iGEM 2023 — Team hzau-china

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{% 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 %}
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## What should this page contain?

---

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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{% block title %}Engineering Success{% endblock %}
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Colonization Module
Blocking Module
NOT-Gate

This year, we have summited five new parts to the registry to prove our Engineering Success.

Colonization module

To make engineering bacteria obtain the capacity of adhesion, we decid to insert *hsa* from *Streptococcus gordonii str. Challis* into pET-28a(+) and transform the plasmid into *E. coli* BL21(DE3) by infusion cloning. Hsa can anchor to the cell wall and bind to sialic acid in the cat's mouth, helping our bacteria to adhere to the salivary pellicle. The *hsa* has a length of 6537 bp. To reduce the weight of the vector, we only selected the regions in *hsa* that can bind to sialic acid. After transforming pET-28a(+) with *hsa* into *E. coli* BL21(DE3), we intend to perform functional validation in terms of both biofilm formation ability and adhesion to saliva-coated cat tooth.

![](https://static.igem.wiki/teams/4645/wiki/csga-hsa/hsa-pcr-8-5.png)

**Figure 1.Gel electrophoretic map after transforming the constructed plasmid into E. coli BL21 (DE3).**

But after further literature review, we found that *hsa* is divided into five regions, NR1, SR1, NR2, SR2, and CWAD, with NR2 being the main salivary acid-binding site, SR2 acting as a molecular stalk and CWAD is the cell wall anchoring region. We only intercepted a portion of NR2, and don't need experimental verification to know that our genes cannot function in bacteria. So after literature references, we chosing *csgA* associated with biofilm from *E. coli* K-12. CsgA is a Curli amyloid fibrils protein secreted extracellularly to mediate host cell adhesion and contributes to biofilm formation. We intercepted the NR2 region of Hsa and linked it to the C-terminus of CsgA with the linker of (GGGGS)4, and linked the fusion gene to pET-28a(+) by homologous recombination, named *csgA-hsa*. And by SDS-PAGE analysis we identified the expression of CsgA-Hsa.

![](https://static.igem.wiki/teams/4645/wiki/csga-hsa/hlc-bl21-d3-8-22-1-1.png)

**Figure 2. Gel electrophoretic map after transforming the pET-28a(+) with csgA-hsa into E.coli BL21(DE3).**

![](https://static.igem.wiki/teams/4645/wiki/csga-hsa/csga-hsa-protein.jpg)

**Figure 3. SDS-PAGE analysis of CsgA-Hsa.**

After completing the vector construction and protein expression validation of CsgA-Hsa, we began adhesion ability on cat tooth and quantitative and qualitative of biofilm experiments.

We covered the induced bacterial debris on the saliva coated cat tooth, and after elution and antibody incubation, there was a significant difference in fluorescence intensity between the experimental group and the control group, fully demonstrating the ability of CsgA-Hsa to bind to sialic acid.

![](https://static.igem.wiki/teams/4645/wiki/csga-hsa/cattoothblot.png)

**Figure 4. The results ofadhesion ability on cat tooth.**

Incubating the induced engineering bacteria in a 96 well plate, giving them time to form a biofilm, then stain with crystal violeted and detected OD600 values using an enzyme-linked immunosorbent assay. The significant difference in results between the experimental group and the control group indicated that our engineered bacteria have formed a biofilm.

![](https://static.igem.wiki/teams/4645/wiki/csga-hsa/biofilm-formation.png)

**Figure 5. OD600 values after crystal violet staining.**

Cultivating and inducing engineered bacteria in a 6-well plate at 37°C, then eluting, fixing, and staining them. We observed the result with a fluorescence microscope after staining engineered bacteria. Under the induction of 0.1mM IPTG, the amount of biofilm formation was the highest and far exceeded that of other concentration groups and control groups.

![](https://static.igem.wiki/teams/4645/wiki/csga-hsa/pi.png)

**Figure 6. Fluorescence images of gradient-induced bacterial biofilm.**

For more details, please click the link below:[http://parts.igem.org/Part:BBa\_K4645003](http://parts.igem.org/Part%3ABBa_K4645003)

Blocking module

NeuA and NeuB are scFvs that we use rigid linker to fuse the light chain and heavy chain, and they have the ability to bind to Fel d 1 and prevent IgE from people who are allergic to cats engagement.

