iGEM 2023 — Team nmu-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

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# March

## HP

1.We went to the nursing home near the school to carry out voluntary activities to learn from the spirit of Lei Feng. During the communication with the elderly, we learned that many elderly are plagued by constipation. So we thought about whether we could use synthetic biology to solve the problem of constipation to benefit the elderly. We first needed a wider sample of data to clarify how many people in the elderly population had constipation.

2.We reviewed a large number of publicly available data on constipation in various provinces and cities in China, and designed a questionnaire to publish online.

3.Moreover, we conducted research and interviews in many provinces and cities across the country, and found that the proportion of constipation in the elderly is quite high, which brings them great pain and increases their risk of developing other diseases. We are more deeply aware that constipation is a very important problem to be solved.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/notemarchgai.jpg)

## Wet Lab

1.We learned some experimental protocols and basic theories of synthetic biology genetic circuits.

2.We are trained in some basic experimental operations, like plasmid construction, cell culture, SDS page, and western blotting.

3.We discussed a pivotal question concerning our project: how engineering bacteria can perceive that the body is in a state of constipation. So we conducted a review of the literature to understand the relevant aspects.

## Safety

Learn from what previous teams did to ensure safety through out the project and gain experience.

## Model

Learn from previous mathematical modeling work and gain experience.

# April

## HP

1.Next, we interviewed Traditional Chinese medicine teacher to learn about the concept of "treating diseases before they occur" in traditional Chinese medicine, which inspired our project: perhaps we can use synthetic biology technology to design a drug to prevent people from developing constipation, rather than waiting until the patient already has a constipation problem.

2.During the communication with teammates in the wet lab, they suggested that they might utilize E. coli as chassis to design a living bacteria drug, which can colonize in the intestine for long after being taken in. Considering that our original design was to effectively solve constipation problems among the elderly, we went to the community again to interview the elderly to obatain their opinions on our project. We found that they concerned a lot about the safety of engineered bacteria. They also conveyed that as long as safety is guaranteed (such as national recognition), they are willing to accept this kind of living bacteria drug that can prevent and solve constipation problems in the long term. In addition, regarding the pharmaceutical form, they preferred food products such as yogurt rather than conventional forms such as capsules.

3.Since the wet lab was puzzled to determine the initial sensing substances between methane and butyrate, we contacted Professor Wang Lianghua, director of the biochemistry teaching and research department of our school, to ask about the selection of constipation markers and whether the orthogonality and robustness of the current circuit design are appropriate. He proposed that the specificity of methane as a receptor may not be strong and suggested that we try to simplify the genetic circuit to make the gene expression more efficient.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/noteapirl.jpg)

## Safety

1.We began to explore the design of kill switch circuits, looking for triggers and effectors via literature review.

2.The first generation of kill switch circuit was designed, consisting of a temperature control promoter and toxin protein MazF, to achieve the purpose of environmental protection.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/imagineb1.png)

## Wet Lab

1.The promoter AOX1 that senses methane changes and the promoter pchA that senses butyrate changes were screened from the literature, and it is intended to use two promoters to preliminarily design genetic circuits.

2.Two genetic circuits were designed. The first one is to sense methane changes, which needs to use granular methane monooxygenase (pMMO) to convert methane into methanol first, and then methanol acts on pAOX1, thus initiating expression. The second one is to sense the change of butyrate, which binds to Lrp protein, acts on PpchA, and then initiates downstream gene expression. However, our engineered bacteria cannot feel these two substances at the same time, so we can only select one as a marker of our engineered bacteria feeling constipation.

3.We reviewed the literature to see which is more appropriate as a marker of constipation in our engineered bacteria.

# May

## HP

1.Through interviews with community residents, we learned that they concerned a lot about the safety of engineered bacteria. In order to obtain professional opinions on the safety of engineering bacteria, including the relevant principles and policy, we interviewed Professor Deng Zixin, an expert in the field of microbial metabolism. He suggested to us that we need to design a safety module from two aspects: on the one hand, we need to consider the environmental protection after engineered bacteria are excreted from the human body; on the other hand, considering the safety of engineering bacteria in vivo, we should add an in vivo inducible containment switch.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/note-revised4.jpg)

2.In previous surveys, we learned that older people in the community preferred to use probiotic products in the forms of foods such as yogurt, rather than the conventional form of capsules and other common medicines. In order to find out whether our project is possible in the current biomedical industry, and get a professional view, we consulted Mr. Liao Xiang, who focuses on investment in the biomedical industry. He proposed that both yogurt and capsules could be used as carriers of engineered bacteria, but if yogurt was used, it would require a special design to resist stomach acid and consider high demands on transportation. In addition, food and drugs have different regulatory requirements, so it is recommended to clarify the product positioning.

## Safety

1.After receiving feedback from the HPers on Professor Deng's suggestions to improve the safety module, we began to review the strategies of the previous team, both in environmental protection and internal containment.

2.Based on the review, a highly operable dual drug control system was selected. The two drug control systems were preliminarily designed: malic acid killing system and rhamnose maintenance system. The effect concentration and induction conditions of the two inducers were then reviewed in the literature to determine the feasibility.

## Wet Lab

1.Through literature investigation and expert consultation, it was found that although methane gas is related to constipation in some cases, not all constipation is accompanied by high methane gas levels, and elevated methane levels may be a normal physiological phenomenon, such as high-fiber diet, active intestinal peristalsis, etc. That is to say, the association between methane and constipation is not specific. We also found that methanol is harmful to the human body and causes poisoning after entering the body. The association between butyrate and constipation is strong, under normal circumstances, the concentration of butyrate in the intestine is maintained at 10-20mM, and when constipation occurs, the concentration of butyrate will be less than 10mM. Therefore, we ended up determining butyrate as the sensory marker for our engineered bacteria.

2.We tried to simplify the genetic circuit to pursue the efficiency and controllability of expression, and designed two genetic circuits, intending to compare the perception and feedback of butyrate between the two designs through bacterial experiments.

3.We searched for the fragment sequence we need in vector builder and NCBI, designed it on snapgene, and submitted it to Azenta Life Science for synthesis.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/note8.png)![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/note9.png)

## Model

Mendelian randomization was used to model and analyze whether E. coli was suitable as chassis.

# June

## HP

1.In order to understand whether our products belong to drugs, health products or food from a legal perspective, so that we can position Gut-sweeper's products and promote them more rigorously, we consulted Mr. Zhang Chengzhong, who engaged in food and drug administrative in the drug supervision and inspection department. He suggested that after passing the safety assessment, our final products should choose the appropriate declaration route according to the scope of application to determine whether the product ultimately belongs to medicine, health products or food.

2.In order to have a more comprehensive understanding of the clinical treatment of constipation, as well as the views and suggestions of front-line doctors on our program, we interviewed Dr. Yu from the Department of Rectal Surgery of our affiliated hospital. He proposed that our color module may affect the doctor's examination of the patient's feces, and at the same time, constipation will develop into some severe symptoms of the gastrointestinal tract in the later stage, and in more severe cases, there will be water-electrolyte imbalance and intestinal obstruction and even life-threatening. Therefore, our engineered bacteria need to feel the state of constipation in a short time and respond in time.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/note-5.jpg)

## Wet Lab

1.After the gene fragment is in place, we introduce it into BL21 and culture it in LB culture medium and LB culture medium containing sodium butyrate, respectively, in a microplate reader with shaking at 37°C, while measuring fluorescence intensity and bacterial growth curve with a microplate reader. It was found that the original gene fragment of Enterohaemorrhagic E. coli had a better perception and feedback of butyrate than our simplified gene line, and finally we decided to follow the existing butyrate receptor network in Enterohaemorrhagic E. coli.

2.Since the leucine response regulatory protein Lrp acts as a sensor to transduce the stimulation of butyrate into the PchA-Ler regulatory network that controls the LEE gene, we hypothesized whether the PchA promoter's ability to sense and respond to butyrate could be enhanced by increasing the expression level of Lrp protein in engineered bacteria, and decided to test this hypothesis by increasing the expression of Lrp in bacteria.

![Gut-sweeper]( https://static.igem.wiki/teams/4656/wiki/build3.png)

3.We found the fragment sequences we need in vector builder and NCBI, designed them on snapgene, and submitted them to Azenta Life Sciences for synthesis.

## Model

According to the growth rate of E. coli in the intestine and the rate of production of its products, the variables and mathematical derivation of the model were discussed.

# July

## Safety

1.A dual-drug control system plasmid was constructed and synthesized by Azenta Life Science.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/buildb2-1.png)

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/buildb2-2.png)

2.Register and refine parts; design dual drug control system experiments.

## Wet Lab

1.The sensory fragments were introduced into BL21, and the Lrp fragments and sensory fragments were simultaneously imported into BL21, and the fluorescence intensity and bacterial growth curve were measured after culture in LB culture medium and LB culture containing sodium butyrate, respectively. It was found that the increased expression of Lrp could indeed increase the sensitivity of PchA promoters to butyrate.

2.Since the concentration of intestinal butyrate is significantly reduced during constipation, the sensory fragment we designed is butyrate-inducible, that is, a certain range of high butyrate concentration can promote the expression of this gene line. Therefore, we envisaged using changes in butyrate concentration to negatively regulate the products of our gene route, for which our genetic circuit needs a negative regulatory element to promote the expression of related genes when the butyrate concentration decreases. Through the investigation of the literature, it was found that Plam is a strong promoter in lamada bacteriophages, while Cl is an inhibitory protein that binds to the Plam promoter, thereby inhibiting downstream gene expression. So we decided to use Cl/Plam for negative regulation, we need to find the fragment sequence we need in vector builder and NCBI, designed it on snapgene, and submitted it to Azenta Life Sciences for gene fragment synthesis.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/build4.png)

3.After the fragment is in place, it is introduced into BL21, gradient LB culture containing sodium butyrate, and cultured in a microplate reader at 37°C with a microplate reader while measuring fluorescence intensity and bacterial growth curve with a microplate reader. It is found that Cl/Plam elements can play a negative regulatory role.

## Model

The E. coli growth model and product production model were fitted to the experimental results of the microplate reader, and the two models were tested and improved.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/note-revised2.jpg)

# August

## HP

1.The experimental group has successfully engineered the bacteria to sensitively feel the declining butyrate concentration in the intestine and enhance the expression of the Plam promoter and its downstream genes through negative regulation, and then we need to choose a therapeutic substance to improve the condition of constipation, so we interviewed Chai Hui, an expert in the field of biomedicine. She recommends that we use substances that occur naturally in the body as therapeutic drugs, rather than synthetic drugs. In addition, she reminds us of the effects of bacterial overgrowth.

## Safety

1.The plasmid is delivered half a month later for extraction, transduction, identification, and the experimental design protocol is improved.

2.Carry out the experiment of dual drug control system, basically obtain the expected results, summarize the experience and shortcomings, sort out the data and visualization; receive safety form feedback, and carry out rectification and improvement.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/note10.png)![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/note11.png)

## Wet Lab

Based on the feedback from the HP group students contacting Professor Chai Hui, we hypothesized whether our engineered bacteria could produce endogenous substances in the human body when they detected constipation, which could be used to treat constipation. Through literature research, it was found that 5-HT can promote intestinal motility and secretion, and is an ideal therapeutic substance. Tryptophan hydroxylase (TPH) and serotophanate decarboxylase (TDC) are two key enzymes in 5-HT synthesis, so we wanted our engineered bacteria to produce these two enzymes. We attached two enzymes to the Plam promoter so that the engineered bacteria can produce them when they sense a decrease in butyrate concentration. We need to find the fragment sequences we need in vector builder and NCBI, designed them on snapgene, and submitted them to Azenta Life Sciences for gene fragment synthesis.

