CTT1 gene
circuit, we found that the performance of the single yeast treatment module still had
limitations: although it had significant effects on ROS clearance (targeting the
upstream signal of JAK2) and intestinal barrier repair (affecting the sustained
activation of IL2), it was still insufficient in terms of the breadth of immune
regulation and long-term stability maintenance. Moreover, from the literature
comparison, it was also clear that there was a knowledge gap: this module had no known
direct connection with the TGFBR2 signaling pathway.

(2) Imagine

In response to this performance gap, the team did not choose to continue stacking more
complex circuits in yeast. Instead, they proposed a new idea: introducing a naturally
immune-regulating partner that can complement the yeast; expanding the engineering goal
from a single cell to a multi-microbial system, achieving a transition from molecular
engineering to micro-ecosystem engineering.

(3) Design and Build

The design concept is to combine the precise elimination ability of engineered yeast
with the broad-spectrum regulatory ability of probiotics to achieve coordinated
intervention at both the upstream and downstream of the inflammatory signals.
**a. The molecular circuitry of the yeast treatment module (precise elimination and
repair)**
The core of the engineered yeast is the condition-inducible therapeutic pathway, as
described above: pGPX2 → NCW2-SOD1 → P2A → NCW2-TFF3 → T2A → NCW2-CTT1.
**b. The natural immune regulation advantages of DT88 (broad-spectrum regulation and
signal weakening)**
The Lactobacillus plantarum DT88, as an auxiliary strain, compensates for the
deficiencies of the yeast module at the ecological and signaling levels through its
natural metabolic and cytokine regulatory capabilities:
**Inhibiting pro-inflammatory factors:**DT88 can significantly reduce the elevated
levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the microplastic-exposed
mouse model, while simultaneously synergistically enhancing the production of
anti-inflammatory factor IL-10.
**Inhibiting co-stimulatory molecules:** The symbiotic system has been proven to be
able to reduce the expression of activation markers CD86 on the surface of monocytes.

**Metabolic products anti-inflammatory:** The short-chain fatty acids (SCFAs)
produced by DT88, especially butyric acid, can inhibit the NF-κB signaling pathway in
immune cells and epithelial cells, reducing the transcription of inflammatory factors at
the nuclear level.

(4) Test

The objective of this section is to combine the evidence from the literature and the
modeling results to verify whether the dual intervention of the symbiotic system can
precisely act on the core target, and to focus on evaluating the micro-regulatory effect
of DT88.
**a. Immunomodulatory effects:**
The co-culture system of Saccharomyces cerevisiae and Lactobacillus exhibits unique
immunomodulatory properties, and its anti-inflammatory effect is significantly superior
to that of a single strain[10]. This synergy is not simply additive; rather,
it
stems
from the host immune system's specific recognition and response to the combined patterns
of microbial molecules (MAMPs). In mice exposed to microplastics, DT88 can restore
elevated levels of TNF-α, IL-6, and IL-1β to normal, and promote an increase in IL-10,
thereby guiding the immune response towards a tolerative state[11].
**b. Inhibition of immune cell activation:**
This system can significantly reduce the expression of activation markers CD14 and CD86
on the surface of monocytes, demonstrating its stable effect in inhibiting excessive
inflammatory signals[10].
**c. Contribution to metabolic homeostasis:**
Microbial metabolites, short-chain fatty acids (SCFAs), are the main energy source for
colonic epithelial cells, promoting their proliferation and repair. At the same time,
SCFAs can inhibit the NF-κB pathway, reduce the release of inflammatory factors, and
stimulate goblet cells to secrete mucin to strengthen the intestinal mucus
barrier[12].
**d. Molecular targeting mechanism:**
JAK2 pathway (upstream weakening): DT88 reduces the levels of TNF-α and IL-6, thereby
indirectly inhibiting the activation of the JAK/STAT pathway and synergistically
promoting the production of IL-10.
IL-2 pathway (co-stimulation inhibition): This system can down-regulate the expression
of CD86 on monocytes, which is equivalent to adding a "molecular brake" in the T cell
activation circuit, alleviating the excessive immune activation induced by
microplastics.
TGFBR2 pathway: Current research shows that there is no known direct molecular
association between this system and TGFBR2.

![img](https://static.igem.wiki/teams/5847/engineering/part3-13.webp)

Table 2. The effect of the symbiotic system on the target

(5) Learn

In conclusion, the therapeutic effect of this symbiotic system is multi-level and
three-dimensional. It has established a "deep defense" strategy. Additionally, we later
discovered that the symbiotic system has a reinforcing effect on the barrier structure:
The symbiotic system achieves structural reinforcement by regulating tight junction
proteins (TJPs). Exposure to microplastics can damage the integrity of the intestinal
barrier. Probiotics interact with epithelial cells and upregulate the expression of
"sealing-type" proteins such as ZO-1, Occludin, and Claudin-1, reducing the permeability
of the paracellular pathway and preventing harmful substances from leaking. Thus, this
module has initially solved the problem of the harm caused by microplastics to the human
body. In the next step, we need more experiments for verification[32].
In conclusion, the therapeutic effect of this symbiotic system is multi-level and
three-dimensional. It has established a "deep defense" strategy. Additionally, we later
discovered that the symbiotic system has a reinforcing effect on the barrier structure:
The symbiotic system achieves structural reinforcement by regulating tight junction
proteins (TJPs). Exposure to microplastics can damage the integrity of the intestinal
barrier. Probiotics interact with epithelial cells and upregulate the expression of
"sealing-type" proteins such as ZO-1, Occludin, and Claudin-1, reducing the permeability
of the paracellular pathway and preventing harmful substances from leaking. Thus, this
module has initially solved the problem of the harm caused by microplastics to the human
body. In the next step, we need more experiments for verification[13].

