iGEM 2024 — Team lzu-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each
day for your project.{% endblock %}
{% block page\_content %}

Notebook

Human practice

Wet Lab

Model

Human Practice

April

To optimize our team's project, we held an offline expert exchange meeting under the leadership of our PI,
Principal Li Xun. During the meeting, we discussed whether thsS/R has specificity for thiosulfate and
PphsA,
as well as how to address the high off-target activity and large editing window of the CBE protein.

![](https://static.igem.wiki/teams/5391/notebook/figure1.jpg)

Figure 1

![](https://static.igem.wiki/teams/5391/notebook/figure2.png)

Figure 2

Ultimately, after considering the teachers’ suggestions, we determined that thsS can only be
phosphorylated
in
a thiosulfate environment, thereby activating its histidine kinase function, which leads to the
phosphorylation and dimerization of thsR, giving it activity and allowing specific action on PphsA. Due to
the
inherently large size of the CBE protein, it exhibits off-target effects, low editing efficiency, and a
large
editing window, making standard CBE unsuitable for our project, which requires more precise and efficient
editing. After reviewing a substantial amount of literature,we identified a small, efficient CBE protein
called TadA-CDd, which was optimized from ABE through the PANCE (Phage-Assisted Non-Continuous Evolution)
system, resulting in significantly lower off-target activity. We introduced the V106W mutation to further
reduce its off-target rate, so we modified the original CBE gene in our system to the V106W mutant gene of
TaDa-CDd.

May

To further refine our project and address the difficulties and uncertainties encountered during our
literature
review, we conducted a second online expert exchange meeting organized by our PI, Principal Li Xun.

![](https://static.igem.wiki/teams/5391/notebook/figure3.jpg)

Figure 3

![](https://static.igem.wiki/teams/5391/notebook/figure4.png)

Figure 4

In this meeting, Teacher Li Yang explained the detailed signaling pathways of inflammatory factors and
guided
us in reading other high-impact literature to clarify our confusions. Teacher Li provided an in-depth
analysis
of the mechanism of action of the parasitic-derived AvCystatin (Av17), the drug protein selected by our
team
to serve as the treatment system in the overall system for IBD therapy. Parasites utilize highly effective
immunomodulatory molecules to alter their host's immune system and reduce the inflammatory response
against
them. This mechanism is the basis for our choice of AvCystatin as the drug protein in the treatment
system.

AvCystatin primarily targets macrophages, interfering with the host's immune system by regulating the
expression level of IL-10, thus downregulating the inflammatory response against them. In the signaling
events
triggered by macrophages, AvCystatin is first absorbed by the macrophages, which subsequently induces the
phosphorylation of mitogen-activated protein kinases (MAPK) ERK 1/2 and p38. The activation of these
kinases
can lead to the production of regulatory type 2 macrophages. The activation of ERK1/2 results in a rapid,
transient phosphorylation of histone H3 at the IL-10 promoter, allowing it to bind to transcription
factors
such as Sp1 and STAT3, which are parallelly activated by p38. Notably, the expression of IL-10 in this
induction is sensitive to tyrosine kinase and relies on the activation of both MAP kinases (which is in
stark
contrast to the expression of IL-12/23p40). Dual-specificity phosphatases (DUSP1 and DUSP2) regulate the
induction of macrophages by AvCystatin, with DUSP1 specifically responsible for regulating the
phosphorylation
of ERK and p38, thereby controlling the expression of IL-10 in AvCystatin-regulated macrophages. In other
words, AvCystatin induces and regulates macrophages via activation and inactivation pathways of MAP
kinases.
Additionally, AvCystatin can also induce the phosphorylation of transcription factors CREB and STAT3,
regulated by phospho-ERK. Phosphoinositide-3-kinase (PI3K) can reduce the release of cytokines induced by
AvCystatin, but downstream target AKT will not be activated after exposure to AvCystatin. We derived
considerable benefit from this meeting and will continue to work hard to improve our project.

June

To understand the public's awareness of inflammatory bowel disease (IBD and analyze the social impact of
education and outreach related to IBD, our team designed a simple survey questionnaire, which was widely
distributed across various provinces in China. We hope to use the most research to improve our project.
Additionally, our IHP group engaged in interviews with clinical and microbiological research institutions,
interviewing senior colleagues. This time, we interviewed Dr. Jiang Lei, a surgeon from the First Clinical
Medical College of Lanzhou University, and Ms. Mao Ting from the Gansu Provincial Institute of Biology.