## Model

According to the metabolic rate of tetracycline in the intestine and the speed of drug module control of suicide, the variables and mathematical derivation of the model were carried out, and the model fitting was carried out based on experimental data for verification and improvement.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/note-revised6.jpg)

# September

## HP

After some improvements to our project product, we determined that the final form of the product was mainly yogurt, positioned as a drug, through which the engineered bacteria were delivered into the intestines of the target population. But at the same time, considering factors such as transportation and preservation, we also retained the design of the capsule carrier in the form of the drug. In order to gain the views and attitudes of the elders after the improvement of our project, we arranged a community return visit. After the improvement of the project, the trust of the elderly in the community in the safety of our project products has increased, and they said that if our products are approved by the state in the future, they will be willing to try.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/note-7.jpg)

## Wet Lab

## Safety

1.On the basis of the skeleton provided by the literature, the new kill switch circuit specialized for yogurt was designed, the plasmid and experimental design were constructed, and the check in form feedback was received, all of which passed the review.

2.Due to the complexity of the plasmid backbone and the high reliability of the literature data, consider postponing the experiment in the future; and review and submit the final safety form.

3.Under the PI suggestion, the crowd sensing module is designed to increase safety; Preliminary text writing.

![Gut-sweeper]( https://static.igem.wiki/teams/4656/wiki/researchb4.png)

## Wet Lab

BL21 was introduced into the gene fragment and liquid-cultured in LB culture medium for 16 h, and then WB detection was performed on the expressed protein. It was found that Escherichia coli BL21 could successfully express TPH and TDC enzymes.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/note12.png)

## Model

1.Write descriptive materials related to modeling, and sort out and examine the process of mathematical modeling.

2.Cooperate with the animation team to visualize the model.

# October

1.Each group performs text organization and wiki upload.

2.All members work together to check wiki text, images, and videos for errors, including the part interface.

3.Conduct presentation video shoots and edits; prepare for the jamboree.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

### Engineering Success

Scroll To Explore

![]()

# Overview

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/eg-cycle.png)

Synthetic biology is the utilization of engineering principles, using synthetic DNA, gene editing and bioengineering technology to design, build and improve biological systems to achieve the goal of application. Therefore, after using these engineering principles to design and build prototypes, it is necessary to go through a series of tests and repeated improvements to ensure that the final product meets the initial expectations and achieves the optimization of application. Our team applied this concept to the project, forming a complete engineering cycle :research, imagine, design, build, test and learn. In the research, we conducted literature review and expert consultation. In imagine, team members carried out brainstorming and put forward creative ideas. In the design, according to the objectives, scope, feasibility and resource requirements of the project, we used engineering principles to design gene fragments and verification methods. In the build, we sent the designed gene fragment to the company for synthesis, and the synthesized plasmid was transferred into the chassis bacteria. In the test, we analyzed the function of the engineering biological system we constructed through bacterial experiments. In the process of learning, we analyzed the experimental results to evaluate whether the engineering biological system we constructed has achieved the expected goal. Through the above several engineering cycles, we have made gut-sweeper into a safe and effective engineering E.coli, which provides a new idea for the treatment of constipation.

## Please click the following tab buttons to shift !

* Sensory and Metabolic module
* Safety module

---

# Abstract

In the initial phase of the project during the first engineering cycle, we endeavored to identify characteristic substance changes in the body when it experiences constipation. Through investigation and research, we observed that an increase in methane gas and a decrease in butyrate concentration occur within the intestines when the body is constipated. Consequently, we initially designated methane and butyrate as sensing biomarkers for our engineered bacteria. However, later findings from interviews with relevant experts and literature research revealed that certain normal physiological phenomena could also lead to elevated methane levels, and the specificity of methane in relation to constipation was not strong. Furthermore, methane needs to be converted into methanol to be sensed by the engineered bacteria, and methanol has toxic effects on the human body. In contrast, butyrate exhibits a stronger specificity in its association with constipation. Consequently, we ultimately designated butyrate as the sensing biomarker for our engineered bacteria.

In the second engineering cycle, we aimed to simplify the genetic circuit to achieve better controllability and expression efficiency. However, through bacterial experiments and comparisons, we found that the existing butyrate sensing circuit in enterohemorrhagic Escherichia coli was more sensitive and efficient. Therefore, we decided to retain the existing butyrate sensing circuit of enterohemorrhagic Escherichia coli.

In the third engineering cycle, we attempted to the increasing expression of Lrp within the bacteria to enhance their sensitivity to butyrate. Through bacterial experiments, we confirmed that increasing the expression of Lrp indeed enhanced the sensitivity of our engineered bacteria to butyrate.

In the fourth engineering cycle, as our designed butyrate sensing circuit is butyrate-inducible, meaning that a certain range of high butyrate concentrations can promote the expression of this gene circuit, and since butyrate concentrations decrease during constipation, we needed to add a negative regulatory element to enable our engineered bacteria to express and secrete therapeutic substances during constipation. Through literature research, we discovered Cl/plam, where Cl is a repressor protein that can bind to the plam promoter and inhibit downstream gene expression. Subsequently, we successfully validated the effectiveness of Cl/plam negative regulation through bacterial experiments.

In the fifth engineering cycle, we attempted to use endogenous substances from the human body as the therapeutic substances expressed and secreted by our engineered bacteria. Through literature searches, we found that 5-HT can effectively promote intestinal motility and secretion. Therefore, we planned to have the engineered bacteria express the two key enzymes during the 5-HT synthesis process: TDC and TPH, utilizing tryptophan in the intestines to synthesize 5-HT. We successfully expressed these two enzymes in our engineered bacteria through subsequent bacterial experiments.

* [**Cycle1**
  Perceiving the constipation](#Research1)
  + [Research](#Research1)
  + [Imagine](#Imagine1)
  + [Design and Build](#BuildDesign1)
  + [Test](#Test1)
  + [Learn](#Learn1)
* [**Cycle2**
  Simplifing the gene circuit](#Research2)
  + [Research](#Research2)
  + [Imagine](#Imagine2)
  + [Design and Build](#BuildDesign2)
  + [Test](#Test2)
  + [Learn](#Learn2)
* [**Cycle3**
  Enhancing the sensitivity](#Research3)
  + [Research](#Research3)
  + [Imagine](#Imagine3)
  + [Design and Build](#BuildDesign3)
  + [Test](#Test3)
  + [Learn](#Learn3)
* [**Cycle4**
  Negative regulatory element](#Research4)
  + [Research](#Research4)
  + [Imagine](#Imagine4)
  + [Design and Build](#BuildDesign4)
  + [Test](#Test4)
  + [Learn](#Learn4)
* [**Cycle5**
  Therapeutic agents](#Research5)
  + [Research](#Research5)
  + [Imagine](#Imagine5)
  + [Design and Build](#BuildDesign5)
  + [Test](#Test5)
  + [Learn](#Learn5)
* [**References**
  Wide brainstorming](#references1)
* ![糖豆人](https://static.igem.wiki/teams/4656/wiki/tangdouren.png)

  ![](https://static.igem.wiki/teams/4656/wiki/gototop.png)

# Cycle1:Research

**How do our engineered bacteria perceive the state of constipation in the body?**

Constipation is a relatively common phenomenon, and unhealthy lifestyles, as well as bad eating habits, can lead to constipation. According to statistics, the prevalence of chronic constipation in Chinese adults is between 4% to 6%, increasing with age and reaching as high as 22% in the population over 60 years old. Currently, there are several main therapies for constipation, including medication, dietary adjustments, biofeedback therapy and surgical treatment. However, these treatments face challenges such as short-term efficacy, significant trauma, and potential side effects. There is an urgent need to explore a new treatment approach. We aim to leverage synthetic biology principles to create an engineered bacteria capable of detecting the body's constipation state and then secreting relevant substances to alleviate constipation.

Through research and investigation, we have found that when the body experiences constipation, there are changes in the concentrations of methane gas and butyrate within the intestinal tract.

Relevant experiments indicated a significant association between methane excretion and chronic constipation. In a glucose breath test (GBT) conducted on 50 patients experiencing gastrointestinal symptoms, it was found that constipated individuals had a higher median CH4 excretion compared to those with regular daily bowel movements (p=0.0406). Constipated patients exhibited an average CH4 emission of 30.3 ppm, while the average CH4 excretion in the normally defecating population was 21.5 ppm. It can be concluded that when the body experiences constipation, there is an elevated concentration of methane gas within the intestinal tract[1].

Short-chain fatty acids (SCFAs), with butyrate making up approximately 60% of them, are primarily produced through fermentation and degradation by the gut microbiota in the human intestinal tract. It is estimated that the concentration of butyrate within the intestinal lumen of humans and animals ranges from 10 to 20 mM. When the body experiences constipation, there is a shift in the gut microbiota, resulting in reduced butyrate production. During constipation, the concentration of butyrate within the intestinal tract can drop below 10 mM. It can be concluded that the concentration of butyrate in the intestinal tract decreases when constipation occurs[2].

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/research1.png)

# Cycle1:Imagine

Can engineered bacteria detect the state of constipation in the body by sensing changes in methane or butyrate concentrations?

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/imagine1.png)

# Cycle1:Design and Build

The AOX1 promoter is a commonly used high-efficiency inducible promoter in Pichia pastoris yeast. When methanol is the sole carbon source, the AOX1 promoter can be induced by methanol to initiate the expression of alcohol oxidase enzymes, allowing for metabolic processes utilizing methanol. Particulate methane monooxygenase (pMMO) is capable of converting methane into methanol. Therefore, we utilize this enzyme to convert methane within the intestinal tract into methanol. Subsequently, by inducing the expression of relevant genes downstream of the AOX1 promoter, we achieved the engineered bacteria's ability to sense methane[3].

The PchA promoter is an inducible promoter found in enterohemorrhagic Escherichia coli (EHEC). When butyrate binds to Lrp, they form a complex that subsequently binds to the PchA promoter, initiating the expression of PchA. PchA then interacts with the Lee promoter, thereby promoting the expression of genes downstream of the Lee promoter. Therefore, our goal is to introduce the PchA promoter into the engineered bacteria, enabling them to sense butyrate[4].

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/design1.png)

# Cycle1:Test

Through HP investigations, literature reviews, and group discussions, the production and emission of methane gas in the intestines are associated with various factors, including the types and quantities of intestinal bacteria, dietary habits, and intestinal motility. While methane gas is correlated with constipation in some cases, it is not always accompanied by elevated methane gas levels in all instances of constipation. An increase in methane levels may be a normal physiological phenomenon, such as with a high-fiber diet or active intestinal peristalsis. Therefore, the specificity of the association between methane and constipation is not strong. Additionally, we discovered that methanol is a colorless, volatile liquid. When ingested by the human body, it is metabolized into formic acid and carbon dioxide, both of which enter the body and can lead to poisoning. Methanol has varying degrees of toxic effects on the central nervous system, cardiovascular system, digestive system, and visual system of the human body. In contrast, butyrate demonstrates a stronger specificity in its association with constipation. Under normal circumstances, the concentration of butyrate in the intestines remains within the range of 10-20 mM, but during constipation, the concentration of butyrate drops below 10 mM.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/spec.png)

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/test1.png)

# Cycle1:Learn

Therefore, we ultimately chose to use butyrate as the sensing marker for our engineered bacteria.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/learn1.png)

# Cycle2:Research

Under the professional guidance and advice of our teachers, we have made diligent efforts to simplify the genetic circuit in order to better optimize gene expression and reduce unnecessary nonspecific reactions, thereby effectively enhancing the controllability of gene expression. This process is not only aimed at achieving higher expression efficiency but also contributes to ensuring more precise control of gene expression levels under different conditions, ultimately providing more reliable results for our research or applications.

# Cycle2:Imagine

Considering that the pchA promoter can sense the complex of Lrp and butyrate, initiating the expression of the pchA gene, is it possible for us to further simplify the genetic circuit? In other words, can we directly connect the protein we want to express after the pchA promoter without the need for additional regulatory elements? Such simplification could potentially enhance the efficiency and controllability of the gene expression system, providing greater convenience and feasibility for our research or applications. This concept holds promise for optimizing the gene expression system, making it more efficient and flexible, while reducing the complexity of building genetic circuits.