References

* 1. Yan Z, Liu Y, Zhang T, Zhang F, Ren H, Zhang Y. Analysis of Microplastics in Human Feces
  Reveals a Correlation between Fecal Microplastics and Inflammatory Bowel Disease Status.
  Environ Sci Technol. 2022;56(1):414-421. doi:10.1021/acs.est.1c03924
* 2. Fournier E, Leveque M, Ruiz P, et al. Microplastics: What happens in the human
  digestive
  tract? First evidences in adults using in vitro gut models. J Hazard Mater.
  2023;442:130010.
  doi:10.1016/j.jhazmat.2022.130010
* 3. Thin ZS, Chew J, Ong TYY, Raja Ali RA, Gew LT. Impact of microplastics on the human
  gut
  microbiome: a systematic review of microbial composition, diversity, and metabolic
  disruptions. BMC Gastroenterol. 2025;25(1):583. Published 2025 Aug 13.
  doi:10.1186/s12876-025-04140-2
* 4. Miao L, St Clair DK. Regulation of superoxide dismutase genes: implications in
  disease.
  Free Radic Biol Med. 2009;47(4):344-356. doi:10.1016/j.freeradbiomed.2009.05.018
* 5. Bissinger PH, Wieser R, Hamilton B, Ruis H. Control of Saccharomyces cerevisiae
  catalase T
  gene (CTT1) expression by nutrient supply via the RAS-cyclic AMP pathway. Mol Cell Biol.
  1989;9(3):1309-1315. doi:10.1128/mcb.9.3.1309-1315.1989
* 6. Thim L, Wöldike HF, Nielsen PF, Christensen M, Lynch-Devaney K, Podolsky DK.
  Characterization of human and rat intestinal trefoil factor produced in yeast.
  Biochemistry.
  1995;34(14):4757-4764. doi:10.1021/bi00014a033
* 7. Tsuzi D, Maeta K, Takatsume Y, Izawa S, Inoue Y. Regulation of the yeast phospholipid
  hydroperoxide glutathione peroxidase GPX2 by oxidative stress is mediated by Yap1 and
  Skn7.
  FEBS Lett. 2004;565(1-3):148-154. doi:10.1016/j.febslet.2004.03.091
* 8. Liu Z, Chen O, Wall JBJ, et al. Systematic comparison of 2A peptides for cloning
  multi-genes in a polycistronic vector. Sci Rep. 2017;7(1):2193. Published 2017 May 19.
  doi:10.1038/s41598-017-02460-2
* 9. Xue S, Liu X, Pan Y, et al. Comprehensive Analysis of Signal Peptides in Saccharomyces
  cerevisiae Reveals Features for Efficient Secretion. Adv Sci (Weinh).
  2023;10(2):e2203433.
  doi:10.1002/advs.202203433
* 10. Nenciarini S, Rivero D, Ciccione A, et al. Impact of cooperative or competitive
  dynamics
  between the yeast Saccharomyces cerevisiae and lactobacilli on the immune response of
  the
  host. Front Immunol. 2024;15:1399842. Published 2024 Oct 10.
  doi:10.3389/fimmu.2024.1399842
* 11. Teng X, Zhang T, Rao C. Novel probiotics adsorbing and excreting microplastics in
  vivo
  show potential gut health benefits. Front Microbiol. 2025;15:1522794. Published 2025 Jan
  10.
  doi:10.3389/fmicb.2024.1522794
* 12. Markowiak-Kopeć P, Śliżewska K. The Effect of Probiotics on the Production of
  Short-Chain
  Fatty Acids by Human Intestinal Microbiome. Nutrients. 2020;12(4):1107. Published 2020
  Apr
  16. doi:10.3390/nu12041107
* 13. Demarquoy J. Microplastics and probiotics: Mechanisms of interaction and their
  consequences for health. AIMS Microbiol. 2025;11(2):388-409. Published 2025 Jun 9.
  doi:10.3934/microbiol.2025018

Engineering

Cycle 1: Selection of chassis strains (safety module)

Cycle 2: The "safety support" function of a symbiotic system

Cycle 3: Anti-acid and bile acid module

Cycle 4: Thoughts on Safety Circuitry

Cycle 5: Extracorporeal safety

Cycle 6: Internal safety

Cycle 7: Spore prevention (knockout gene)

References

Cycle 1: Selection of chassis strains (safety module)

(1) Research

The chassis bacteria must meet the following conditions in order to exert therapeutic
effects in the intestinal tract:
**a. Safety and modifiability:** They must be safe for human use and have mature
modification tools;
**b. Ability to colonize the intestine:** They must be able to survive in the complex
intestinal environment for a long time;
**c. Expression ability:** They must ensure the effective operation of the synthetic
biological circuit;
**d. Natural therapeutic properties (plus points):** such as anti-inflammatory and
adsorption capabilities.

Based on this, the team compared several candidate strains:
**a. E. coli (K-12, Nissle 1917):** The tools are mature, but the safety and
colonization
ability are insufficient;
**b. Bifidobacteria, Bacteroides, Clostridium:** They have colonization or metabolic
advantages, but the tools are not mature or their safety is questionable;
**c. Lactobacillus:** Food-grade probiotics, resistant to acid and bile, able to
survive
for a long time, but the tools and expression efficiency are limited;
**d. Saccharomyces cerevisiae:** GRAS certified, mature tools, capable of secreting
complex proteins, but limited colonization.

![img](https://static.igem.wiki/teams/5847/engineering/safe1.webp)

Table 3. Comparison of adaptability of candidate chassis strains

Conclusion: No single strain can fully meet the requirements. However, E. coli Nissle
1917
and Saccharomyces cerevisiae demonstrate excellent safety and engineering capabilities,
while Lactobacillus plantarum has unique anti-inflammatory and adsorption advantages.

(2) Imagine

After a preliminary comparison, we realized that no single strain could simultaneously
meet
all the requirements of safety, engineering feasibility, intestinal colonization ability
and
expression efficiency. This led us to propose a new idea - to construct a "symbiotic
system", hoping to select two food-grade strains to make up for their respective
deficiencies.

We hypothesized that the symbiotic system might be a more ideal choice than a single
chassis
strain, as it not only enhances the overall safety and stability of the system, but also
takes advantage of the intestinal therapeutic properties of Lactobacillus plantarum
itself.

(3) Design and Build

During further literature research, we discovered a unique strain of Lactobacillus
plantarum, L. plantarum DT88. Previous studies have shown that it can survive in the
intestinal tract for an extended period and has the ability to adsorb microplastics,
which
perfectly aligns with the goals of our project[1].
By reviewing the relevant literature on symbiotic systems, we found that yeast and
Lactobacillus plantarum often coexist and mutually benefit, exchanging nutrients to
promote
each other's growth and enhance the stability of the community[2].However, in
most
environments, Escherichia coli is inhibited by Lactobacillus plantarum - because it
secretes
organic acids, lowers the pH, competes for resources and space - making it difficult for
it
to maintain stability and functionality as a symbiotic system[3].
So, our team members immediately agreed and we designed a system of division of labor
and
cooperation:
**Saccharomyces cerevisiae:** The main base strain, responsible for logic gate
control,
multi-enzyme co-secretion and treatment modules;
**Lactobacillus DT88:** The auxiliary strain, utilizing its natural microplastic
adsorption ability and probiotic characteristics to enhance overall safety and
application
potential.
Eventually, we selected the "yeast + DT88" symbiotic consortium as the engineering
bacterial
chassis.