![](https://static.igem.wiki/teams/5391/notebook/figure5.png)

Figure 5

![](https://static.igem.wiki/teams/5391/notebook/figure6.png)

Figure 6

![](https://static.igem.wiki/teams/5391/notebook/figure7.png)

Figure 7

![](https://static.igem.wiki/teams/5391/notebook/figure8-11.png)

Figure 8-11

![](https://static.igem.wiki/teams/5391/notebook/figure12.png)

Figure 12

![](https://static.igem.wiki/teams/5391/notebook/figure13.png)

Figure 13

We collected a total of 500 questionnaires. Based on the data results, we reached the following
conclusions:
the public's understanding of inflammatory bowel disease is poor, with a generally low level of awareness,
and
the channels for promoting IBD need to be strengthened. Therefore, it is essential to enhance public
education
about IBD to improve awareness and understanding of the disease, thereby promoting health and preventive
awareness among the public. For those who believe that awareness of IBD is unnecessary or manageable, more
intuitive and easily understandable educational methods should be employed to help more people recognize
the
importance of IBD. Moreover, Dr. Jiang expressed his support for our use of engineered bacteria and
synthetic
biology to treat IBD. He is optimistic about molecular targeted research and hopes that emerging research
findings can be applied to reduce severe cases, alleviate patient suffering, and avoid additional surgical
trauma. From Ms. Mao, we gained valuable insights into the application of synthetic biology in the
treatment
of IBD. She also holds high hopes for molecular targeted therapy for IBD but emphasized the importance of
biosafety.

July

In conjunction with the interviews from clinical and biological research institutions, our team held the
third
online expert discussion meeting. Due to the lengthy nature of our gene sequence, the probability of
failure
during gene insertion into plasmids is significantly high; thus, we mainly discussed how to address the
oversized nature of our system. At the same time, we continued our interviews with clinical teachers, this
time interviewing Dr. Ji Rui and Ms. Ren Qian from the First Clinical Medical College of Lanzhou
University.

![](https://static.igem.wiki/teams/5391/notebook/figure14.jpg)

Figure 14

![](https://static.igem.wiki/teams/5391/notebook/figure15.png)

Figure 15

![](https://static.igem.wiki/teams/5391/notebook/figure16.png)

Figure 16

![](https://static.igem.wiki/teams/5391/notebook/figure17.png)

Figure 17

Based on the teachers’ suggestions, we decided to split the plasmid, distributing the original large
plasmid
onto two high-copy plasmids with different resistance genes, each under 8000 bp, to reduce the difficulty
of
gene synthesis and electroporation, thereby ensuring the smooth progress of the experiment to some extent.
During the interview, Dr. Ren provided detailed and valuable responses: whether the strains are colonized
in
the intestine, determining the concentration of anti-inflammatory factors released based on the severity
of
the patient's condition, and considerations regarding the accuracy of drug release locations, among
others.
Antibiotics and other medications can lead to many adverse reactions, and long-term use can reduce an
individual's resistance and immunity. The tolerance level of individuals to medications also varies,
making
drugs unsuitable as a long-term solution. Additionally, there are significant challenges in the selection
of
microbial flora for fecal microbiota transplantation.

From our conversation with Dr. Ji Rui, we learned that most patients and their families are open to
emerging
treatments, and their acceptance of new therapies was an unexpected result prior to our interviews. As a
result, we are more confident in optimizing our project and promoting our ideas and new treatment pathways
to
the public. We hope to ignite patients' confidence in recovery and provide them with encouragement and
comfort.

August

In order to understand the TCM concept of inflammatory bowel disease, familiarize us with its prevention
strategies.

Dr. Yao provide us with future improvement suggestions for our project from the perspective of TCM.Drawing
on
TCM therapeutic ideas has inspired us to delve deeper into researching gastrointestinal treatment targets,
while also considering the possibility of looking further afield to explore the ultimate sites of
inflammatory
bowel disease.

![](https://static.igem.wiki/teams/5391/notebook/figure18.png)

Figure 18

To gain a deeper understanding of the perspective of the integration of traditional Chinese and Western
medicine on IBD, analyze the differentiation and treatment of UC and CD, and comprehensively understand
the
treatment approach of the integration of traditional Chinese and Western medicine for IBD, we interviewed
Director Li Xuefeng of the Lanzhou University Traditional Chinese Medicine Culture Dissemination and
Innovation Research Center to learn about his understanding of IBD.