# Cycle2:Design and Build

Designing two gene fragments: one directly attaching EGFP downstream of the pchA promoter, and the other appending EGFP after the existing gene segment in enterohemorrhagic E. coli. We compared the responsiveness and feedback levels of these two fragments to butyrate.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/build2.png)

# Cycle2:Test

Bacterial Experiment: We cloned the synthesized A segment and B segment onto the pet-28a plasmid. Subsequently, we individually transformed these plasmids into Escherichia coli BL21. After plating and overnight incubation, we extracted an equal volume of bacterial culture and inoculate it into a 96-well plate containing LB medium and sodium butyrate. Then the plate was incubated at 37°C with shaking in a microplate reader, simultaneously measuring fluorescence intensity and bacterial growth curve.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/test2.png)

# Cycle2:Learn

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/learn2.png)

E. coli strains transfected with either A segment or B segment were cultivated in the presence of 10mM and 20mM sodium butyrate. The results revealed that the sensitivity of A segment to sodium butyrate and its ability to enhance the expression of downstream genes were superior to those of B segment.

In the end, we opted for the design of the A segment.

# Cycle3:Research

Leucine responsive regulatory protein (Lrp) is a crucial transcriptional regulatory factor in prokaryotes, widely expressed throughout the prokaryotic world. Lrp can function as a global or local transcriptional regulator, participating in the control of various essential physiological processes in microorganisms, including amino acid metabolism, flagellum formation, heavy metal transport, peptide transport, energy metabolism, and more. It also serves as a "feast/famine" regulatory protein, assisting microorganisms in adapting to diverse external environments[4].

As a sensor, Lrp transduces the stimulus of butyrate into the regulatory network controlling the LEE genes through the PchA-Ler system.

# Cycle3:Imagine

Is it possible for us to enhance the sensitivity and responsiveness of the PchA promoter to butyrate in engineered bacteria by increasing the expression level of the Lrp protein? By elevating the expression of Lrp, we aim to bolster the engineered bacteria's ability to detect butyrate, allowing them to quickly sense the state of constipation within the body. Subsequently, this enhanced sensitivity could trigger the expression of downstream genes to address constipation, thus preventing potential harm caused by prolonged constipation to the body.

# Cycle3:Design and Build

We simultaneously introduced the pBAD33 plasmid carrying Lrp and the pet-28a plasmid containing the receptor into Escherichia coli. Bacteria that received only the receptor plasmid but not the Lrp plasmid were used as a control group. We compared the sensitivity of these two groups to butyrate sensing.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/build3.png)

# Cycle3:Test

Bacterial Experiment: The experiment was divided into two groups. In one group, the pBAD33 plasmid carrying the Lrp gene and the pet-28a plasmid containing the PpchA-pchA-Plee-EGFP segment were simultaneously introduced into Escherichia coli BL21. In the other group, only the pet-28a plasmid containing the PpchA-pchA-Plee-EGFP segment was introduced into Escherichia coli BL21. After plating and overnight incubation, an equal volume of bacterial culture was extracted and inoculated into a 96-well plate containing LB medium and sodium butyrate. Each well was supplemented with arabitol to a final concentration of 3.2 mM. The plate was then incubated at 37°C with shaking in a microplate reader, simultaneously measuring fluorescence intensity and bacterial growth curve.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/test3.png)

# Cycle3:Learn

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/learn3.png)

Both groups were cultured in 0mM, 10mM, and 20mM sodium butyrate, and the results demonstrated that increased expression of Lrp does indeed enhance the sensitivity of the PchA promoter to butyrate .

# Cycle4:Research

According to the literature, it is evident that the concentration of butyrate in the intestinal tract significantly decreases during constipation. Our PpchA-pchA-Plee-gene, on the other hand, is butyrate-inducible, meaning that higher concentrations of butyrate within a certain range can enhance the expression of this gene circuit. Therefore, we envision utilizing changes in butyrate concentrations to negatively regulate the products of our genetic circuit. To achieve this, our genetic circuit requires a negative regulatory element. This would enable us to promote the expression of relevant genes when butyrate concentrations decrease.

# Cycle4:Imagine

Is it possible for us to consider introducing a repressor protein to achieve negative regulation of the gene circuit? By introducing this repressor protein, we aim to suppress gene expression when the body is in a normal state and, conversely, promote gene expression when the body is in a constipated state. This would enable our engineered bacteria to switch their function between normal and constipated states, allowing for a dynamic response to the body's condition.

# Cycle4:Design and Build

Plam is a potent promoter found in the lambda bacteriophage, while Cl is an inhibitory protein that can bind to the Plam promoter, thereby repressing downstream gene expression. Cl/Plam is a well-known component of a genetic circuit involving a "NOT" logic gate[5].

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/build4.png)

# Cycle4:Test

The synthesized PpchA-pchA-Plee-cl-plam-EGFP gene fragment was cloned into the pet-28a plasmid. Subsequently, it was co-transformed into Escherichia coli BL21 along with the pBAD33 plasmid carrying the Lrp gene. After plating and overnight incubation, an equal volume of bacterial culture was extracted and inoculated into a 96-well plate containing LB medium and sodium butyrate. Each well was supplemented with arabitol to a final concentration of 3.2 mM. The plate was then incubated at 37°C with shaking in a microplate reader, simultaneously measuring fluorescence intensity and bacterial growth curve.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/test4.png)

# Cycle4:Learn

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/learn4.png)

The results indicate that at a concentration of 0mM sodium butyrate, EGFP exhibits high expression, whereas at a concentration of 20mM sodium butyrate, EGFP is hardly expressed. This suggests that the Cl/Plam element effectively serves as a negative regulatory component.

# Cycle5:Research

Based on interviews and research, naturally occurring substances produced within the body possess the following potential advantages as therapeutic agents compared to chemically synthesized drugs.

Biocompatibility: Naturally occurring substances produced within the body are often more readily accepted by the human body due to their closer alignment with the body's biochemical processes. This reduces the risk of triggering severe side effects or immune reactions.

Lower Toxicity: Naturally occurring substances typically exhibit lower toxicity as they are widespread in nature and interact with biological systems. In contrast, some chemically synthesized drugs may require more chemical processing to ensure their safety.

Bioavailability: Naturally occurring substances produced within the body are often more readily absorbed and utilized because they are closely aligned with the body's metabolic pathways. This can enhance the bioavailability of drugs, reducing the required dosage and frequency of treatment.

Reduced Allergic Reactions: Naturally occurring substances are generally less likely to trigger allergic reactions, as the body has already developed a certain degree of immune tolerance towards them. In contrast, certain components in synthetic drugs may lead to allergic reactions in some patients.

# Cycle5:Imagine

Is it possible to enable our engineered bacteria to produce endogenous substances when they detect constipation in the body, which can be used to treat constipation? By harnessing engineered bacteria, we can design a gene circuit with negative regulation, ensuring the release of the necessary bioactive substances only when the body is experiencing constipation. This strategy not only helps us achieve more precise treatment but also minimizes interference with and side effects on the human body to the greatest extent possible.

# Cycle5:Design and Build

Serotonin (5-HT) is a vital neurotransmitter that plays a crucial role in regulating emotions, memory, appetite, gut homeostasis, and metabolism in the body. While serotonin plays a key role in the central nervous system, the majority of serotonin (up to 95%) is produced by enterochromaffin cells (EC cells) in the gut. Research suggests that the stimulation of 5-HT in the gastrointestinal tract can activate either the 5-HT2 receptors in smooth muscles or the 5-HT4 receptors in enteric neurons, leading to gastrointestinal smooth muscle contractions and promoting intestinal motility and secretion. As a result, it is widely used in the treatment of gastrointestinal disorders[6].

TDC and TPH are two crucial enzymes in the synthesis of 5-HT. They use tryptophan as a precursor, and through the action of tryptophan hydroxylase (TPH) and serotonin decarboxylase (TDC), produce serotonin (5-HT)[7].

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/build5.png)

# Cycle5:Test

The synthesized fragments mentioned above were ligated into the pet-28a plasmid, which was subsequently introduced into Escherichia coli BL21. After plating and overnight incubation, bacterial cultures were collected and grown in liquid LB medium for 16 hours. The expressed proteins were then subjected to Western blot (WB) analysis.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/test5.png)

# Cycle5:Learn

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/learn5.png)

Escherichia coli BL21 can effectively express both TPH and TDC enzymes.

# References

1. Furnari M, Savarino E, Bruzzone L, Moscatelli A, Gemignani L, Giannini EG, et al. Reassessment of the role of methane production between irritable bowel syndrome and functional constipation. J Gastrointestin Liver Dis. 2012;21(2):157-63. Epub 2012/06/22. PubMed PMID: 22720304.
2. Koo CW, Tucci FJ, He Y, Rosenzweig AC. Recovery of particulate methane monooxygenase structure and activity in a lipid bilayer. Science. 2022;375(6586):1287-91. Epub 2022/03/18. doi: 10.1126/science.abm3282. PubMed PMID: 35298269; PubMed Central PMCID: PMCPMC9357287.
3. Nakanishi N, Tashiro K, Kuhara S, Hayashi T, Sugimoto N, Tobe T. Regulation of virulence by butyrate sensing in enterohaemorrhagic Escherichia coli. Microbiology (Reading). 2009;155(Pt 2):521-30. Epub 2009/02/10. doi: 10.1099/mic.0.023499-0. PubMed PMID: 19202100.
4. Salvi PS, Cowles RA. Butyrate and the Intestinal Epithelium: Modulation of Proliferation and Inflammation in Homeostasis and Disease. Cells. 2021;10(7). Epub 2021/08/08. doi: 10.3390/cells10071775. PubMed PMID: 34359944; PubMed Central PMCID: PMCPMC8304699.
5. Wang B, Kitney RI, Joly N, Buck M. Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nat Commun. 2011;2:508. Epub 2011/10/20. doi: 10.1038/ncomms1516. PubMed PMID: 22009040; PubMed Central PMCID: PMCPMC3207208.
6. Li B, Li M, Luo Y, Li R, Li W, Liu Z. Engineered 5-HT producing gut probiotic improves gastrointestinal motility and behavior disorder. Front Cell Infect Microbiol. 2022;12:1013952. Epub 2022/11/08. doi: 10.3389/fcimb.2022.1013952. PubMed PMID: 36339343; PubMed Central PMCID: PMCPMC9630942.
7. Park S, Kang K, Lee SW, Ahn MJ, Bae JM, Back K. Production of serotonin by dual expression of tryptophan decarboxylase and tryptamine 5-hydroxylase in Escherichia coli. Appl Microbiol Biotechnol. 2011;89(5):1387-94. Epub 2010/11/17. doi: 10.1007/s00253-010-2994-4. PubMed PMID: 21080162.

Please click here to see our safety modeule

# Abstract

In the field of therapeutic synthetic biology, safety also involves designing measures to prevent engineered cells from harming host cells and environment. Alongside conventional methods like sterilization, the kill-switch circuit has been widely used as the first choice by previous researchers and iGEMers.

In our project, engineered ECN producing 5-HT were utilized to alleviate constipation, sensing changes in butyrate concentration in the intestine to release 5-HT, aiming to provide long-term preventive effects for individuals prone to constipation. In the four generations of our safety design, which will be mentioned later, we have considered the following concerns:

1.The uncontrolled proliferation of engineered 5-HT ECNs results in excessive production of 5-HT, which disrupts intestinal physiology, even leading to alterations in the microbial composition.

2.Engineered 5-HT ECNs have the ability to survive and replicate for a certain time outside the body after being excreted in feces, which raises concerns about potential environmental hazards, especially in aquatic environments.

3.The efficacy of engineered 5-HT ECNs is compromised as they are significantly depleted by gastric juices, preventing the desired effect from being achieved.