(4) Test

We verified through literature and existing studies: 1. Lactobacillus DT88 demonstrated
excellent microplastic adsorption ability in vitro and could survive stably in the
intestinal tract.

![img](https://static.igem.wiki/teams/5847/engineering/safe2.webp)

Fig 1. Scanning electron microscopy (SEM) images of bacteria aggregated with
microplastics[1]

**a. Saccharomyces cerevisiae** has a long-term application history in the food and
pharmaceutical industries (GRAS certification), and can efficiently secrete complex
proteins.
**b. The two** can support each other under a symbiotic condition, enhancing the
stability and functionality of the community. The rationality of the symbiotic system
has
been further confirmed at this stage.

(5) Learn

Each single strain has its own drawbacks: it cannot simultaneously achieve safety, tool
maturity or functionality; the symbiotic system, which combines safety, functionality
and
scalability, better meets the application requirements of intestinal treatment.
At this stage, the team realized that the transition from single-bacterial engineering
to
symbiotic system engineering is the key to improving the performance and safety of the
project. In the future, we will further explore the functional potential of the
symbiotic
system to make it a more powerful and stable treatment platform.

Cycle 2: The "safety support" function of a symbiotic system

(1) Research

In the first safety cycle, we identified the limitations of a single strain in terms of
safety, functionality, and intestinal colonization ability. Although engineering
modifications can confer strong functions to the strain, they also bring potential
biosafety
risks, such as stability and controllability issues in a complex intestinal environment.
Therefore, we studied how to enhance overall safety through systematic design.
Literature
indicates that introducing an unmodified probiotic at the GRAS (Generally Recognized as
Safe) level as a companion not only utilizes its natural probiotic properties but also
enhances the stability and predictability of the entire system through a mutually
beneficial
symbiotic relationship.

(2) Imagine

We envision that the symbiotic system itself can serve as a "security barrier", using
natural probiotics to "support" and "restrict" the engineered yeast, thereby enhancing
the
biological safety of the entire product. This is the original intention behind our
construction of the symbiotic system.

(3) Design and Build

We designed and constructed a symbiotic system consisting of engineered Saccharomyces
cerevisiae (GRAS-certified) and the natural probiotic Lactobacillus plantarum DT88. We
conducted an in-depth analysis of the advantages in the previous strain selection
process.
The core safety logic of this design is "responsibility separation":
**a. Function and modification separation:** We concentrated the complex genetic
circuits (such as adsorption, anti-inflammatory modules) in the modified Saccharomyces
cerevisiae, while Lactobacillus plantarum DT88 was completely retained in its wild type
and
underwent no genetic modification. This enabled most risks to be concentrated in a
chassis.

**b. Utilizing natural probiotic properties:** Lactobacillus plantarum DT88 is
itself a
verified probiotic with the abilities to resist inflammation, regulate the intestinal
microbiota, and adsorb microplastics. Its addition not only does not introduce new
safety
risks but also adds additional therapeutic benefits and safety guarantees to the system.
**c. Enhancing system stability:** The two organisms form a more stable
micro-ecosystem
through nutritional complementation and stress sharing, reducing the risk of the
engineered
bacteria losing control or failing in the intestinal tract.

![img](https://static.igem.wiki/teams/5847/engineering/safe3.webp)

Fig 2. Detailed Description of Symbiotic Systems

The symbiotic system composed of Saccharomyces cerevisiae and Lactobacillus plantarum
can
establish metabolic interdependence and mutual provision of nutrients. In an environment
rich in nitrogen sources such as the intestinal tract, Saccharomyces cerevisiae will,
through an active metabolic strategy regulated by the TORC1 signaling pathway, secrete
excessive amino acids. The amino acids secreted by the yeast provide essential growth
factors for many amino acid-deficient lactic acid bacteria (such as Lactobacillus
plantarum). This targeted nutrient supply significantly promotes the growth and survival
of
Lactobacillus plantarum in the co-culture system, with its biomass being much higher
than
that in a single culture condition. Although the main nutrient flow is from yeast to
bacteria, this relationship can develop into mutualism. For example, lactic acid
bacteria
can hydrolyze some complex carbohydrates that yeast cannot directly utilize (such as
lactose), breaking them down into monosaccharides like glucose and galactose, which can
be
absorbed and utilized by yeast.
Saccharomyces cerevisiae can secrete some extracellular peptide substances known as
"reactivation factors". These substances have been proven to protect the coexisting
Lactobacillus from the stress damage caused by bile salts, which is crucial for the
survival
of probiotics in the upper digestive tract.The deep cavity of the intestine is an
anaerobic or micro-aerobic environment. Saccharomyces cerevisiae mainly undergoes
alcohol
fermentation here, producing ethanol. High concentrations of ethanol are also toxic to
the
yeast itself. Studies have found that co-culture with Lactobacillus plantarum can
significantly improve the ethanol tolerance of Saccharomyces cerevisiae, enabling it to
persist longer and function more effectively in the intestinal
environment[4-7].

(4) Test

Although the symbiotic system can provide mutual support and enhance stability in the
intestinal environment, the team found that it still faces severe challenges when
passing
through the gastrointestinal tract. Saccharomyces cerevisiae must first withstand the
harsh
acidic gastric juice and bile salts, but its survival rate will significantly decrease.
This
directly affects the number of effective bacteria that can reach the intestine and
function
properly. The team realized that if most of the engineered yeast were inactivated before
reaching the target site, then no matter how sophisticated the symbiotic system design
was,
it could not achieve the expected therapeutic effect. Therefore, although the symbiotic
system provided important safety support for the project, it itself could not completely
solve the physical and chemical challenges brought by the digestive tract environment,
and
additional protective measures were urgently needed.

(5) Learn

Through this round of safety cycle, the team has clearly recognized the value of the
symbiotic system as an endogenous security strategy. However, we also realize that
relying
solely on the biological symbiosis and mutual benefit cannot completely address the
stringent challenges of the digestive tract environment. To ensure that the engineered
yeast
can reach the intestine stably and efficiently and exert its therapeutic effect, we must
develop additional protective mechanisms. Our next step will be to focus on researching
and
comparing various protection strategies against gastric acid and bile salts, in order to
find the safest, most effective and feasible solution.