Overall, TCM is an indispensable part of treating IBD. The future of integrative treatment for IBD is
promising, but there is still a need for ongoing exploration and innovation in scientific research and
clinical practice, continuously perfecting and optimizing treatment plans, with the fundamental starting
point
and ultimate goal always being to alleviate patients' suffering.

![](https://static.igem.wiki/teams/5391/notebook/figure19.png)

Figure 19

In order to better focus on the physical and mental health of IBD patients, we interviewed Dr. Liu
Jianbin,
an
experienced director of the mental health department, to explore how to build a solid mental health
fortress
for IBD.

![](https://static.igem.wiki/teams/5391/notebook/figure20.png)

Figure 20

Looking ahead, we can enhance mental health education by using lectures, informational materials, online
psychological support groups, and community outreach activities to raise awareness among patients and
their
families about the importance of psychological care. This can help create a caring and inclusive social
environment, reduce discrimination and misunderstandings, and potentially offer personalized psychological
intervention plans to provide multifaceted support and improve patients’ quality of life.

To broaden the team's knowledge of inflammatory bowel disease and better integrate the team's research
into
clinical practice, guided by the principle of responsibility towards IBD patients and the world, our team
participated in the Gansu Province Inflammatory Bowel Disease Specialist Alliance Launch Meeting, the
Gansu
Province IBD Standardized Diagnosis and Treatment Guidelines Learning Session, and the 9th Gansu Province
Intestinal Disease Forum and IBD Youth Forum, hosted by the Gastroenterology Department of the Second
Affiliated Hospital of Lanzhou University on August 9-10, 2024.

This meeting primarily focused on expert lectures, classic case sharing, and case discussions, offering a
rich
and compact content experience and a strong learning atmosphere. We realized the importance of increased
communication and learning. Promoting multidisciplinary collaboration is crucial for inflammatory bowel
disease, and we hope to have the opportunity to participate in similar seminars in the future, gaining
further
insights into inflammatory bowel disease and contributing our strength to patients.

![](https://static.igem.wiki/teams/5391/notebook/figure21.png)

Figure 21

In consideration of the deaf and hard of hearing community,Lanzhou University, in collaboration with
Beijing
Institute of Technology, Sichuan University, Jilin University, Hainan University, Nanjing University of
Technology, and Zhejiang University of Technology, has launched an iGEM sign language science
communication
activity.

Through cooperation with universities in specific regions, the aim is to form a comprehensive network
coverage
model from east to west, south to north, and center, thereby expanding influence and promoting the
enhancement
of science popularization and innovation awareness.

![](https://static.igem.wiki/teams/5391/notebook/figure22.png)

Figure 22

September

To raise awareness of inflammatory bowel disease (IB) among the elderly the northwest region, we chose
Kang
Le
Hospital in Chengguan District, Lanzhou, as the venue for our outreach activities. This hospital serves a
large number of elderly and caregivers, making it an ideal location to reach target audience. During these
events, our team members provided detailed explanations of IBD to the elderly and their caregivers.

![](https://static.igem.wiki/teams/5391/notebook/figure23-26.png)

Figure 23-26

We focused on explaining the symptoms, causes, and prevention methods of the disease, as well as the
importance of early diagnosis and treatment. By increasing caregivers' awareness, we hope they can provide
better care for elderly patients in their daily work. Our goal is to enhance societal awareness of IBD,
help
patients manage their chronic conditions more effectively, and ultimately improve their quality of life.

As a chronic intestinal disease, inflammatory bowel disease (IBD) has long course, and long-term
medication
can seriously affect the physical and mental health of patients In addition to general treatment,
specialized
care should not be overlooked. To understand the positive significance of nursing care related to IBD we
interviewed Nurse Director Zhen Xiaoling from the Gastroenterology Department of Lanzhou University First
Hospital.

![](https://static.igem.wiki/teams/5391/notebook/figure27.png)

Figure 27

For IBD patients, effective nursing measures include psychological support, lifestyle management,
medication
management, and pain management. Providing appropriate and quality care is significant for patients with
inflammatory bowel disease.
To strengthen research collaboration within the gastrointestinal research team and to mutually learn from
the
new ideas and concepts of different teams, we initiated the Gut Alliance in collaboration with Tsinghua
University.

![](https://static.igem.wiki/teams/5391/notebook/figure28.png)

Figure 28

The Gut Alliance aims to:
1.Promote Academia:Enhance the depth and breadth of research on intestinal diseases and accelerate
scientific
discoveries.