In our initial engineering cycle, we considered environmental protection first and foremost. So we added a temperature-controlled kill switch to ensure that the engineered bacteria will die after being expelled from the body. Then when conducting questionaries to the general public, we found that people were afraid of the bacteria and questioned the safety of the engineered bacteria in vivo. At the same time, Prof. Deng also pointed out the necessity of adding an in vivo containment system.

In our second engineering cycle, we followed Prof. Deng's suggestion, and after absorbing the experience of our previous teams, we designed a short and compact dual-drug containment system and conducted relevant proof-of-concept experiments with preliminary results. In a new round of public research, we found that yogurt, as a laxative food, was favored by the elderly, who preferred yogurt rather than capsules to be used as a carrier for the engineered bacteria.

So, in our third engineering cycle, considering the complex transportation and preservation conditions of yogurt, we specifically designed the kill switch and anti-acid encapsulation for yogurt to adapt to various scenarios from transportation to excretion. Although not ready for experimentation, the skeleton of our route has been fully characterized by the original article. At the same time, it is worth mentioning that, adhering to the principle of local people solving local problems, we do not have a one-size-fits-all approach to the positioning of our product carriers, as well as the product itself, and in the future, we will design our product in a comprehensive manner, taking into account the policy and geographic characteristics of the area.

In our fourth engineering cycle, at PI's suggestion, we made a simple design for quorum sensing. In our design, the colony is only allowed to grow rapidly when both the colony density and butyrate concentration are below the threshold, which greatly ensures in vivo safety. We will validate this part of the design in the future.

* [**Cycle1**
  circuit for environmental protection](#ResearchB1)
  + [Research](#ResearchB1)
  + [Imagine](#ImagineB1)
  + [Design and Build](#DesignbuildB1)
  + [Test](#TestB1)
  + [Learn](#LearnB1)
* [**Cycle2**
  ideas of double drug control](#ResearchB2)
  + [Research](#ResearchB2)
  + [Imagine](#ImagineB2)
  + [Design and Build](#DesignbuildB2)
  + [Test](#TestB2)
  + [Learn](#LearnB2)
* [**Cycle3**
  yogurt specialized kill-switch circuit](#ResearchB3)
  + [Research](#ResearchB3)
  + [Imagine](#ImagineB3)
  + [Design and Build](#DesignbuildB3)
  + [Test](#TestB3)
  + [Learn](#LearnB3)
* [**Cycle4**
  Quorum Sensing Modules](#ResearchB4)
  + [Research](#ResearchB4)
  + [Imagine](#ImagineB4)
  + [Design and Build](#DesignbuildB4)
  + [Test](#TestB4)
  + [Learn](#LearnB4)
* [**References**
  Wide brainstorming](#referencesB-1)
* ![糖豆人](https://static.igem.wiki/teams/4656/wiki/tangdouren.png)

  ![](https://static.igem.wiki/teams/4656/wiki/gototop.png)

# Cycle1:Research

## Kill-switch for environmental protection

In our group discussion, we firstly considered the need to exterminate engineered bacteria upon their release into the environment thus preventing potential contamination in future application scenarios, namely, a kill-switch circuit for environmental protection.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/researchb1.png)

Despite the powerful sterilization capacity of conventional physical and chemical methods, they are inconvenient to use and may pose risks of accidental harm to users, making the kill-switch circuit a better choice[1]. (Detailed information can be found on our [Safety](https://2023.igem.wiki/nmu-china/safety%20) page).

We conducted research on previous teams' kill-switch strategies, and common circuits found include temperature control, oxygen control, light control, and pH control, all based on inherent differences between the internal and external environments.

# Cycle1:Imagine

## Temperature control strategy

We decided to choose the currently maturest strategy, temperature control strategy. We envisioned that at 37℃ in the human body, bacteria can grow normally. However, when they are discharged into the sewage system and the temperature drops below 30℃, the expression of toxic proteins (such as mazF, ccdB, rel, etc.) was elevated, leading to extensive bacterial death[2,3].

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/imagineb1.png)

# Cycle1:Design and Build

## PCspA-mazF as our circuit

The cold-acting promoter CspA can only be efficiently translated at a low temperature[4], thereby inhibiting the expression of downstream genes, namely toxin proteins in our design. We made a comparison between the most commonly used toxic proteins, mazF, ccdB, relE, finding that both mazF and ccdB are suitable while relE have the potential to cause cell death in human cell lines according to team Fudan\_2020 on part BBa\_K185047[5]. We finally chose mazF as our toxic protein considering patent issue of ccdB.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/buildb1.png)

# Cycle1:Test

## Questionnaire survey

Our HPers conducted a questionnaire survey mainly targeting the elderly to explore their acceptance of our engineered bacteria. Unfortunately, the survey results were discouraging as the elderly expressed concerns regarding the safety of the bacteria.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/testb1-revised1.jpg)

# Cycle1:Learn

## Inducible containment system

During the discussion with Professor Deng Zixin, an expert in the field of microbial metabolism, we discovered a flaw in our safety module, the lack of consideration for urgent containment within the body. Additionally, as mentioned before, our HPers conducted a questionnaire survey among the elderly, revealing that they harbor preconceived notions about bacteria and lack sufficient understanding of engineered bacteria, leading to concerns regarding the use of engineered bacteria for constipation treatment.

To overcome this obstacle, it is essential not only for the HP group to promote and educate about probiotics but also for us to incorporate an inducible containment system for probiotics. Having a self-controlled system will instill a stronger sense of security among individuals.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/learnb1.png)

# Cycle1:Improve

## Drug control strategy

We decided to introduce a more flexible and autonomous system, drug control system, widely used in in-vivo containment system, and conducted research and exploration.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/improveb1.png)

# Cycle2:Research

## In-vivo urgent containment

In response to the concerns regarding the in-vivo safety of bacteria, we conceived the idea of designing an in-vivo containment system by introducing drug control system. With the advice from Professor Deng Zixin, an expert in the field of microbiology, who suggested incorporating an emergency containment for our engineered bacteria, we became more determined in pursuing this idea, collecting and screening drug control strategies.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/researchb2.jpg)

# Cycle2:Imagine

## Ideas of double drug control

Common drug control circuits are designed to be induced by rhamnose, arabinose, and tetracycline. While browsing through previous wikis, we came across an intriguing switch called the l-malic acid switch[6,7,8]. It offers a better taste flavor compared to rhamnose and arabinose, making it more readily accepted. We planned to utilize the l-malic acid-sensing switch to induce bacteria death, alongside the rhamnose switch for environmentally exposed death, which constitute a double drug control system.

In practical application scenarios, it would be necessary to take an appropriate amount of rhamnose to maintain a certain concentration in the intestine, ensuring normal bacterial growth. When it becomes necessary to stop the treatment, l-malic acid intake can be utilized to halt the growth of engineered bacteria within the body.

# Cycle2:Design and Build

## L-malic acid and rhamnose to constitute double drug control

The double drug control strategy is implemented through the induction of rhamnose and l-malic acid. In an environment lacking rhamnose, the engineered bacteria will not express the tetR, and the ptet promoter activates the expression of the toxic protein mazF, thereby triggering apoptosis, which makes rhamnose a necessary addition for their survival. The l-malic acid switch is activated by l-malic acid and initiates the expression of downstream toxic proteins, allowing patients to independently eradicate the bacteria in their bodies by taking l-malic acid.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/buildb2-1.png)

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/buildb2-2.png)

**Circuit design of our double drug control**

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/designb2.png)

**Sketch of the working mechanism**

# Cycle2:Test

## Bacterial experiment|Verification of the circuit pRha-tetR-ptet-mazF

Rhamnose kill-switch is for environment protection, that is to say, when bacteria is excreted outside and rhamnose is diluted, bacteria will die due to the kill-switch, which is triggered by low concentration of rhamnose.

Pre-experimentation via plate spreading showed that rhamnose addition or not had a significant effect on the survival of bacterial and subsequent OD evaluation under different concentration of rhamnose co-culture for 36 hrs showed that groups with higher concentration of rhamnose grew better, which met our expectations.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/testb2-revised1.jpg)

**Fig. 1 |****Rhamnose addition maintains the growth viability of engineered bacteria with rhamnose kill switch circuit**
a.Pre-experimental plate spreading results, with rhamnose addition or not
b.Visualization of (a)
c.Growth curve measured by enzyme-labeled Instrument, co-cultured for 36 hrs with different concentrations of rhamnose

## Bacterial experiment|Verification of the circuit pT7-mleR-p\_mleS-mazF

Malic acid kill-switch is for in-vivo urgent containment, which means the patient can control the process as their wish by taking in some malic acid with good flavor.

Pre-experimentation via zone of inhibition showed that malic acid addition could inhibit the bacteria growth. Then we carried out OD evaluation, which was unsatisfactory for OD evaluation could not distinct living and dead individuals well. So we performed interval plate spreading at 2h, 4h, 6h, 8h, 10h, 12h under different concentrations of malic acid co-culture, which showed that malic acid addition could exert killing effect.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/testb2-rrevised2.png)

**Fig. 2 |****Malic acid addition exerted killing effect on engineered bacteria with malic acid kill switch circuit**
a.Anti-bacterium-circles testing as a pre-experimentation, different concentrations of malic acid treatment
b.Visualization of (a).
c.Growth curve measured by enzyme-labeled Instrument, co-cultured for 36 hrs with different concentrations of malic acid
d.Interval plate spreading counting results and visualization

# Cycle2:Learn

## Deficiencies of current system in future use

Our experimental results demonstrate the feasibility of the circuit. However, we still found two deficiencies if put into practice. Firstly, l-malic acid also presents in many fruits, which may accidentally trigger the kill-switch after eating abundant fruits. Further investigation is needed to compare the l-malic acid sensing threshold with the concentration of l-malic acid in the intestines after consuming abundant fruits to determine the viability of this strategy.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/learnb2.png)

Secondly, in addition to the engineered bacteria, the intestines harbor a vast population of other microbial communities. Currently, it is uncertain how these communities may affect the concentration of rhamnose. Furthermore, the complex environment of the intestines could potentially lead to rapid depletion of rhamnose to a lower concentration than the threshold required to maintain the activity of the engineered bacteria. One possible solution is to incorporate a positive feedback switch, which would allow even lower concentrations of rhamnose to sustain microbial activity. Moreover, another questionnaire survey carried out by our HPers showed that yogurt as the carrier was more preferred than capsules among the elderly, considering that products like yogurt demand higher transportation and preservation conditions, we may design a yogurt specialized kill-switch circuit.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/learnb2-2.png)

# Cycle2:Improve

## Yogurt specialized kill-switch circuit

We redesigned the a yogurt specialized kill-switch circuit, well adapt to transportation and preservation conditions.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/improveb2.png)

# Cycle3:Research

## Yogurt: a preferred delivery methods among the elderly

Another questionnaire survey targeting the elderly was conducted. The survey specifically compared two delivery methods, yogurt and capsules. The results indicated that the elderly preferred the form of yogurt. We then had a discussion concerning above results with biomedical expert Liao Xiang . He held the points that both yogurt and capsules can be used as carriers for engineered bacteria, but if yogurt is used, the packages need to be designed to withstand stomach acid and transportation issues need to be considered.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/researchb3-1.png)

Therefore, we explored new kill-switch circuit specialized for yogurt and acid resistant coats[10] for bacteria, for yogurt can not be made into an enteric form like capsules, which can been found in our cycle of coats. Due to the excessive contents in this cycle, we've listed cycle of coats in the supplementary pdf [Cycle of coats](https://static.igem.wiki/teams/4656/wiki/cycle-of-coats.pdf).