Cycle 3: Anti-acid and bile acid module

(1) Research

Before the Saccharomyces cerevisiae enters the intestine as the host strain, it must
overcome the challenges of strong acidic gastric juice (pH 1.5–3.5) and bile salts. To
this
end, we systematically investigated various protective strategies and classified them
into
three categories:
a. Intrinsic biological strategies: enhancing tolerance through genetic engineering (PMA1
overexpression, strengthening the CWI pathway, ALE evolution).
b. Extrinsic physical strategies: providing a barrier through microcapsule encapsulation
(alginate, SA-chitosan complex, functional composite materials).
c. Exogenous and symbiotic strategies: buffering acid and bile salts by using food
matrices
or co-cultured microorganisms.
After comparison, we found that the sodium alginate-chitosan composite microcapsule
achieved
the best balance point in terms of protective effect, feasibility, cost and safety.
Therefore, it was used as the anti-gastric acid and anti-bile acid module.

![img](https://static.igem.wiki/teams/5847/engineering/safe4.webp)

Table 4. Comparison of yeast protection strategies

(2) Imagine

We envision using the alginate-chitosan composite microcapsules to provide an external
"armor" for the Saccharomyces cerevisiae:
The yeast cells are first encapsulated in the alginate gel matrix; through Ca²⁺
cross-linking,a stable gel network is formed；an outer layer of chitosan is then coated
to
form a dense poly-electrolyte composite membrane；after entering the small intestine,the
microcapsules gradually disintegrate, releasing the active yeast.

(3) Design and Build

Based on the literature and experimental feasibility, we have formulated the following
plan:

Core matrix: 3% (w/v) sodium alginate solution;
Crosslinking agent: 2% (w/v) CaCl₂ solution, cured for 30 minutes;
Protective shell: 0.38% (w/v) chitosan solution, coated for 60 minutes;
Post-treatment: Sodium citrate treatment is carried out to obtain a "liquid core",
avoiding
physical compression and improving survival rate.
Preparation method: Gel beads are formed by syringe drop method, with aseptic operation
throughout the process

(4) Test

We verified the protective effect of microcapsules through in vitro simulation
experiments:
free yeast was basically inactivated; single-layer SA embedding provided partial
protection;
SA-chitosan composite embedding showed a significantly higher survival rate.

![img](https://static.igem.wiki/teams/5847/engineering/safe5.webp)

Fig 3. Microcapsule coating experiment

(5) Learn

Through this round of cycle, we have gained the following experiences:
a. The SA-chitosan composite system effectively enhanced the survival rate of yeast.
b. The liquid core design prevented cells from being compressed and improved the
long-term
survival rate.
c. The subsequent optimization directions are:
adjusting the concentrations of SA and chitosan to improve acid tolerance;
adding prebiotics (such as inulin) to the matrix to achieve a "synbiotic" effect;
combining genetic engineering strategies (such as overexpression of PMA1) with physical
encapsulation to construct a dual-layer defense system[8-13].

Cycle 4: Thoughts on Safety Circuitry

(1) Research

Just like most oral medications and health supplements, when addressing a certain issue
in
the human body, we should minimize the harm to the body as much as possible. In our
previous
design and construction, we took this into consideration. However, we felt that a safe
and
effective way was still needed to directly eliminate the harm of the product to the
human
body. We conducted extensive literature research and reviewed the safety control systems
of
past iGEM teams, from which we gained inspiration.

(2) Imagine

We believe that the safest and most effective method is through a kind of
"signal-controlled
sterilization". It consists of sensors and effectors, just like the reflexes in the
human
body. It is simple yet highly effective.

![img](https://static.igem.wiki/teams/5847/engineering/safe6.webp)

Fig 4. Our security strategy direction

(3) Design and Build

Since this product is used through the human body, we initially selected the sensors of
the
applicable safety lines for this project: 1. Input signal from the front of the body 2.
Changes in internal signals 3. Input signal from the rear of the body. The input signal
from
the front of the body refers to when the product is inside the body, it activates the
safety
line by receiving signals from certain substances taken orally; the change in internal
signals means that some inherent conditions within the human body act as signals, and
this
signal change is activated by similar "0-1" input changes in the engineering process;
the
input signal from the rear of the body refers to the need to apply external factors to
activate the safety line when a small number of engineering bacteria are excreted
outside
the body and there may be a risk of leakage.
As for the effectors, we designed them from the very beginning using the
anti-toxin-toxin
system and nucleic acid metabolism perspectives. This part of the review is mainly
conducted
from these two perspectives, and in the future, our method will also be referred to by
other
teams' methods for further optimization.

(4) Test

After conducting a systematic review, we verified that our idea was feasible. Moreover,
we
also applied this perception solution in other modules. This part reflects our
contribution
to the engineering cycle in our safety review. For the specific review, please refer to
the
[safety section.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27safety%27%29%20%7D%7D)

(5) Learn

Through a systematic review of the sensing signals and in combination with the
characteristics of our own project, we have initially understood how to select the
conditions for regulating the safe circuit. However, most of our review was focused on
prokaryotes. The activation expression elements for the corresponding conditions in
Saccharomyces cerevisiae still require further exploration by us.

Cycle 5: Extracorporeal safety

(1) Research

The first type of chassis bacteria we consider to be in a "suicide scenario" is the
issue of
preventing leakage when they are excreted out of the body.
1. Environmental persistence: The engineered bacteria survive unexpectedly in the soil
or
water.
2. HGT risk: The genetic circuits of the engineered bacteria transfer to the natural
bacterial community, causing unpredictable ecological impacts.
So, how can we ensure that the engineered bacteria do not spread to the environment when
they are in the action environment? How can we achieve "elimination upon exhaustion"?

(2) Imagine

We reviewed the results of the previous engineering cycle and wondered: Isn't it
precisely
by sensing the changes within the body that we can achieve the effect of "exhaustion
leads
to extinction" in a specific environment?

(3) Design and Build

The most reliable external safety switch must utilize the inherent and inevitable
physical
or chemical differences between the internal environment (the intestinal tract) and the
external environment (waste). Research has found that the temperature difference is one
of
the simplest and most reliable signals.
Internal safety state: The core body temperature of the human body remains at 37 ℃ ± 1
℃.

External clearance state: After excretions enter the environment (such as a laboratory
or
environmental water bodies), the temperature usually drops rapidly to the environmental
temperature (usually below 30 ℃).
This stable temperature gradient can meet the ideal control logic of "OFF in the body,
ON
outside the body". Based on this, we chose to construct a cold-triggered kill switch.