![](https://static.igem.wiki/teams/4645/wiki/feld/f-a-b.png)

**Figure 7. SDS-PAGE analysis of purifed FelD, NeuA and NeuB.**

![](https://static.igem.wiki/teams/4645/wiki/feld/antigen-result1.png)

**Figure 8. OD450-Concentration curve of block ability of FelD to IgE from allergic human donor sera or Sstandard IgE sample.**

To verify the blocking activity of our scFvs, we first expressed recombinant Fel d 1, FelD (Figure 7, A). The ability to bind to allergic IgE was also verified (Figure 8). Subsequently, we successfully expressed and purified NeuA and NeuB (Figure 7, B), and verified by blocking ELISA that they have blocking activity and better blocking ability when used together (Figure 9).

![](https://static.igem.wiki/teams/4645/wiki/neua-neub/block-result.png)

**Figure 9. The result of blocking ELISA. Values represent the effect of binding allergen and preventing IgE engagement.**

For more details, please click the link below:  [BBa K4645000](http://parts.igem.org/Part%3ABBa_K4645000), [BBa K4645001](http://parts.igem.org/Part%3ABBa_K4645001),[BBa\_K4645002](http://parts.igem.org/Part%3ABBa_K4645002)

NOT-Gate

At first, we intented to use VqmA and Pqtip to sense the concentration of bacteria. By SDS-PAGE analysis we identified the expression of VqmAphage.

![](https://static.igem.wiki/teams/4645/wiki/quorum-sensing/vqma-sds.jpg)

**Figure 10. SDS-PAGE analysis of VqmAphage**

However, Pqtip didn't work. We found that the sequence of qtip promoter contains more than one initiation codon. This may lead to the losing of efficiency of ECFP. So, we tried to delete sequence between eCFP and the last initiation codon and between eCFP and the penult one. Sad to say, we were not able to make this part work finally.

So, we use AHL quorum sensing system to regulate the circuit. 50 μL *E. coli* BL21(DE3) with 50μL luxR ([BBa\_K4115039](http://parts.igem.org/Part%3ABBa_K4115039)) + 50 μL *E. coli* BL21(DE3) and 50 μL luxR ([BBa\_K4115039](http://parts.igem.org/Part%3ABBa_K4115039)) + 50 μL *E. coli* BL21(DE3) with luxI ([BBa\_C0161](http://parts.igem.org/Part%3ABBa_C0161)) was respectively mixed with 5 mL LB medium. Then they were cultured in microplate reader 37°C, 220 rpm for 8 hours and detected OD600, fluorescence intensity (458 nm excitation light, 489 nm emission light) every 10 minutes.

![](https://static.igem.wiki/teams/4645/wiki/quorum-sensing/luxr-luxi.png)

**Figure 11. Fluorescence intensity in different value of OD600.**

Later, we built a circuit ([BBa\_K4645019](http://parts.igem.org/Part%3ABBa_K4645019)) to test whether Not-Gate could workor not. Blank E.coli BL21(DE3), engineered E.coli BL21(DE3) with this biobrick were cultured in microplate reader 37°C, 220 rpm for 10 hours and detected OD600, fluorescence intensity (458 nm excitation light, 489 nm emission light) every 10 minutes.

![](https://static.igem.wiki/teams/4645/wiki/quorum-sensing/luxr-tetr-chara.jpg)

**Figure 12. Changes of fluorescence intensity / OD600 in blank E.coli BL21(DE3), engineered E.coli BL21(DE3) with this biobrick over time.**

The result shows that in the early growth stage, transformed bacteria expressed amcyan fluorescent protein continually. When the group density reached the threshold value, the expression of amcyan fluorescent protein began to be blocked up.

For more details, please click the link below: [BBa\_K4645004](http://parts.igem.org/Part%3ABBa_K4645004), [BBa\_K4645005](http://parts.igem.org/Part%3ABBa_K4645005), [BBa\_K4645016](http://parts.igem.org/Part%3ABBa_K4645016), [BBa\_K4645019](http://parts.igem.org/Part%3ABBa_K4645019)

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## What should this page contain?