Note: The iGEM spirit emphasizes "local people solve local problems". Considering the vast territory of our country, transportation cost is a crucial factor to consider when designing a product. Specifically, yogurt has strict requirements for transportation conditions, and the high cost of cold chain transportation poses a challenge to nationwide promotion of our product. Therefore, we have not completely abandoned the capsule carrier. Additionally, due to its lower transportation cost, it may be suitable for promotion in the central and western regions of our country.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/researchb3-revised2.png)

# Cycle3:Imagine

## Kill-switch circuit specialized for yogurt

Yogurt products offer excellent flavor, but they require strict storage and transportation conditions, including refrigeration. Building upon the existing literature's framework, we have redesigned a set of inducible suicide environmental exposure switches to adapt to various scenarios: transportation, in-vivo environments, and excretion scenarios. These modifications aim to further enhance the safety of the engineered bacteria.

# Cycle3:Design and Build

## Different endings in different scenarios

We envisioned three scenarios for engineering bacteria carried by yogurt: transportation scenario, in-vivo scenario, and excretion scenario. Engineering bacteria should be kept alive in transportation scenario and in-vivo scenario and exterminated in excretion scenario.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/designb3.png)

## Circuit specialized for yogurt

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/buildb3.png)

In the first route, when a small amount of tetracycline (tet) is introduced, it undergoes a conformational change upon binding to Tet repressor (tetR), causing the inhibition of Ptet to be lifted and resulting in LacI expression. This, in turn, suppresses the synthesis of tetR, further weakening the inhibition on Ptet. As a result, a positive feedback loop is formed, leading to the high expression of mazF. Therefore, only a minimal intake of tet is required to exert strong repression on the engineered bacteria[11,12].

In the second route, based on the study by Wang et al[13], we envisioned 3 scenarios:

**Scenario One:** Transportation scenario, where the temperature is below 30℃. In this scenario, Cl857 inhibits pR, resulting in no expression of mazE but expression of mazF. However, the addition of yogurt containing rhamnose allows for simultaneous expression of mazE in the engineered bacteria, preventing bacterial death.

**Scenario Two:** In-vivo scenario, where the temperature is 37℃, Cl857 releases the inhibition on pR, leading to the expression of phIF and suppression of downstream mazF expression. At the same time, mazE is expressed. Regardless of whether there is a sufficient concentration of rhamnose to induce mazE, the bacteria can survive.

**Scenario Three:** Excretion scenario, where the temperature in the sewer system is below 30℃, mazE is not expressed while mazF is expressed. Additionally, rhamnose is diluted, insufficient to induce mazE expression. As a result, the bacteria die.

# Cycle3:Test

Currently, we have only conducted experimental designs for this system. Although the framework of the circuits has been well characterized in the literature and stood the test of time, we will strive to complete and refine our validation in the future. At the same time, the current design is overly complicated, so simplification is also one of the tasks we will undertake in the future.

# Cycle3:Learn

## Drawbacks of current system and lessons

Tetracycline, as an antibiotic, has certain side effects and may have negative impacts on other gut microbiota. Although the designed positive feedback amplification system allows for the induction of engineered bacteria death at very low concentrations of tet, exploring alternative systems is highly necessary for safety considerations. Furthermore, during our review of the previous team's wiki, we discovered that in the case of inflammatory constipation, there may be damaged microvessels in the intestine. Could the engineered bacteria potentially take advantage of this opportunity for invasion? SZU2021 has already provided a good response to this question. Meanwhile, our PI Changhai Lei raised a question that whether we can construct a Quorum Sensing Module to further ensure the safety by controlling the population density, which better reflects the preventive role of our engineered bacteria.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/learnb3-revised2.jpg)

# Cycle3:Improve

## Quorum Sensing Modules

We designed a set of Quorum Sensing Modules to control the population density, making sure that only when the population and butyrate concentration are both below the threshold, can our bacteria quickly grow.

# Cycle4:Research

## Quorum Sensing for population control

In nature, for multicellular groups as well as single-cell populations, there is a very simple yet crucial function: cell community control. However, in unnatural environments, E. coli community populations are not so easily stabilized. And in such a special environment as the human gut, in order to ensure the homeostasis of the intestinal flora and thus the safety of our project within the dynamic range, we must add a quorum sensing module to the Gut Sweeper.

When searching for information concerning Quorum Sensing, we were impressed by the work done by professor You in 2004 published on Nature, laying the framework for our design[14].

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/researchb4.png)

# Cycle4:Imagine

## Colony density and butyrate concentration to be the indicators

We envisioned that bacterial growth capacity is strictly kept under control in two scenarios: 1) colony density exceeded threshold, 2) butyrate concentration surpassed the threshold, which means that the colony is only allowed to grow rapidly when both norms are below the threshold.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/imagineb4.png)

# Cycle4:Design and Build

## Colony density sensing module and Butyrate concentration sensing module

Our quorum sensing module is divided into two sub-modules.

1)Colony density sensing module

2)Butyrate concentration sensing module

Controlled by the two sub-modules, the colony is only allowed to grow rapidly when both norms are below the threshold

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/designb4.png)

## Circuit of our two sub-modules

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/buildb4.png)

When the population density is low, the dominant promoter expresses the downstream genes LuxI and LuxR, which express a small amount of AHL protein and LuxR protein, respectively, and due to the low concentration of AHL protein, it is not enough to bind all LuxR protein. The free LuxR protein cannot induce the luxl promoter to express tetR and bga2 proteins, and can only induce the tetR promoter to express GDH (glutamate dehydrogenase), so it does not lead to bacterial death.

When the population density is high, the dominant promoter increases the expression of the downstream gene Lux, which expresses a large amount of AHL protein. And the high concentration of AHL protein binds to LuxR and forms a dimer. This dimer has the opposite effect to the free LuxR protein, inducing the luxl promoter to express tetR and bga2 proteins, and failing to induce the tetR promoter to express GDH (Glutamate dehydrogenase), while at the same time the induced expression of the tetR protein also inhibits the expression of GDH. And GDH is a protein that must be expressed for cell growth, so inhibition of GDH expression will lead to bacterial death.

Therefore, this sub-module I can prevent the dysregulation of bacterial community homeostasis caused by excessive population density.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/buildb4-2.png)

In normal people, the concentration of butyrate is high, which leads to the high expression of tetR downstream of PLEE. No matter how the population density, tetR’s sufficient inhibition to the promoter PtetR prevents the expression of GDH in the downstream of PtetR. Besides, the effect is more obvious when the population density is high (because tetR can also be produced in the downstream of PluxI promoter), so the bacteria can not proliferate.

In people with constipation, the concentration of butyrate is low, so the amount of tetR expression is also low. If the population density is high, the PluxI promoter will be active, so enough tetR still can be produced, then bga2 expresses but GDH is suppressed. Only when butyrate and population density are both low that makes PtetR active due to insufficient inhibition of tetR production. Then the expression of downstream GDH is promoted and the number of bacteria grows.

# Cycle4:Test

Quorum sensing based on the AHL system has been widely used in synthetic biology since its proof-of-concept by You et al. in 2004. As for the butyrate sensing backbone and tetR backbone in module II, we have already covered them in our previous experiments, and both of them achieved the expected results. Complete tests will be carried out in the future.

# Cycle4:Learn

## Colonization ability is yet to be improved

The Quorum Sensing system keeps the population under control and allows the colony to multiply rapidly in certain conditions. An important prerequisite for this system is that the colonies are able to colonize over a period of time rather than pass through the gut. In cycle3, we have designed a mucin and tannin encapsulation with reference to the literature. According to the report, bacteria can colonize for several days after encapsulation[10]. So such an encapsulation can be used in conjunction with a Quorum Sensing system to achieve colonization and population control simultaneously over a period of time. However, how to further extend the colonization time to better demonstrate the constipation preventive role of our engineered bacteria is one of the directions of our future work.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/learnb4.png)

# Cycle4:Improve

## Summary of our design

In the above four cycles, with the support from human practice, we envisioned and designed double drug kill-switch, yogurt specialized kill-switch, encapsulation for acid resistance and Quorum Sensing system, but most of the tests are still at the stage of literature reference, and more experiments are yet to be carried out.

# References

1. Lee, J. W., Chan, C. T. Y., Slomovic, S., & Collins, J. J. (2018). Next-generation biocontainment systems for engineered organisms. Nature chemical biology, 14(6), 530–537.
2. Amitai, S., Yassin, Y., & Engelberg-Kulka, H. (2004). MazF-mediated cell death in Escherichia coli: a point of no return. Journal of bacteriology, 186(24), 8295–8300.
3. Jurėnas, D., Fraikin, N., Goormaghtigh, F., & Van Melderen, L. (2022). Biology and evolution of bacterial toxin-antitoxin systems. Nature reviews. Microbiology, 20(6), 335–350.
4. Stirling, F., Bitzan, L., O'Keefe, S., Redfield, E., Oliver, J. W. K., Way, J., & Silver, P. A. (2018). Rational Design of Evolutionarily Stable Microbial Kill Switches. Molecular cell, 72(2), 395.
5. Focht, C. M., & Strobel, S. A. (2022). Efficient quantitative monitoring of translational initiation by RelE cleavage. Nucleic acids research, 50(18), e105.
6. Renault, P., Gaillardin, C., & Heslot, H. (1989). Product of the Lactococcus lactis gene required for malolactic fermentation is homologous to a family of positive regulators. Journal of bacteriology, 171(6), 3108–3114.
7. Denayrolles, M., Aigle, M., & Lonvaud-Funel, A. (1994). Cloning and sequence analysis of the gene encoding Lactococcus lactis malolactic enzyme: relationships with malic enzymes. FEMS microbiology letters, 116(1), 79–86.
8. Lemme, A., Sztajer, H., & Wagner-Döbler, I. (2010). Characterization of mleR, a positive regulator of malolactic fermentation and part of the acid tolerance response in Streptococcus mutans. BMC microbiology, 10, 58.
9. Yang, X., Yang, J., Ye, Z., Zhang, G., Nie, W., Cheng, H., Peng, M., Zhang, K., Liu, J., Zhang, Z., & Shi, J. (2022). Physiologically Inspired Mucin Coated Escherichia coli Nissle 1917 Enhances Biotherapy by Regulating the Pathological Microenvironment to Improve Intestinal Colonization. ACS nano, 16(3), 4041–4058.
10. Chan, C. T., Lee, J. W., Cameron, D. E., Bashor, C. J., & Collins, J. J. (2016). 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nature chemical biology, 12(2), 82–86.
11. Gardner, T. S., Cantor, C. R., & Collins, J. J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature, 403(6767), 339–342.
12. Wang, X., Han, J. N., Zhang, X., Ma, Y. Y., Lin, Y., Wang, H., Li, D. J., Zheng, T. R., Wu, F. Q., Ye, J. W., & Chen, G. Q. (2021). Reversible thermal regulation for bifunctional dynamic control of gene expression in Escherichia coli. Nature communications, 12(1), 1411.
13. You, L., Cox, R. S., 3rd, Weiss, R., & Arnold, F. H. (2004). Programmed population control by cell-cell communication and regulated killing. Nature, 428(6985), 868–871.

Please click here to see our sensory and metabolic modeule

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{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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### Experiments

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Protocols

Experiment results

# 1. Configuration of LB solid medium

Prepare 200ml medium, weigh 2g of tryptone, 1g of yeast extract, 2g of NaCl, 3g of agar powder in a beaker, add 200ml of purified water, and stir with a glass rod until the solutes are dissolved. Adjust the pH to 7.0~7.4 with 5mol/L NaOH (usually 40μl), and then dispense the mixture into a 250ml conical flask. Seal the flask with sealing film, and then wrap it with double-layered kraft paper. Then steam-sterilize it under high pressure for 20 min at 121℃, 103.4kPa. Take it out and put it on the ultra-clean bench and cool it down to 50~55℃, and then add the antibiotic (kanamycin) to reach the final working concentration of 50μg/ml.

# 2. PCR amplification

1. Take an ice box, put dNTP, prime star enzyme, 5X prime star buffer, upstream and downstream primers, template plasmid, double distilled water, into the ice box and wait for melting. Take the corresponding number of microtubules and put them into the ice box.