To achieve the logic of "surviving at 37℃ (OFF), killing at low temperatures (ON)", we
cannot simply use cold-inducing promoters because many cold promoters have the defect of
low
background expression, which may lead to leakage at 37℃. We conceived a reverse logic
circuit based on heat shock promoters to ensure the highest control accuracy:
Utilizing the heat-activated promoter (PHSP) to drive a transcriptional repressor.
At 37℃ (inside the body): PHSP activity is high → A large number of inhibitory factors
are
expressed → The killing system is completely inhibited → Safe State (OFF).
At low temperature (outside the body): PHSP activity is low → The expression of
inhibitory
factors decreases → The killing system is un-inhibited and activated → Kill State
(ON)

![img](https://static.igem.wiki/teams/5847/engineering/safe7.webp)

Fig 5. Temperature-killing design direction

Regarding the comparison and selection of promoters, Saccharomyces cerevisiae possesses
multiple heat shock protein promoters (such as PHSP104, PHSP82, PHSP26). After comparing
the
characteristics of the promoters in the literature, we chose PHSP26. The PHSP26 promoter
is
renowned for its high sensitivity to heat stress and fast response. We hypothesized (and
will verify in the tests) that: PHSP26 can produce sufficient high concentrations of
inhibitory protein (TetR) at 37°C (equivalent to a mild heat shock for yeast), ensuring
the
inhibition and suppression of the downstream killing pathway (with extremely low
background
leakage). When the temperature drops, the transcriptional activity of PHSP26 will
rapidly
decline, releasing the inhibition and triggering the killing process.
In terms of the effectors, we start from the genetic material DNA to design the killing
mechanism. We have divided it into two schemes: inhibiting and blocking DNA synthesis,
and
directly destroying the DNA structure. First, we identified the key gene ADE2 in the
nucleic
acid metabolism of Saccharomy cescerevisiae.The ADE2 gene encodes
phosphoribosylaminoimidazole carboxylase, which is a key enzyme in the de novo
biosynthesis
pathway of purines (such as adenine)[14].
Then we identified the nucleases nucA, which, as an heterologous protein, has been
experimentally demonstrated in the literature to be capable of exerting a lethal effect
on
yeast cells[15].
Regarding this effector aspect, we proposed two schemes:
Scheme One (ADE2): (Fig 6. A)
Design the lox66 and lox71 sequences, and insert them into the upstream and downstream
of
the ADE2 gene using the CRISPR/Cas9 technology, so that ADE2 is in a "ready for
deletion"
state that Cre can recognize.
Construct the PtetR→Cre Recombinase expression plasmid → express the knockout of ADE2

Scheme Two (NucA): (Fig 6. B)
Construct the PtetR→nucA expression plasmid
By combining the sensor and the effector in this way, we obtained the in vitro safe
circuit
that we designed.

![img](https://static.igem.wiki/teams/5847/engineering/safe8.webp)

Fig 6. Construction of external safety circuits

(4) Test

In this part, we conducted an intensity experiment to verify the important promoter
HSP26.
We placed EGFP downstream of the HSP26 promoter and performed an enzyme reader analysis.
The
final results showed that HSP26 is indeed an excellent temperature promoter.
Additionally, due to the heterogeneity of nucA, we needed to be cautious. We discovered
that
nucA without a signal peptide segment in the literature still has functionality.
Therefore,
we predicted the site of signal peptide cleavage and finally removed the signal peptide
segment to obtain the target protein. The predicted 3D structure has high confidence and
is
close to the real structure. This provided a strong foundation for our laboratory's
construction.

![img](https://static.igem.wiki/teams/5847/engineering/safe9.webp)

Fig 7. Protein acquisition of nucA

(5) Learn

In this part, we conducted an intensity experiment to verify the important promoter
HSP26.
We placed EGFP downstream of the HSP26 promoter and performed an enzyme reader analysis.
The
final results showed that HSP26 is indeed an excellent temperature promoter.
Additionally, due to the heterogeneity of nucA, we needed to be cautious. We discovered
that
nucA without a signal peptide segment in the literature still has functionality.
Therefore,
we predicted the site of signal peptide cleavage and finally removed the signal peptide
segment to obtain the target protein. The predicted 3D structure has high confidence and
is
close to the real structure. This provided a strong foundation for our laboratory's
construction.

Cycle 6: Internal safety

(1) Research

Following the previous cycle, how can we ensure that the engineered bacteria can be
effectively removed after completing their tasks, avoiding their prolonged retention in
the
intestinal tract and thereby reducing the risk of entering the bloodstream?
Referring to our review of the sensors, we discussed that the "drug" control system has
great potential for regulating the safe pathways within the body. When the product is
put
into use, this sensing method can fully exert its flexibility to ensure personal safety
after consumption. For the effector part, we investigated various biological safety
control
strategies, including traditional antibiotic killing, nutritional defect types, and
inducible suicide switches. Among them, the exogenous small molecule-induced
toxin-antitoxin
system is highly regarded due to its strong controllability, rapid response, and
independence from host endogenous signals.

(2) Imagine

We envision that a highly controllable "suicide switch" can be designed, which will be
activated when the engineered bacteria reach a specific stage within the host body. This
switch should be like a secure "key", only activating the clearance program upon
receiving a
clear external instruction, thereby minimizing the potential risks to the host to the
greatest extent.

(3) Design and Build

Based on the above concept, we learned from the GreatBay-SCIE 2023 team and designed a
set
of an Araboside-induced K1 toxin expression system as a safe clearance pathway in
vivo:
Sensor: We use Araboside to induce the safety system. When oral Araboside is taken, the
engineered bacteria will sense this safety signal.
Effectors: Use the expression system of K1 killer toxin. The precursor of K1 toxin is
processed in the cells to form a mature αβ heterodimer, which can specifically bind and
lyse
the cell wall of sensitive yeast cells, causing cell death.
Working principle:
a. During normal operation, the repressive promoter pNOTAra inhibits toxin expression,
and
the system is in a safe state;
b. When it is necessary to clear the engineered bacteria, the patient orally takes
Araboside. The Araboside enters the intestine and is perceived by the engineered
bacteria;
c. The inducible promoter pAra is activated, driving the expression of the K1 toxin
precursor protein;
d. The toxin precursor is processed and cut in the endoplasmic reticulum and Golgi
apparatus, and finally secreted to the extracellular space;
e. The mature toxin binds to the β-1,6-glucan of the yeast cell wall through its α
subunit,
and the β subunit forms a channel, causing the engineered yeast cells to lyse and die.