---

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

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

## Inspirations

---

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

{% endblock %}

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{% block title %}Results{% endblock %}
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![](https://static.igem.wiki/teams/4645/wiki/wet-lab/head/head-result1.png)

Overview
Engineered bacteria demonstration
Colonization & Blocking
Sterilization
Suicide
Reference

Overview

The goal of our project is to alleviate allergy symptoms caused by cats using the engineered *E. coli* Nissle 1917 which can neutralize antigen to colonize in cat mouth. Until now, we have confirmed the feasibility of our project in *E. coli* DH5α and BL21(DE3). As is described in our design page, our project has three core modules and a suicide module. The "Colonization" module and the "Blocking" module are connected by an NOT-Gate, which allow engineered bacteria to survive in the cat mouth for a long time and better achieve the prevention of allergy to cat. The "Sterilization" module ensure the health of cat mouth. The final product is a probiotic nutritional supplement.

In order to prove our concept, we have conducted many experiments in the last 3 months after entering the lab. Most of the core modules of our project have been validated so far. However, due to time and equipment constraints, our project has not yet been validated on rats. With the preliminary experimental results available so far, we have determined that our project is practical and feasible for further optimization and extension in the future.

Demonstration of engineered bacteria

The primary experiments were implemented in common *E. coli* strain DH5α or BL21(DE3). The "Colonization" module can produce CsgA-Hsa which help engineered bacteria adhere to tooth. Then the "Blocking" module can express two scFvs which can bind antigens and prevent IgE engagement. Moreover, The "Sterilization" module can function better to prevent *S. mutans* from growing by expressing ClyR. Around these core components, we have demonstrated our engineered bacteria through experiments and other methods.

![](https://static.igem.wiki/teams/4645/wiki/project/design/111circuit.png)

**Figure 1. The outline of genetic circuit.**

Colonization & Blocking

# Colonization

In order to ensure that the engineered bacteria can stay in the cat's oral cavity for a period of time to fulfill its role, our first concern is the bacterial colonization ability. After literature references, we chose *csgA* associated with biofilm from *E. coli* K-12 and *hsa* associated with adhesion protein from *Streptococcus gordonii str.Challis*. CsgA is a Curli amyloid fibrils protein secreted extracellularly to mediate host cell adhesion and contributes to biofilm formation, which promotes bacterial resistance to environmental stressors. Hsa anchors to the cell wall and binds to salivary acids in the cat's mouth, helping our bacteria to adhere to the salivary pellicle.

To balance the biofilm forming ability of CsgA and the salivary acid adhesion ability of Hsa, we designed the CsgA-Hsa fusion protein, the specific information of this component is at (http://parts.igem.org/Part: BBa\_K4645003). Theoretically, the engineered bacteria secrete the fusion protein extracellularly, the amyloid fibrillar protein CsgA portion of the fusion protein accumulates around the bacteria to form a biofilm, and Hsa firmly binds the salivary acid molecule. The Hsa is divided into five regions, NR1, SR1, NR2, SR2, and CWAD, with NR2 being the main salivary acid-binding site, SR2 acting as a molecular stalk and CWAD is the cell wall anchoring region [1]. We intercepted the NR2 region of Hsa and linked it to the C-terminus of CsgA with the linker of (GGGGS)4 [2].

Using different concentrations of IPTG to induce *E. coli* BL21(DE3) containing the target gene, and then incubate the target protein with cat tooth to detect the fluorescence intensity of cat tooth [3].The results showed that the fluorescence intensity of the experimental group added with 0.1 mM IPTG and 1 mM IPTG was significantly different from the two control groups and other concentration induction groups (**Figure 2**). We repeated this experiment several times and obtained the same results. From this, it is determined that the fusion protein CsgA-Hsa indeed has an adhesive effect on saliva coated cat tooth.

![](https://static.igem.wiki/teams/4645/wiki/csga-hsa/cattoothblot.png)

**Figure 2. The results of Cat tooth blot.**Control1 represents a blank control without the addition of protein fragmentation solution, Control2 represents the negative control of the broken solution of bacteria that were not transformed into the plasmid.

After *E. coli* BL21(DE3) was induced with 0.1 mM IPTG, the OD600 values of the group induced by IPTG showed significant differences compared to the other two groups [4]. We can determine that the biofilm formation ability of the engineered bacteria containing the target protein is very significantly different from the two control groups at an inducible concentration of 0.1 mM IPTG, which confirms that *CsgA-Hsa* indeed enhances the formation of bacterial biofilm.

![](https://static.igem.wiki/teams/4645/wiki/csga-hsa/biofilm-formation.png)

**Figure 3. The results of induction with IPTG after 10 hours.**Because the difference between the experimental group and the two control groups was too large, the Y-axis is expressed in logarithmic format.