2. Configure the reaction system as required by primestar. Note the amount of template used (concentration determines volume)

**Total volume: 50μL**

|  | Volume | Final concentration |
| --- | --- | --- |
| 5 X PrimeSTAR Buffer (Mg2+Plus) | 10μl | 1 X |
 dNTP Mixture (2.5mM each) | 4μl | 200μM each || Primer1 | 10-15pmol | 0.2-0.3μM |
| Primer2 | 10-15pmol | 0.2-0.3μM |
| Template | <200ng |  |
| PrimeSTAR HS DNA Polymerase (2.5U/μl) | 0.5ul | 1.25units/50μl |
| DdH2O | Up to 50 μl |  |

3. Turn on the PCR instrument and set up the program according to the reaction conditions.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/pcr2.png)

# 3. Electrophoretic verification of PCR results

1. Gel dispensing: weigh 0.35g agar powder, add 30ml 1X TAE into the flask, microwave heat for 2.5 min, then cool it down to 50℃. Take gelred from 4 degrees refrigerator, aspirate 3ul with a special gun for electrophoresis and add it with the gun tip sticking out under the liquid surface, rotate it slightly to mix well. Pour the liquid into the grid for dispensing gel, insert the comb and wait for solidification.

2. Sample preparation: After PCR, take out each reaction tube, take 5μl from each tube, add each into a new microtube, label it well, add 1ul 6X loading buffer to each tube, mix well.

3. Run the gel: take out the Marker from the -20℃ refrigerator, pull out the comb from the solidified gel, put it into the electrophoresis tank full of 1X TAE according to the direction, add the prepared samples into each gel well, and add 5ul of melted Marker, record the order of the samples in the wells. Run the gel at 150V constant voltage.

4. Observe under UV light and compare the position of the fluorescent bands with the Marker instructions.

5. Take Photographs.

# 4. DNA purification and recovery

1. Column equilibration: Add 500μl of equilibrium solution BL to the adsorption column CB2 (adsorption column in the collection tube) and centrifuge for 1 min at 12,000 rpm.

Centrifuge the column for 1 min, pour out the waste liquid from the collection tube and put the column back into the collection tube.

2. Cut a single target DNA strip from the agarose gel (remove as much excess as possible) into a clean centrifuge tube

3. Add an equal volume of solution PC to the gel block (if the gel weighs 0.1g and its volume can be considered as 100pl, add 100μl of PC solution. Use a gel cutter to cut 1% agarose gel, the weight of a single block is about 0.06g, the actual weight of the gel block is related to the concentration and thickness of the gel), water bath at 50℃ for about 10 min, during which time the centrifuge tube is constantly and gently up and down to ensure that the gel block is fully dissolved.

4. Add the solution obtained in the previous step to an adsorbent column CB2 (adsorbent column in a collection tube), centrifuge at 12,000rpm (~13,400xg) for 1 min, pour off the waste liquid in the collection tube, and place the adsorbent column CB2 in a collection tube.

5. Add 600μl of rinse solution PW to the adsorbent column CB2, centrifuge at 12,000rpm for 1 min, pour off the waste liquid in the collection tube, and place the adsorbent column CB2 into the collection tube.

6. Repeat step 5

7. Place the column CB2 in a collection tube and centrifuge at 12,000rpm for 2 min to remove as much rinse solution as possible. Leave the column at room temperature for a few minutes and dry thoroughly.

Note: Ethanol residue in the rinse solution will affect subsequent enzyme reaction (digestion, PCR, etc.) experiments.

8. Place the column CB2 into a clean centrifuge tube and add an appropriate amount of Elution Buffer EB to the center of the adsorbent membrane (if the recovered target fragment is >4kb, the Elution Buffer EB should be warmed up in a water bath at 65-70°C) and leave it at room temperature for 2 min. Centrifuge the column at 12,000rpm for 2 min and collect the DNA solution.

# 5. Enzymatic digestion of PCR products and templates

1. The enzymatic reaction system is as follows:

| Reaction System Composition | Volumes |
| --- | --- |
| DdH2O | Up to 50μl |
| 10x NE Buffer(cutsmart) | 5μl |
| HindIII | 1μl(Ensure that the glycerol concentration does not exceed 5% of the total reaction solution) |
| EcoRI | 1μl |
| Plasmid or PCR recovery product | 1μg (Adjust the volume according to the concentration) |

2. Put into the water bath and react at 37℃ for 15 min

3. Inactivate at 65℃ for 20 min, or use DNA Purification Recovery Kit for the recovery of the digested product.

# 6. Enzymatic reaction of fragment carriers

1. The molar ratio of carrier to fragment is approximately 1:3~4. Calculate the desired volume ratio based on concentration and fragment length.

2. Prepare the following reaction system on ice:

| Reaction System Composition | Volumes |
| --- | --- |
| Solution l | The sum of the volumes used for the carrier and the fragment, the 4~5X |
| Plasmid gel recovery products | X |
| PCR digestion of recovered products | 3~4X |

3. The reaction solution is collected to the bottom of the tube by brief centrifugation. React for 120 min at 16°C and may be repeat overnight at 4°C.

# 7. Conversion of enzyme-linked products and coating plates

1. Take the cloned receptor cells from the -80°C refrigerator and thaw them on ice.

2. Take 2-5μl of recombinant product and add it to 50μl of receptor cells, flick a few times to mix, ice bath for 30 min.

3. Heat-excite in 42℃ water bath for 30-45s.

4. Immediately place on ice to cool for 2-3 min.

5. Add 500μl LB medium (without antibiotics) and shake the bacteria for 1h at 37°C, 180rpm.

6. Preheat Amp-resistant LB plate solid medium in a 37°C incubator.

7. Centrifuge at 12000rpm for 1 min and discard 200-400μl of supernatant. Resuspend the bacteria with the remaining medium and spread it gently on the plate with a pasteurized tube.

8. Invert the culture in 37℃ incubator for 12-16h (first positive for 5 min to evaporate the water, then invert the culture).

# 8. Bacterial liquid amplification

1. Turn on the UV light of the aseptic table 10 minutes in advance. (Place the alcohol-wiped guns to be used into the table together).

2. Turn off the UV light and turn on the fluorescent light, wipe the table with alcohol and spray alcohol in the air, light the alcohol lamp and turn on the first level of air.

3. Put all items into the table and sterilize hands with alcohol.

4. Aspirate 8.3μl of kanamycin with a 10ul gun. Hold the gun in the right hand, take the test tube of LB liquid in the left hand, bake the mouth of the tube with the alcohol lamp fire for 2-3 seconds, remove the stopper with the right little finger and palm, tilt the test tube, hit the Amp into the inner wall of the mouth of the tube, and parallel the test tube so that the kanamycin will be immersed in the LB. Alcohol lamp flame grilled the mouth of the tube and plugged back the stopper.

5. Do the same for the other tubes.

6. Burn tweezers, take a white tip, open the lid of the plate, pick a single larger colony, the tip of the gun can be stained with bacteria.

7. Hold the forceps in the right hand and hold the tip of the gun with bacteria, take the test tube of LB liquid in the left hand, bake the mouth of the tube with alcohol lamp for 2-3 seconds, use the little finger and palm of the right hand to pull out the stopper and put the tip of the gun into the test tube. Alcohol lamp fire grilled the mouth of the tube and stuffed back the stopper.

8. Do the same operation for the other tubes.

9. When all tubes are done, put them together in a shaker, at 180rpm at 37℃ for 16-18h.

# 9. Western Blot

## (i) Protein sample preparation

1. Amplify the bacteria.

2. Discard medium and rinse.

3. Add lysis solution to lysate for 10 min (operated on ice), transfer to Ep tube and continue lysis for 20 min.

4. Centrifuge at 4℃ at 14000rpm for 15 min and remove the supernatant.

## (ii) SDS-PAGE electrophoresis

1. Washing the glass plate

2. Filling and Sampling

a) The glass panes are aligned and placed in the clamps and stuck tightly. After leak checking, clamp the glass plate vertically on the rack and prepare for filling.

b) Dispense 10% Separation Gel, add TEMED and shake well immediately before filling. When filling, use a 10ml gun to draw 5ml of glue along the glass, and wait until the surface of the glue rises to the height of the center line of the green band. Then add a layer of water to the glue.

c) When there is a line of refraction between the water and the glue, it means the glue has solidified. Wait for another 3 minutes for the glue to fully solidify, pour off the water on the layer of glue and absorb the water with blotting paper.

d) Prepare 5% gel concentrate, add TEMED and shake immediately to fill the gel. Fill the remaining space with concentrate and insert the comb into the concentrate. After the gel concentrate has solidified, gently pull out the comb by squeezing each side of the comb with both hands and holding it vertically upwards.

e) Rinse the gel concentrate with water and put it into the electrophoresis tank.

f) Add 1×Tris-glycine to the upper and lower tanks of the electrophoresis tank and top sample.

g) Connect the electrophoresis tank to the electrophoresis instrument. The upper tank is connected to the negative pole, the lower tank is connected to the positive pole, energized with a starting voltage of 80 V. When the bromochromophenol blue indicator reaches the junction of the separated gel and the concentrated gel, switch to a constant voltage of 120 V, and stop electrophoresis after running to the bottom layer.

## (iii) Transferring the membrane

1. Immerse the gel in transfer buffer and equilibrate for 10 min.

2. According to the size of the glue cut PVDF membrane and filter paper 6 pieces, into the transfer buffer equilibrium 10 min. (PVDF membrane need to be soaked in pure methanol first 1 min).

3. Assemble the transfer sandwich: sponge 3 layers of filter paper film, 3 layers of filter paper sponge, after each layer is placed, use a test tube to drive away the air bubbles. The gel is placed on the negative side (black side).

4. Place the transfer bath in an ice bath, put sandwiches (black side to black side), add transfer buffer, insert electrodes, 100V, 2h (current 0.25A).

5. After transferring the membrane, cut off the power and remove the hybridization membrane.

## (iv) Confinement and hybridization

1. Prepare 5% skimmed milk powder with TBST as solvent.

2. Soak the membrane in 5% skimmed milk powder, shake it at low speed on a shaker, seal it for 2h, discard 5% skimmed milk powder, shake it at high speed on a shaker with TBST and wash it for 5 min, repeat three times.

3. Primary antibody incubation: add primary antibody and incubate at 4℃ overnight.

4. Secondary antibody incubation: Recover primary antibody, wash with TBST for 15 min, repeat three times. Add the corresponding secondary antibody, incubate on shaking table at room temperature for 2h, wash with TBST for 15 min, repeat three times.

# 10. Plasmid extraction (plasmid extraction kit)

1. Take 1-4ml of the overnight culture, add it to a centrifuge tube, and centrifuge it for 1 min at 12,000 rpm using a conventional tabletop centrifuge, aspirating the supernatant as much as possible.

2. Add 150μl of Solution P1 to the centrifuge tube with the bacterial precipitate, and use a pipette or vortex shaker to thoroughly suspend the bacterial precipitate.

3. Add 150pl of Solution P2 to the centrifuge tube and gently turn it up and down 6-8 times to fully lyse the organisms.

Due to the use of TIANRed, the color of the solution will be a clear purple after the addition of solution P2 and thorough mixing. If there is a cloudy red color mixed with the purple color, the lysis is insufficient. Continue mixing until the color of the solution has completely changed to a clear purple.

4. Add 350μl of Solution P5 to the centrifuge tube and immediately and rapidly mix up and down 12-20 times, after mixing well enough, a flocclent precipitate will appear. Centrifuge at 12,000rpm (~13,400xg) for 2 min.

5. Transfer the supernatant collected in the previous step into the adsorbent column CP3 with a pipette (the adsorbent column is placed into the collection tube), taking care to try not to aspirate the precipitate. centrifuge at 12,000 rpm (~13,400xg) for 30 sec, pour off the waste liquid in the collection tube, and place the adsorbent column CP3 into the collection tube.