2.Support the Medical Community:Provide clinical doctors with the latestresearch findings to optimize
treatment plans and improve patient outcomes.

3.Influence Society:Raise public awareness of gut health, advocate for healthy lifestyles, and reduce the
incidence of intestinal diseases.

To facilitate in-depth communication and sharing of expertise and the latest research findings in the
field
of
synthetic biology, and to promote academic integration and collaboration between our two universities, we
created content in a question-and-answer format based on synthetic biology and our respective project
themes.
We hope that this engaging and interesting format will stimulate a strong interest in synthetic biology
and
promote its development in practical applications

![](https://static.igem.wiki/teams/5391/notebook/figure29.png)

Figure 29

This collaborative recording activity has built a bridge for academic communication between the
universities,
allowing both sides to fully integrate their advantageous resources and form a powerful alliance to
explore
the unknown mysteries in the field of synthetic biology together. The question-and-answer format of the
podcast content makes it easier to convey specialized knowledge clearly and accurately, enabling listeners
to
better understand complex scientific concepts and making a positive contribution to the popularization of
science and scientific education.

The team has conducted solid theoretical research on inflammatory bowel disease (IBD) and has gained a
comprehensive understanding of its pathogenesis and prevention strategies. As a result, we created an IBD
knowledge manual to out public science education in communities, helping the public to correctly
understand
IBD.

![](https://static.igem.wiki/teams/5391/notebook/figure30.png)

Figure 30

Our work has not only increased public awareness of IBD but also highlighted the importance of early
diagnosis
and timely treatment. By dispelling public misconceptions and biases about IBD, we have promoted societal
support and understanding for patients. In addition, we encourage the public to adopt healthy lifestyles
to
facilitate early prevention of IBD. These efforts will contribute to reducing the overall prevalence of
IBD
in
society and alleviating the medical burden on the community.

In order to popularize synthetic biology and IBD, we have successively conducted cloud teaching, Western
Sky
Association teaching, Teaching Association teaching, Lanzhou Baishuxiang teaching, Luotuoxiang teaching,
Linjiazhuang teaching, Yuzhong County Picture Book Museum teaching, Zhangye City teaching,Longnan City
teaching, Lanzhou University Affiliated Primary School teaching, etc.

![](https://static.igem.wiki/teams/5391/notebook/figure31.png)

Figure 31

To gain a deeper understanding of advancements in intestinal technology, companies in the field of IBD
research, and how they leverage technology to drive the development of innovative therapies, we collected
valuable information regarding research findings, technology applications, and future directions through
Professor Lu's expertise. Additionally, we drafted a preliminary business plan in conjunction with our
project.

![](https://static.igem.wiki/teams/5391/notebook/figure32.png)

Figure 32

![](https://static.igem.wiki/teams/5391/notebook/figure33.jpg)

Figure 33

During the series of activities and interviews with numerous stakeholders, the team members will promptly
summarize the meaningful content (in both Chinese and English) and then publish it on various online
platforms
in the form of tweets or videos for public education. As of September 25, 2024, the total viewership
across
all platforms has exceeded 10,000.This season, the team has released the following content:24 posts on the
WeChat public account platform9 videos on platforms such as Douyin, Bilibili, and Xiaohongshu, including 6
popular science experiment videos, 2 interview videos, and 1 sign language video.

![](https://static.igem.wiki/teams/5391/notebook/figure34.png)

Figure 34

![](https://static.igem.wiki/teams/5391/notebook/figure35.png)

Figure 35

Wet Lab

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Model

July:

After gaining an understanding of the mechanisms behind inflammatory bowel disease and biological
experiments,
we have preliminarily outlined a plan to establish a mathematical model. This model, based on fluid mechanics,
biostatistics, and pharmacokinetics, aims to explain the entire process of drug flow in the patient’s body,
the
proliferation of engineered bacteria, and the therapeutic effects of the drug on the disease. Additionally, we
have assigned each team member specific tasks to learn different related subjects.

August:

We began by studying the application of fluid mechanics in the digestive system, the role of biostatistics in
the proliferation of ’EcN‘ bacteria, and the pharmacokinetic mechanisms of the ’AvCystatin‘ protein in the
human
body. Furthermore, we explored simulation software such as OpenFOAM and ANSYS Fluent to conduct simulations,
with each team member establishing their own foundational model.

September:

We discussed and reviewed the knowledge acquired over the previous two months and addressed challenges
encountered during the modeling process. Subsequently, we integrated our individual models into a cohesive new
model that simulates the entire drug administration process for patients and provides recommendations for drug
dosage.