![img](https://static.igem.wiki/teams/5847/description/description10-4.webp)

Fig 8. Construction of internal safety circuits

(4) Test

We verified the feasibility of this safety circuit through literature and existing iGEM
projects, and also identified its potential shortcomings: Feasibility verification: The
arabinose induction system has been successfully applied in both Escherichia coli and
yeast,
with rapid response and high sensitivity. The K1 toxin has a highly specific killing
effect
on Saccharomyces cerevisiae. We have conducted preliminary experiments so far, but still
need optimization.

![img](https://static.igem.wiki/teams/5847/engineering/f11.avif)

Fig 9. Arabinose-induced preliminary experiment

(5) Learn

At present, we have also identified some potential deficiencies in the system: 1.
Leakage
expression risk: There is background leakage expression of the two promoters in the
arabinose regulatory system, and the dynamic relationship between them needs further
study.
2. Individual differences impact: Different individuals have different rates of
absorption
and metabolism of arabinose in the intestine, which may lead to unstable induction
efficiency. In the future, many aspects need to be considered and optimized.

Cycle 7: Spore prevention (knockout gene)

(1) Research

Firstly, we conducted a thorough literature review on the spore formation mechanism of
Saccharomyces cerevisiae, and focused on referring to the official safety guidelines of
iGEM. According to the White List (White List | iGEM
Responsibility)published on the iGEM website, although Saccharomyces cerevisiae
is
classified as a microorganism with biosafety level 1, since it has the potential to form
spores, the team must formulate measures to reduce the associated risks. The official
clearly stated that spore formation, due to its difficulty to be controlled, may lead to
"Release Beyond Containment", which would directly violate the core safety policy of
iGEM.

Studies have shown that spore formation is a complex process that is strictly regulated,
and
its initiation depends on specific environmental signals - extreme nitrogen deficiency
and
the presence of non-fermentable carbon sources. Among the key regulatory genes, the
transcriptional activator Ime1p encoded by the IME1 gene is the "master switch" for
meiosis
and spore formation; the IME4 gene determines the accumulation of IME1 transcripts; and
the
SPO11 gene is responsible for the DNA double-strand breaks required for
meiosis[16].

(2) Imagine

Based on the above research, we envision using genetic engineering techniques to
fundamentally eliminate the spore-forming ability of Saccharomyces cerevisiae. Our goal
is
to establish a "spore-free" chassis that permanently resists the environmental signals
that
induce spore formation. Compared to relying solely on external environmental control
(which
is unreliable outside the laboratory), directly disrupting the genetic pathway is a more
thorough and controllable safety strategy.

(3) Design and Build

Among all the candidate genes, we finally chose IME1 as the knockout target. As the
highest
regulator of spore formation, the deletion of IME1 can completely block the entire
cascade
reaction from its source, and has the least impact on the normal growth of yeast.

(4) Test

**Literature verification**
Multiple authoritative studies have shown that the deletion of IME1 can specifically and
stably eliminate the ability to form spores. IME1 has been confirmed to be the "master
switch" for meiosis and spore formation. After deleting this gene, cells are completely
insensitive to induction signals such as nitrogen source deficiency. Relevant
experimental
evidence indicates that the diploid strain ime1Δ/ime1Δ is completely unable to form
spores
in the spore formation medium. Additionally, the same study also proved that the ime1Δ
mutation does not affect mitotic reproduction and basic metabolism. For example, some
related applications have successfully constructed sporeless ime1Δ strains while
maintaining
excellent biotechnological characteristics[15].
**Experimental Design**
Knockout of the IME1 gene: We used the CRISPR/Cas9 technology to design and implement
the
precise knockout of the IME1 gene.
Spore formation experiment: The modified yeast and the wild-type yeast were placed
together
in standard spore formation media (such as potassium acetate medium), and observed under
a
microscope for several days. The wild-type should show typical tetrad spores, while the
modified strain would completely fail to form any spore structures.

![img](https://static.igem.wiki/teams/5847/engineering/safe12.webp)

Fig 10. The experimental design for the verification after knocking out IME1

(5) Learn

Through this round of cycle, we envision having constructed a safe platform that cannot
form
spores, effectively addressing the significant biological safety risks posed by spore
dispersion in engineered bacteria.
However, we also recognize that merely achieving "spore-free" status is not sufficient
to
establish a comprehensive safety system. Although the spore risk has been eliminated,
the
nutrient cells may still survive for a short period after being released into the
environment, and there is a very low probability of horizontal gene transfer (HGT)
risk.
In the future, we will conduct more exploration of target genes and carry out related
wet
experiments to select the most suitable knockout genes for this project.