6. Add 300μl of rinse solution PWT to the adsorbent column CP3, centrifuge at 12,000rpm (~13,400xg) for 30 sec, pour off the waste liquid in the collection tube.

7. Place the adsorbent column CP3 into the collection tube and centrifuge at 12,000rpm for 1 min, in order to remove the residual rinse solution from the adsorbent column.

8. Place the adsorbent column CP3 in a clean centrifuge tube, add 50-100μl of elution buffer TB dropwise to the middle part of the adsorbent membrane, and centrifuge at 12,000rpm for 30 sec to collect the plasmid solution into the centrifuge tube.

# 11. Enzyme Labeling Instrument Operation

## (i) Preparation of initial liquid culture of bacteria

1. Prepare liquid LB medium, then autoclave (120°C, 41 min) and cool to room temperature.

2. Inoculate individual colonies of bacterial strains into 5ml of LB medium in culture tubes with kanamycin.

3. Note: If more replicates are needed, several colonies in separate culture tubes can be used.

## (ii) Pretreatment of 96-well plate and lid

1. Prepare 10% Triton X-100 in ethanol by mixing 1ml of Triton X-100 with 9ml of ethanol.

2. Pour 10ml of the diluted Triton X-100 (10% Triton X-100) to a new microplate lid.

3. Incubate the lid with diluted Triton X-100 for 15 seconds at room temperature.

4. Take a new 96-well plate and fill each well with 100% ethanol (400μl per well). Incubate for 15 minutes at room temperature.

5. Discard ethanol from the microplate wells and diluted Triton X-100 from the microplate lid.

6. Air-dry the microplate wells and microplate lid in a biosafety cabinet for 30 minutes.

7. Turn on the UV light of the biosafety cabinet to further sterilize the microplate wells and lid for 15 minutes.

## (iii) Sample Testing

| Plasmids | Sodium butyrate (mM) | Bacteria solution(μl) | Total volume (μl) | Replicates | Resistance |
| --- | --- | --- | --- | --- | --- |
| Pet-28a (sensor1) | 20 | 1 | 200 | 3 | KN+CM |
| Pet-28a (sensor1) | 10 | 1 | 200 | 3 | KN+CM |
| Pet-28a (sensor1) | 0 | 1 | 200 | 3 | KN+CM |

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/e11-2.png)

| Plasmids | Sodium butyrate (mM) | Bacteria solution(μl) | Total volume (μl) | Replicates | Resistance |
| --- | --- | --- | --- | --- | --- |
| Pet-28a (sensor2) | 20 | 1 | 200 | 3 | KN+CM |
| Pet-28a (sensor2) | 10 | 1 | 200 | 3 | KN+CM |
| Pet-28a (sensor2) | 5 | 1 | 200 | 3 | KN+CM |
| Pet-28a (sensor2) | 0 | 1 | 200 | 3 | KN+CM |

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/e11-4.png)

## (iv) Enzyme marker and software setup

1. Turn on the enzyme marker and open the Gen5 software.

2. Adjust the parameters and measure the fluorescence intensity and bacterial growth curve.

a. Plate type: 96-well plate.

b. Lid using: checked.

c. Temperature: 37°C.

d. Shake: orbital for 0:10.

e. Kinetic measurement for a desired total time (e.g., 16h, 24h, or 48h) and interval of 10 min with continuous orbital shaking.

f. Reading: absorption at 600 nm, fluorescence at excitation = 488 nm and emission = 525 nm.

## (v) Collating results

1. After completing data collection, remove the 96-well plate from the enzyme labeler.

2. Sterilize the sample and discard the microplate properly.

3. Save the results in the Gen5 software and export the data to an Excel spreadsheet file.

4. Close the Gen5 software and then turn off the enzyme marker.

# 12. Anti-bacterium-circles testing

1. Configure different concentrations of L-malic acid solution according to the effect concentration from previous literature: 25μM, 50μM, 75μM.

2. Dilute the bacterial solution with LB solid medium cooled to about 55°C at a ratio of 1:10, mix thoroughly and take 15ml into a sterile plate, shake gently, solidify and make a plate

3. Gently put the sterile Oxford cups into the previously made plates respectively, and then use a sterile pipette to suck 200μl of malic acid solution of different concentrations into the Oxford cups, and add 200μl of malic acid-free solution as the blank control. Three replicates were performed for each group.

4. Incubate the plates overnight at the temperature of around 37°C and measure the diameters using a circle of inhibition meter.

# 13. Growth curve measuring by enzyme-labeled instrument

## (i) Preparation of initial liquid culture of bacteria

1. Prepare liquid LB medium, then autoclave (120°C, 41 min) and cool to room temperature.

2. Inoculate individual colonies of bacterial strains into 5ml of LB medium in culture tubes with kanamycin.

3. Note: If more replicates are needed, several colonies in separate culture tubes can be used.

## (ii) Pretreatment of 96-well plate and lid

1. Prepare 10% Triton X-100 in ethanol by mixing 1ml of Triton X-100 with 9ml of ethanol.

2. Pour 10ml of the diluted Triton X-100 (10% Triton X-100) to a new microplate lid.

3. Incubate the lid with diluted Triton X-100 for 15 seconds at room temperature.

4. Take a new 96-well plate and fill each well with 100% ethanol (400μL per well). Incubate for 15 minutes at room temperature.

5. Discard ethanol from the microplate wells and diluted Triton X-100 from the microplate lid.

6. Air-dry the microplate wells and microplate lid in a biosafety cabinet for 30 minutes.

7. Turn on the UV light of the biosafety cabinet to further sterilize the microplate wells and lid for 15 minutes.

## (iii) Sample Testing

| L-malic acid(mM) | Mother liquor(μl) | Bacteria solution(μl) | Total volume (μl) | Replicates | Resistance |
| --- | --- | --- | --- | --- | --- |
| 0 | 0 | 200 | 200 | 3 | KN |
| 25 | 5 | 195 | 200 | 3 | KN |
| 50 | 10 | 190 | 200 | 3 | KN |
| 75 | 15 | 185 | 200 | 3 | KN |

**Mother liquor: 1M L-malic acid**

| Rhamnose(mM) | Mother liquor(μl) | Bacteria solution(μl) | Total volume (μl) | Replicates | Resistance |
| --- | --- | --- | --- | --- | --- |
| 0 | 0 | 200 | 200 | 3 | KN |
| 5 | 1 | 199 | 200 | 3 | KN |
| 10 | 2 | 198 | 200 | 3 | KN |
| 15 | 3 | 197 | 200 | 3 | KN |

**Mother liquor: 1M Rhamnose**

## (iv) Enzyme marker and software setup

1. Turn on the enzyme marker and open the Gen5 software.

2. Adjust the parameters and measure bacterial growth curves.

a. Plate type: 96-well plate.

b. Lid using: checked.

c. Temperature: 37°C.

d. Shake: orbital for 0:10.

e. Kinetic measurement for a desired total time (e.g., 16h, 24h, or 48h) and interval of 10 min with continuous orbital shaking.

f. Reading: absorption at 600 nm

## (v) Collating results

1. After completing data collection, remove the 96-well plate from the enzyme labeler.

2. Sterilize the sample and discard the microplate properly.

3. Save the results in the Gen5 software and export the data to an Excel spreadsheet file.

4. Close the Gen5 software and then turn off the enzyme marker.

# 14. Interval plate spreading

1. Prepare sufficient solid plate containing kanamycin.

2. Inoculate the engineering bacteria into 5ml LB liquid medium in the shaker at the temperature of around 37°C and take it out when the OD value is around 0.4-0.5.

3. Add a certain amount of malic acid into 5ml of LB liquid medium, making the concentration of malic acid to be 25mM, 50mM, 75mM. Continue to culture them in the shaker at the temperature of around 37°C.

4. Every 2h, spread the plate until the 12h, a total of 6 time points. Pre-measurement of OD value can be used to roughly determine dilution gradient. Each group contains three replicates.

5. Plate clony can be counted after 12-16h of incubation, and the counting is done by jingdian camera.

6. Analyze and process the data, and draw the killing curves using graphpad.

# 1. Validating the feasibility of metabolic module

Using untreated pET28a as a control group, we performed experiments such as WB, fluorescence intensity (OD600) measurement, and gray value analysis to verify the feasibility of the metabolic module pathway.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/metabolic.png)

**Fig. 1 |****TPH and TDC can be expressed by engineered bacteria and exert no effect on their proliferation.**
a.Original image of SDS-PAGE presenting the TPH and TDC expression, beat-actin as a control
b.Grayscale stripe extraction from the original image of SDS-PAGE
c.Histogram of grayscale values presenting the TPH and TDC expression
d.OD evaluation of the control and engineered bacteria throughout 36 hrs culture

# 2. Verifying the feasibility of the sensory module

The feasibility of our sensory module pathway was verified - the increase in butyrate concentration indeed induced an increase in the expression of tagged proteins in the engineered bacteria, i.e., it was verified that our engineered bacteria could indeed be more sensitive to the increase in butyrate concentration.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/sensor1.jpg)

**Fig. 2 |****Fluorescence Intensity(OD480/OD600) with 0mM, 10mM and 20mM butyrate cocultured in PpchA-pchA-PLEE1-EGFP engineered bacteria**
a.Fluorescence observation of PpchA-pchA-PLEE1-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate through fluorescence microscopic
b.Fluorescence intensity of PpchA-pchA-PLEE1-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate

# 3. Reverse thinking to verify the feasibility of the sensory module

The feasibility of our sensory module pathway was verified - a rise in butyrate concentration did induce an increase in the expression of the engineered bacterial tagged protein Cl, i.e., it was verified that our engineered bacterium was indeed more sensitive to the rise in butyrate concentration.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/sensor2.jpg)

**Fig. 3 |****Fluorescence Intensity(OD480/OD600) with 0mM, 5mM, 10mM and 20mM butyrate cocultured in PpchA-pchA-PLEE1-CI-Plam-EGFP engineered bacteria**
a.Fluorescence observation of PpchA-pchA-PLEE1-CI-Plam-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate through fluorescence microscopic
b.Fluorescence intensity of PpchA-pchA-PLEE1-CI-Plam-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate

# 4. Validating the feasibility of malic acid kill-switch circuit

Malic acid kill-switch is for in-vivo urgent containment, which means the patient can control the process as their wish by take in some malic acid with good flavor.

Pre-experimentation via zone of inhibition showed that malic acid addition can inhibit the bacteria growth. Then we carried out OD evaluation, which is unsatisfactory for OD evaluation cannot distinct living and dead individuals well. So we performed interval plate spreading at 2h, 4h, 6h, 8h, 10h, 12h under different concentrations of malic acid co-culture, which showed that malic acid addition can exert killing effect.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/malic-acid-kill-switch.jpg)

**Fig. 4 |****Malic acid addition exerted killing effect on engineered bacteria with malic acid kill switch circuit**
a.Anti-bacterium-circles testing as a pre-experimentation, different concentrations of malic acid treatment
b.Visualization of (a).
c.Growth curve measured by enzyme-labeled Instrument, co-cultured for 36 hrs with different concentrations of malic acid
d.Interval plate spreading counting results and visualization

Note: Since our sequence has a high number of reverse repeat sequences and the routes involving the toxin proteins are likely to kill the bacteria outright due to short circuit, we encountered numerous difficulties in constructing and extracting this plasmid. Future teams should carefully consult the sequence and construct the plasmid when designing similar kill switch circuit.

# 5. Validating the feasibility of rhamnose kill-switch circuit

Rhamnose kill-switch is for environment protection, that is to say, when bacteria is excreted outside and rhamnose is diluted, bacteria will die due to the kill-switch, which is trigger by low concentration of rhamnose.