![](https://static.igem.wiki/teams/5391/lzu-logo.png)

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{% endblock %}

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

Engineering

Overview

Cycle 1

Cycle2

Cycle3

Cycle4

Overview

With the goal of better characterizing the principles of biobricks in diagnosing and treating inflammatory
bowel disease (IBD) that we have used in the system, and to ensure the security of our system, we designed
four experiment cycles under the guidance of DBSL rules to study our system's practicability.
The goals of the three groups of engineering modifications are:

1. To study the sensitivity of the IBD detecting system,

2. To test how the signal amplifying system works,

3. To verify the complete treatment system,

4. To examine the system’s safety by detecting the suicide switch.

Cycle 1

Design

In order to characterize the sensitivity of the IBD detecting system controlled by two key molecules ThsS and
ThsR, we decided to design a signal-reacting system controlled by ThsS and ThsR and place it in an environment
with thiosulfate--an important biomarker for inflammatory bowel disease. We will detect and study the system's
response to different concentrations of thiosulfate.

Build

We added ThsS and ThsR biobricks into the pSB1C3 plasmid and named it as pSB1C3-ThsS/R. After the ThsS and
ThsR components, we added a sfGFP tag to detect the responsiveness of the IBD detecting system.

![](https://static.igem.wiki/teams/5391/engineer/cycle1-plasmid.png)

Test

We transformed the plasmid into the chassis organism, Nissle 1917, and induced it with thiosulfate solutions
of different concentration gradients. Later,we detected the fluorescence intensity by the microplate reader.

Learn

We found out that our IBD detecting system can diagnose IBD sensitively.

Cycle2

Design

Considering levels of many disease-related biomarkers are dynamic and difficult to capture and monitor in
native environments, we decided to add a CRISPR-based genome(CBE) editing system to amplify the inflammatory
signal reaction.

Build

We added a cytosine base editor, TadA-CDd V106W into the pSB1C3-ThsS/R plasmid and named it as pSB1C3-
TadA-CDd V106W. And in this added system, the promoter of sfGFP was replaced with a CRISPR-editable ACG-tag
sequence, which silenced its translation, TadA-CDd V106W was expressed under the control of ThsS/R to respond
to thiosulfate levels in environment.

![](https://static.igem.wiki/teams/5391/engineer/cycle2-plasmid.png)

Test

We transformed the plasmid into the chassis organism, Nissle 1917, and induced it with thiosulfate solutions
of different concentration gradients. Later, we detected the fluorescence intensity with the microplate
reader.

Learn

The newly added CRISPR-based editing system works out well under stimulated IBD environments.

Cycle3

Design

With the goal of verifying the complete treatment system, based on the first two cycles of experiments, we
have also incorporated drug proteins and their secretion system. As our drug protein, AvCystatin plays a
crucial role in regulating macrophage immune responses. It can activate cellular MAP kinase and induce the
expression of IL-10 and IL-12/23p40, thereby effectively alleviating gut inflammation.

Build

Based on previously built systems, we have built up the complete treatment systems. And because the plasmid
will be too big if we put the drug protein and its secretion system into the same plasmid, we divided our
therapeutic system into two plasmids that could work together, with AvCystatin, the drug protein in the first
plasmid pSB1C3-TadA-CDd V106W and HlyB, HlyD, the secretion system into plasmid pET-9a.

Plasmid pSB1C3-TadA-CDd V106W added with AvCystatin was named pSB1C3-AvCystatin, plasmid pET-9a carried HlyB
and HlyD system was named pET-9a-Hly

![](https://static.igem.wiki/teams/5391/engineer/cycle3-plasmid1.png)

![](https://static.igem.wiki/teams/5391/engineer/cycle-plasimd2.png)

Test

We transformed the plasmid into the chassis organism, Nissle 1917, and induced it with thiosulfate solution
from the optimal response concentration in the previous cycles. Later, we assessed the expression of drug
proteins and the synergistic effect of the dual plasmid system using by western-blot.

Learn

The complete IBD treatment system works can diagnose and treat IBD effectively.

Cycle4

Design

In order to ensure the system’s safety by controlling drug expression, we designed a suicide switch and put it
into the IBD treatment system. The key molecule in the suicide activating part is PnorV promoter and the
bacterial enhancer binding protein NorR, which could be inhibited when the level of another promising IBD
detecting biomarker--nitric oxide decreased. This leads to the inhibited expression of the CI protein. Thus,
the PL promoter is derepressed, inducing the expression of the MazF toxin protein, which will lead to cell
apoptosis. Moreover, studies have shown that MazF has no toxin to healthy human cells so it can be used in
vivo.