References

* 1. Teng X, Zhang T, Rao C. Novel probiotics adsorbing and excreting microplastics in vivo
  show potential gut health benefits. Front Microbiol. 2025;15:1522794. Published 2025 Jan
  10.
  doi:10.3389/fmicb.2024.1522794
* 2. Ponomarova O, Gabrielli N, Sévin DC, et al. Yeast Creates a Niche for Symbiotic Lactic
  Acid Bacteria through Nitrogen Overflow. Cell Syst. 2017;5(4):345-357.e6.
  doi:10.1016/j.cels.2017.09.002
* 3. Carvalho FM, Teixeira-Santos R, Mergulhão FJM, Gomes LC. Effect of Lactobacillus
  plantarum
  Biofilms on the Adhesion of Escherichia coli to Urinary Tract Devices. Antibiotics
  (Basel).
  2021;10(8):966. Published 2021 Aug 11. doi:10.3390/antibiotics10080966
* 4. Nenciarini S, Rivero D, Ciccione A, et al. Impact of cooperative or competitive
  dynamics
  between the yeast Saccharomyces cerevisiae and lactobacilli on the immune response of
  the
  host. Front Immunol. 2024;15:1399842. Published 2024 Oct 10.
  doi:10.3389/fimmu.2024.1399842
* 5. Ponomarova O, Gabrielli N, Sévin DC, et al. Yeast Creates a Niche for Symbiotic Lactic
  Acid Bacteria through Nitrogen Overflow. Cell Syst. 2017;5(4):345-357.e6.
  doi:10.1016/j.cels.2017.09.002
* 6. Vorob'eva LI, Khodzhaev EY, Rogozhin EA, Cherdyntseva TA, Netrusov AI.
  Characterization of
  Extracellular Yeast Peptide Factors and Their Stress-Protective Effect on Probiotic
  Lactic
  Acid Bacteria. Mikrobiologiia. 2016;85(4):393-402.
* 7. He X, Liu B, Xu Y, Chen Z, Li H. Effects of Lactobacillus plantarum on the ethanol
  tolerance of Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2021;105(6):2597-2611.
  doi:10.1007/s00253-021-11198-x
* 8. Lee Y, Nasution O, Lee YM, Kim E, Choi W, Kim W. Overexpression of PMA1 enhances
  tolerance
  to various types of stress and constitutively activates the SAPK pathways in
  Saccharomyces
  cerevisiae. Appl Microbiol Biotechnol. 2017;101(1):229-239.
  doi:10.1007/s00253-016-7898-5
* 9. Sánchez-Adriá IE, Sanmartín G, Prieto JA, Estruch F, Fortis E, Randez-Gil F. Adaptive
  laboratory evolution for acetic acid-tolerance matches sourdough challenges with yeast
  phenotypes. Microbiol Res. 2023;277:127487. doi:10.1016/j.micres.2023.127487
* 10. Chávarri M, Marañón I, Ares R, Ibáñez FC, Marzo F, Villarán Mdel C.
  Microencapsulation of
  a probiotic and prebiotic in alginate-chitosan capsules improves survival in simulated
  gastro-intestinal conditions. Int J Food Microbiol. 2010;142(1-2):185-189.
  doi:10.1016/j.ijfoodmicro.2010.06.022
* 11. Deng N, Du H, Xu Y. Cooperative Response of Pichia kudriavzevii and Saccharomyces
  cerevisiae to Lactic Acid Stress in Baijiu Fermentation. J Agric Food Chem.
  2020;68(17):4903-4911. doi:10.1021/acs.jafc.9b08052
* 12. Levin DE. Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol Mol
  Biol
  Rev. 2005;69(2):262-291. doi:10.1128/MMBR.69.2.262-291.2005
* 13. Kokina A, Kibilds J, Liepins J. Adenine auxotrophy--be aware: some effects of adenine
  auxotrophy in Saccharomyces cerevisiae strain W303-1A. FEMS Yeast Res.
  2014;14(5):697-707.
  doi:10.1111/1567-1364.12154
* 14. Balan A, Schenberg AC. A conditional suicide system for Saccharomyces cerevisiae
  relying
  on the intracellular production of the Serratia marcescens nuclease. Yeast.
  2005;22(3):203-212. doi:10.1002/yea.1203
* 15. Clancy MJ, Shambaugh ME, Timpte CS, Bokar JA. Induction of sporulation in
  Saccharomyces
  cerevisiae leads to the formation of N6-methyladenosine in mRNA: a potential mechanism
  for
  the activity of the IME4 gene. Nucleic Acids Res. 2002;30(20):4509-4518.
  doi:10.1093/nar/gkf573
* 16. Ramírez M, Ambrona J. Construction of sterile ime1Delta-transgenic Saccharomyces
  cerevisiae wine yeasts unable to disseminate in nature. Appl Environ Microbiol.
  2008;74(7):2129-2134. doi:10.1128/AEM.01840-07

{% endblock %}

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

### Experiments

Scroll To Explore

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %} {% block title %}Result{% endblock %} {% block lead
%}You can describe the results of your project and your future plans here.{%
endblock %} {% block page\_content %}

### Results

Scroll To Explore

Result

Verification of Degradation Module Feasibility

Verification of Adsorption Module Feasibility

Validation of Anti-inflammatory and Antioxidant Module

Validation of Safety Modules

Verification of Degradation Module Feasibility

1.Construction of Degradation Enzyme Expression Strains

Using genome editing to construct A MHETase-expressing yeast strain. First, a linear repair
template
containing the MHETase gene was amplified and fused, then integrated into the genome via
CRISPR-mediated
homologous recombination. Subsequently, a plasmid carrying the FAST-PETase gene was
introduced into the
yeast, generating the final degradation enzyme expression strain.

![img](https://static.igem.wiki/teams/5847/results/res1.webp)

Figure 1. Construction of degradation enzyme expression strains.

A.Design of plasmids and fusion fragments;
B.Gel electrophoresis of the fused MHETase repair template;
C.Plate culture of degradation enzyme expression strains.

2.Verification of Enzyme Expression Strains

First, extract genomic DNA of the recombinant strains. Colony PCR was performed then using
primers
targeting the fusion junctions and degradation enzyme genes, followed by gel electrophoresis
to verify
integration and expression.

![img](https://static.igem.wiki/teams/5847/results/res2.webp)

Figure 2. Verification of PCR amplified FAST-PETase gene and MHETase fragment junction
by gel
electrophoresis.

Target colonies with correct integration were selected for subsequent experiments.

3.Measurement of Microplastic Degradation Efficiency

TPA, MHET, and BHET standards were mixed in equal ratios to prepare a gradient concentration
mixture.
Retention times were recorded and standard curves was generated via HPLC. The degradation
enzyme
expression strains were cultured in 20 mL of medium containing microplastics for 72 hours.
2mL of
fermentation broth were sampled, processed, and analyzed via HPLC. The chromatographic data
were compared
with standards for quantification.

![img](https://static.igem.wiki/teams/5847/02/11.avif)

Figure 3. Quantitative analysis of microplastic degradation using HPLC.

A.Chromatogram of experimental groups, control groups, and standards under 254 nm UV
detection;
B.Standard curve generated from mixed standards;
C.Chromatographic peak areas of degradation products in each group;
D.Calculated concentrations of degradation products based on the standard curve.
The chromatographic data indicated that the degradation enzyme expression strains
effectively degraded PET
microplastic powder.

4.Validation of Glucose-Responsive Logic Gate Promoter

A fluorescent reporter strain was constructed via genome editing by integrating a linear
repair template
carrying HXT1p-EGFP into the yeast genome. Verified colonies were cultured in
media containing
0.1%, 0.2%, and 4% glucose (equal total carbon source) for 24 hours. Fluorescence and OD600
were measured
using a microplate reader to assess glucose-induced expression.

![img](https://static.igem.wiki/teams/5847/results/resf4.avif)

Figure 4. Construction and fluorescence validation of glucose-inducible promoter strain.

A.Design of linear repair template and primers;
B.96-well plate containing experimental cultures;
C.Normalized fluorescence intensity detected by microplate reader.