Pre-experimentation via plate spreading showed that rhamnose addition or not has a significant effect on the survival of bacterial
and subsequent OD evaluation under different concentration of rhamnose co-culture for 36 hrs showed that groups with higher concentration of rhamnose grew better, which met our expectations.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/rhamnose-kill-switch.jpg)

**Fig. 5 |****Rhamnose addition maintains the growth viability of engineered bacteria with rhamnose kill switch circuit.**
a.Pre-experimental plate spreading results, with rhamnose addition or not.
b.Visualization of (a).
c.Growth curve measured by enzyme-labeled Instrument, co-cultured for 36 hrs with different concentrations of rhamnose

Although our results are largely as expected. However, in our results, no matter in the plate spreading or the growth curves, it can be observed that some of the engineered bacteria could not be killed despite the absence of rhamnose.
This is probably due to the insufficient expression of mazF in some individuals, and in the future, attempts could be made to replace the toxin proteins or introduce an amplifier to make up for this deficiency.
Meanwhile, it should be noted that since the OD curves could not distinguish the survival state of the bacteria, the downward trend could not be demonstrated, even though the bacteria were in a state of mass apoptosis in this period of time.

# 6. Validating the feasibility of tannin and mucin encapsulation

We produced a layer-by-layer encapsulation with tannin and mucin for bacteria and verified its ability to resist gastric juices by using SGF (simulated gastric juices).

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/encapsulation.jpg)

**Fig. 6 |****Plate spreading results of tannin and mucin multilayer encapsulated E. coli after co-cultured with simulate gastric fluid (SGF).**
a.Tannic acid mucin multilayer-coated E. coli and control E. coli were co-cultured with 25% and 50% concentrations of SGF for 5 min, with no significant difference due to either too strong or weak killing effect.
b.Setting up a gradient for exploration of SGF concentration and treatment time, and 10 min and 25% performed best.
c.Tannic acid mucin multilayer-coated E. coli and control E. coli were co-cultured with 25% concentration of SGF for 10 min with significant difference.
d.Counting the colonies in (c). and visualization

The results showed that the encapsulation only had an acceptable impact on the growth viability of Escherichia coli and exhibited significant protective effects when co-cultured with SGF. Therefore, the encapsulation scheme aligns closely with our expectations. However, encapsulated bacteria also suffer a lot by SGF, which means our protocol and process needs further refinement by paying attention to some details in the referred study.

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

Scroll To Explore

![]()

# Overview

Through one year's unremitting efforts, the circuit we designed was basically verified successfully, and the experimental data were further fitted and validated through modeling. We also made plans for the future product design, proposing the future treatment process.

---

* [**Experiment**
  Experiment results](#r1)
* [**Model**
  Model Results](#r2)
* [**Product**
  Product design and future plans](#r3)
* [**Future treatment**
  The envisioned process of treatment](#r4)
* ![糖豆人](https://static.igem.wiki/teams/4656/wiki/tangdouren.png)

  ![](https://static.igem.wiki/teams/4656/wiki/gototop.png)

# Experiment

## 1.Verifying the feasibility of the sensory module

The feasibility of our sensory module was verified - the increase in butyrate concentration indeed induced an increase in the expression of tagged proteins in the engineered bacteria, i.e., it was verified that our engineered bacteria could indeed be more sensitive to the increase in butyrate concentration.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/sensor1.jpg)

**Fig. 2 |****Fluorescence Intensity(OD480/OD600) with 0mM, 10mM and 20mM butyrate cocultured in PpchA-pchA-PLEE1-EGFP engineered bacteria**

## 2.Reverse thinking to verify the feasibility of the sensory module

The feasibility of our sensory module was verified - a rise in butyrate concentration did induce an increase in the expression of the engineered bacterial tagged protein Cl, i.e., it was verified that our engineered bacterium was indeed more sensitive to the rise in butyrate concentration.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/sensor2.jpg)

**Fig. 3 |****Fluorescence Intensity(OD480/OD600) with 0mM, 5mM, 10mM and 20mM butyrate cocultured in PpchA-pchA-PLEE1-CI-Plam-EGFP engineered bacteria**
a.Fluorescence observation of PpchA-pchA-PLEE1-CI-Plam-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate through fluorescence microscopic
b.Fluorescence intensity of PpchA-pchA-PLEE1-CI-Plam-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate

## 3.Validating the feasibility of metabolic module

Using untreated pET28a as a control group, we performed experiments such as WB, fluorescence intensity (OD600) measurement, and gray value analysis to verify the feasibility of the metabolic module.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/metabolic.png)

**Fig. 1 |****TPH and TDC can be expressed by engineered bacteria and exert no effect on their proliferation.**
a.Original image of SDS-PAGE presenting the TPH and TDC expression, beat-actin as a control
b.Grayscale stripe extraction from the original image of SDS-PAGE
c.Histogram of grayscale values presenting the TPH and TDC expression
d.OD evaluation of the control and engineered bacteria throughout 36 hrs culture

## 4.Validating the feasibility of malic acid kill-switch circuit

Malic acid kill-switch is for in-vivo urgent containment, which means the patient can control the process as their wish by take in some malic acid with good flavor.

Pre-experimentation via zone of inhibition showed that malic acid addition can inhibit the bacteria growth. Then we carried out OD evaluation, which is unsatisfactory for OD evaluation cannot distinct living and dead individuals well. So we performed interval plate spreading at 2h, 4h, 6h, 8h, 10h, 12h under different concentrations of malic acid co-culture, which showed that malic acid addition can exert killing effect.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/malic-acid-kill-switch.jpg)

**Fig. 4 |****Malic acid addition exerted killing effect on engineered bacteria with malic acid kill switch circuit**
a.Anti-bacterium-circles testing as a pre-experimentation, different concentrations of malic acid treatment
b.Visualization of (a).
c.Growth curve measured by enzyme-labeled Instrument, co-cultured for 36 hrs with different concentrations of malic acid
d.Interval plate spreading counting results and visualization

Note: Since our sequence has a high number of reverse repeat sequences and the routes involving the toxin proteins are likely to kill the bacteria outright due to short circuit, we encountered numerous difficulties in constructing and extracting this plasmid. Future teams should carefully consult the sequence and construct the plasmid when designing similar kill switch circuit.

## 5.Validating the feasibility of rhamnose kill-switch circuit

Rhamnose kill-switch is for environment protection, that is to say, when bacteria is excreted outside and rhamnose is diluted, bacteria will die due to the kill-switch, which is triggered by low concentration of rhamnose.

Pre-experimentation via plate spreading showed that rhamnose addition or not has a significant effect on the survival of bacterial
and subsequent OD evaluation under different concentration of rhamnose co-culture for 36 hrs showed that groups with higher concentration of rhamnose grew better, which met our expectations.

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/rhamnose-kill-switch.jpg)

**Fig. 5 |****Rhamnose addition maintains the growth viability of engineered bacteria with rhamnose kill switch circuit.**
a.Pre-experimental plate spreading results, with rhamnose addition or not.
b.Visualization of (a).
c.Growth curve measured by enzyme-labeled Instrument, co-cultured for 36 hrs with different concentrations of rhamnose

Although our results are largely as expected. However, in our results, no matter in the plate spreading or the growth curves, it can be observed that some of the engineered bacteria could not be killed despite the absence of rhamnose.
This is probably due to the insufficient expression of mazF in some individuals, and in the future, attempts could be made to replace the toxin proteins or introduce an amplifier to make up for this deficiency.
Meanwhile, it should be noted that since the OD curves could not distinguish the survival state of the bacteria, the downward trend could not be demonstrated, even though the bacteria were in a state of mass apoptosis in this period of time.

## 6.Validating the feasibility of tannin and mucin encapsulation

We produced a layer-by-layer encapsulation with tannin and mucin for bacteria and verified its ability to resist gastric juices by using SGF (simulated gastric juices).

![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/encapsulation.jpg)

**Fig. 6 |****Plate spreading results of tannin and mucin multilayer encapsulated E. coli after co-cultured with simulate gastric fluid (SGF).**
a.Tannic acid mucin multilayer-coated E. coli and control E. coli were co-cultured with 25% and 50% concentrations of SGF for 5min, with no significant difference due to either too strong or weak killing effect.
b.Setting up a gradient for exploration of SGF concentration and treatment time, and 10 min and 25% performed best.
c.Tannic acid mucin multilayer-coated E. coli and control E. coli were co-cultured with 25% concentration of SGF for 10 min with significant difference.
d.Counting the colonies in (c). and visualization

The results showed that the encapsulation only had an acceptable impact on the growth viability of Escherichia coli and exhibited significant protective effects when co-cultured with SGF. Therefore, the encapsulation scheme aligns closely with our expectations. However, encapsulated bacteria also suffer a lot by SGF, which means our protocol and process needs further refinement by paying attention to some details in the referred study.

# Model

| Model | Method or Equation | Results |
| --- | --- | --- |
| Host strain selection | bidirectional Mendelian randomization | E.coli was a safe and efficient host strain for our engineered bacteria. |
| E. coli growth | ![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/model-5.jpg) | Well fitted for prediction of E. coli intestinal concentrations. |
| Product production | ![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/model-16.jpg) | Well fitted for prediction of 5-HT intestinal concentrations. |
| Kinetic model of drug elimination | ![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/model-25.png) | Useful for personalizing dosing schedules and calculating steady-state concentrations in the gut. |
| Elimination model of E. coli | ![Gut-sweeper](https://static.igem.wiki/teams/4656/wiki/model-35r.jpg) | Gained the crucial parameter for prediction of E. coli elimination. |

# Product design and future plans

## **Step1 Optimize and test quorum sensing module**

In the course of the project, we hypothesized that the amount of engineered bacteria can be automatically controlled by the quorum sensing system of the bacteria themselves, and that the amount of engineered bacteria can be automatically maintained at the proper amount for a long time, thus performing the preventive, monitoring and therapeutic functions of engineered bacteria without manual control. We will then improve and test our quorum sensing module.

## **Step2 Improve the sensitivity of current modules**

In order to maintain the delicate balance of healing and intestinal homeostasis, the sensitivity of the sensory module, the efficiency of the metabolic module, and the stability of the quorum sensing module still require further improvements, calling for more detailed exploration in the future.

## **Step3 Integrate modules into acid-tolerant strains**

Due to the defective nature of the E. coli system, we intend to conduct further experiments in the future using suitable acidophilic strains (e.g. Lactobacillus acidophilus) as chassis bacteria.

## **Step4 Find a more suitable way for transportation and restoration**

We envisioned that the engineered bacteria we constructed would be added to foods such as yogurt. When people consumed these foods, the engineered bacteria would colonize their intestines and carry out their work. But how to maintain the survival of the engineered bacteria after transportation, restoration and consumption before they arrived at the intestines still needs to be explored in more detail.

## **Step5 Carry out experiments on animals**

Application of a medical product must be tested in animals. Due to conditions and time constraints, we were unable to conduct in vivo validation experiments. Subsequently, we plan to conduct more tests using acid-resistant bacteria and carry out in vivo experiments to validate the therapeutic effect in constipated mice model,to obtain more convincing experimental data in vivo.

# The envisioned process of treatment

Once the genetic machine has been successfully designed, we are ready to conduct further experiments in the future using acidophilus as the chassis bacteria due to the defective nature of the E. coli system. We expect to add engineered bacteria to foods such as yogurt, and when people consume these foods, our engineered bacteria will enhance their competitiveness in the intestines with the help of the quorum sensing module until they reach an appropriate population density. When possible constipation occurs, the engineered bacteria will sense the decreasing butyrate level and secrete serotonin to regulate intestinal function, thus promoting intestinal peristalsis, enhancing intestinal secretion, and finally relieving constipation. At the same time, when the bacteria are expelled along with the fecal, we do not need to worry about contaminating the environment because once the engineered bacteria are out of the environment at 37°C, the sophisticated kill switch circuit will exterminate them in time.

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