Build

Similarly, considering the plasmid size and the synergistic effects of different systems, we chose to add the
suicide switch to the pET-9a-Hly plasmid. We sequentially added the PnorV promoter, the NorR binding protein,
the CI protein, the PL promoter, and the MazF protein after the HlyD component. The newly developed plasmid
was named pET-9a-suicide.

![](https://static.igem.wiki/teams/5391/engineer/cycle4-plasmid1.png)

![](https://static.igem.wiki/teams/5391/engineer/cycle4-plasmid2.png)

Test

We transformed the plasmid into the chassis organism, Nissle 1917, and induced it with thiosulfate solution
from the optimal response concentration in the previous cycles. All experiments were conducted in a nitrogen
monoxide environment to simulate the intestinal environment of patients with IBD. We then assessed the
expression of drug proteins and the synergistic effect of the dual plasmid system using by western-blot.
Later, we removed the DETA-NO solution used to provide nitric oxide to simulate the intestinal environment
after the improvement of IBD, and after removing the solution, we collected colonies onto slides at 1 hour, 2
hours, and 3 hours, performed trypan blue staining, and observed under the microscope to count dead cells in
order to evaluate the efficacy of the suicide system.

Learn

We found that the suicide system can effectively respond to and eliminate chassis cells in a non-IBD
environment, thereby preventing drug overexpression and ensuring the safety of the system.

![](https://static.igem.wiki/teams/5391/lzu-logo.png)

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{% 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 %}

## 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 %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

Results

Overview

Experiment

Overview

After a period of relentless effort, we have successfully verified the sensing part of our engineered
bacteria, marking the completion of the first DBTL cycle experiment.

Experiment

cycle1——Verification of the two—component
system thsS/R

The result of electrotransformation

![](https://static.igem.wiki/teams/5391/results/ele1.jpg)

![](https://static.igem.wiki/teams/5391/results/ele2.jpg)

Fig.1│After electroporation and recovery, the sample was diluted and spread onto plates containing 100 μg/mL
chloramphenicol. The plates were then inverted and placed in an incubator. The appearance of single colonies
indicated a preliminary successful transformation of the plasmid into the cells.

Extract plasmids from bacteria and send them to a company for full plasmid sequencing.

![](https://static.igem.wiki/teams/5391/results/smallbox.jpg)

![](https://static.igem.wiki/teams/5391/results/samllpage.jpg)

Fig.2│Plasmid extraction was performed using the M5 HiPer Multi-color Plasmid Miniprep Kit (with column), and
the concentration of the extracted plasmid was measured.

As of before the wiki freeze, the sequencing results were delayed due to internal communication issues at the
sequencing company.

After adding the inducer sodium thiosulfate, the fluorescence intensity of the engineered bacteria was
measured using a fluorescence microplate reader.

After inducing with different concentrations of thiosulfate and shaking for 12 hours, the OD600 and
fluorescence value (Flo) of the bacterial suspension samples were measured respectively to obtain the
fluorescence intensity Flo/OD600, which characterizes the activity of the two-component system thsS/R under
induction by different concentrations of thiosulfate.

![](https://static.igem.wiki/teams/5391/results/figure.png)

Fig.3│The relationship between the measured fluorescence intensity and the concentration of the inducer sodium
thiosulfate is shown in the figure. The F-value of 14.37 is significantly greater than the critical value of
4.95, demonstrating that different concentrations of sodium thiosulfate have a significant impact on
fluorescence intensity.

Based on the response results, we can infer that even in the intestinal environment with low concentrations of
thiosulfate, the engineered bacteria can still activate and effectively function, while avoiding excessive
release of the drug protein. This characteristic enables our engineered bacteria to be extended to the
diagnosis and treatment of patients with mild enteritis in practical applications, with higher safety and
efficacy.

Furthermore, it can be observed that as the concentration of sodium thiosulfate increases, the fluorescence
intensity of the reporter gene gradually decreases. The possible reason for this is that excessively high
concentrations of thiosulfate affect bacterial activity, resulting in reduced reporter gene expression.
However, at this point, the concentration of thiosulfate has reached above 1mM, which is absolutely beyond the
concentration that can be achieved in patients' bodies.

![](https://static.igem.wiki/teams/5391/lzu-logo.png)

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{% endblock %}