The data measured by the microplate reader showed that the fluorescence expression activity of the engineered strain increased with the rising glucose concentration. This result demonstrates that the promoter can effectively sense glucose levels and achieve the recognition of feeding signals.

Verification of Adsorption Module Feasibility

1.Prescreening of Surface Display Fusion Proteins

Three linker between HFBI and Aga2, (G4S)3,
(G4S)5, and
(G4S)8, were tested. TMHMM and ProtScale were used to analyze
transmembrane
domains and hydrophobicity. AlphaFold Server was used to predict structural fidelity of
HFBI and Aga2
domains. Docking simulations with PET were performed using AutoDock and visualized in
PyMOL.

![img](https://static.igem.wiki/teams/5847/results/res5.webp)

Figure 5. Pre-experimental feasibility analysis of HFBI surface display fusion
proteins.

A.Genetic map of HFBI fusion constructs;
B.Analysis of transmembrane structures and hydrophobicity;
C.RMSD-based structural fidelity comparison of fusion proteins;
D.Docking energy analysis of protein-PET interactions.
Results showed all three fusion constructs were feasible for surface display. Constructs
5-1 and 8-1
displayed higher structural fidelity, while 3-1 showed greater deviation. Docking
analysis confirmed
strong PET-binding potential.

2.Design and Construction of Adsorption Module Plasmids

Two plasmids linking AGA2 and HFBI with (G4S)3 and
(G4S)8
were designed and synthesized. The plasmids were transformed into yeast, and colony PCR
was used for
verification.

![img](https://static.igem.wiki/teams/5847/results/res6.webp)

Figure 6. Construction and preliminary validation of adsorption strains.

A.Designed plasmids;
B.Transformation and plating results;
C.Colony PCR verification.
Gel electrophoresis confirmed correct plasmid integration for subsequent experiments.

3.Validation of Surface Display

To verify V5-tag surface display, yeast cells were harvested, washed, blocked, incubated
with
fluorescent antibody, and mounted in 80% glycerol for confocal microscopy observation.

![img](https://static.igem.wiki/teams/5847/results/res7.webp)

Figure 7. Confocal microscopy of immunofluorescently labeled adsorption strains.

Confocal imaging revealed orange-red fluorescence in experimental groups but not in
controls, confirming
surface localization of fusion proteins without cell permeabilization.

4.Validation of Adsorption Function

Adsorption strains were cultured in 20 mL of medium containing microplastics for 36
hours. Cells were
collected, washed, embedded, and sectioned for TEM imaging.

![img](https://static.igem.wiki/teams/5847/results/res8-1.webp)

Figure 8. TEM observation of microplastic adsorption on yeast cell surfaces.

TEM images showed microplastic accumulation around yeast cells in experimental groups,
but not in
controls, demonstrating effective adsorption capability.

Validation of Anti-inflammatory and Antioxidant Module

1.Construction of ΔSEC72 Strain

A gRNA plasmid targeting SEC72 was designed and assembled via multi-fragment PCR and
one-step cloning.
The plasmid was amplified in E. coli and confirmed by sequencing.

![img](https://static.igem.wiki/teams/5847/results/res9.webp)

Figure 9. Design, construction, and verification of SEC72-targeting plasmid.

A.Primers targeting SEC72;
B.PCR amplification of gRNA fragments;
C.Recombinant plasmid plating on selective media;
D.Sequencing confirmation.

2.CRISPR-mediated Knockout of SEC72

A linear repair template containing upstream and downstream homologous arms of SEC72
was generated via
overlap PCR. The repair template and gRNA plasmid were co-transformed into yeast to
induce homologous
recombination, followed by colony PCR verification.

![img](https://static.igem.wiki/teams/5847/results/res10-1.webp)

Figure 10. Construction of linear repair template and verification of SEC72
knockout.

A.Primer design based on SEC72 flanking regions;
B.PCR amplification of repair template;
C.Colony PCR confirmation of SEC72 deletion.

3.Design and Construction of the Anti-inflammatory and Antioxidant Module

Functional elements were amplified and fused to form a linear repair template,
co-transformed with
gRNA plasmids for CRISPR-mediated genome integration.

![img](https://static.igem.wiki/teams/5847/results/res11.webp)

Figure 11. Design and construction of the anti-inflammatory and antioxidant
strain.

A.Template and primer design;
B.Homologous recombination to obtain full-length template;
C.Transformation and colony screening.

4.Verification of Constructed Strains

Colonies were expanded briefly, genomic DNA extracted, and colony PCR performed to
identify correctly
constructed strains.

![img](https://static.igem.wiki/teams/5847/results/res12.webp)

Figure 12. Colony PCR validation of anti-inflammatory and antioxidant strains.

Validation of Safety Modules

1.Alginate-Chitosan Encapsulation for Acid Resistance

To enhance acid tolerance, sodium alginate–chitosan microcapsules were prepared and
tested in
simulated gastric fluid. Free and encapsulated yeasts were incubated for 2 hours,
plated, and analyzed
for survival.

![img](https://static.igem.wiki/teams/5847/results/res13-1.webp)

Figure 13. Preparation of microcapsules and validation of acid resistance.

Encapsulated yeast showed high survival rates, while unencapsulated yeast died in
simulated gastric
conditions, demonstrating effective protection.

2.Temperature-Responsive Safety Module

A fluorescent strain carrying HSP26p-EGFP was constructed via CRISPR. Strains were
cultured at 30°C,
37°C, and 42°C for 24 hours, and fluorescence was measured.

![img](https://static.igem.wiki/teams/5847/results/res14-1.webp)

Figure 14. Construction and fluorescence validation of temperature-inducible
promoter strain.

A.Design of HSP26p-EGFP template and primers;
B.Colony PCR validation;
C.Fluorescence intensity under different temperatures.
Results showed that HSP26p activity significantly increased at 37°C and 42°C,
confirming its
temperature sensitivity and effective induction.

3.Arabinose-Responsive In Vivo Safety Validation

Yeast engineered with toxin–antitoxin plasmids were cultured in media containing 0%,
0.1%, and 1.0%
arabinose. Viable cell counts were determined at 0, 6, 12, and 24 hours.

![img](https://static.igem.wiki/teams/5847/results/res15.webp)

Figure 15. Construction and validation of toxin–antitoxin safety strain.

A.Arabinose concentration setup;
B.Experimental and control groups;
C.Plate results after incubation.
At 1.0% arabinose, colony formation was greatly reduced compared to controls,
indicating effective
arabinose-dependent safety regulation.

{% endblock %}