After induction and staining with IPTG with gradients of 0 mM, 0.1 mM, 0.5 mM, and 1 mM, the samples were observed under a fluorescence microscope [5]. Under all concentrations of induction, the biofilm formation effect of 0.1 mM IPTG was the best, with significant differences between other groups, and the biofilm formation effect was inversely proportional to the IPTG concentration between 0.1-1 mM IPTG. The results of this experiment are consistent with those of Cat dental imprinting, and both are optimal under conditions induced by 0.1 mM IPTG.

![](https://static.igem.wiki/teams/4645/wiki/csga-hsa/ad-fluorescence.jpg)

**Figure 4. Fluorescence images of gradient-induced bacterial biofilm.**(A) 0.1 mM IPTG-induced, viewed under fluorescence microscope at 10x (B) 0.5 mM IPTG-induced, viewed under fluorescence microscope at 10x (C) 1 mM IPTG-induced, viewed under fluorescence microscope at 10x (D) 0 mM IPTG-induced, viewed under fluorescence microscope at 10x (E) Medium with uninoculated bacteria, viewed under fluorescence (F) PI (propidium iodide) stained slides at 4 degrees Celsius, then used for fluorescence microscopy.

# Blocking

The function of "Blocking" module is to produce and secrete scFvs (NeuA amd NeuB) to bind Fel d 1 and prevent IgE from people who are allergic to cats engagement. Then preventing allergy reaction [6,7].

![](https://static.igem.wiki/teams/4645/wiki/feld/f-a-b.png)

**Figure 5. SDS-PAGE analysis of purifed FelD, NeuA and NeuB.**

To test this module, firstly we need to purify FelD expressed by *E. coli* BL21(DE3) and verify it can bind to IgE from people who are allergic to cats. By SDS-PAGE analysis we identified the expression of FelD which can be seen in lane 2 (**Figure 5, A**). To verify the ability of FelD bind to IgE from people who are allergic to cats, FelD was coated and IgE from allergic human donor sera or strandard IgE was incubated at 37℃ for 90 min. Then we Added biotin-labeled IgE antibody and HRP-conjugated Streptavidin. Finally we measured absorbance at 450 nm. Absorbance value present the effect of FelD bind to IgE. So the result of ELISA verified that our FelD has the ability to bind IgE from people who are allergic to cats (**Figure 6**). Additionally, we also tested the binding of IgE to different concentrations of FelD.

![](https://static.igem.wiki/teams/4645/wiki/feld/antigen-result1.png)

**Figure 6. OD450-Concentration curve of block ability of FelD to IgE from allergic human donor sera or Sstandard IgE sample.**

Subsequently, we verified whether NeuA, NeuB, and the combination of NeuA and NeuB had a blocking effect on FelD and IgE from people who are allergic to cats. Firstly, the result of SDS-PAGE analysis determined that NeuA and NeuB were expressed and purified and we can see them in lane and lane (**Figure 5, B**). Then through blocking ELISA, we found that the blocking effect of NeuA and NeuB on FelD is limited when they are used alone, and the blocking effect is very obvious when NeuA and NeuB are combined to FelD (**Figure 7**). The specific scheme of this blocking ELISA is to coated FelD on Microtiter plates and incubate different scFvs combinations at 37℃. The IgE from allergic human donor sera IgE from allergic human donor sera and then biotin-labeled IgE antibody and HRP-conjugated Streptavidin were added successively. Finally we measured absorbance at 450 nm. Absorbance value present the effect of FelD bind to IgE. Therefore, we confirm "Blocking" module can work.

![](https://static.igem.wiki/teams/4645/wiki/neua-neub/block-result.png)

**Figure 7. The result of blocking ELISA. Values represent the effect of binding allergen and preventing IgE engagement.**

# QS system

*Vibrio cholerae* uses a quorum-sensing (QS) system composed of the autoinducer 3,5-dimethylpyrazin- 2-ol (DPO) and receptor VqmA, which together bind to Promoter qtip and activate transcription [8]. According to reports in the literature, we found that *E. coli* itself can produce DPO [9], and the concentration of DPO is positively correlated with the amount of E. coli. We made use of this QS system to realize regulation of downstream circuit. To make sure this system could work as expected, we built this circuit (**Figure 8**) and tramsformed into E.coli.

![](https://static.igem.wiki/teams/4645/wiki/quorum-sensing/v-tus.png)

**Figure 8. The circuit we built to make sure it could work as expectedand.**

Firstly, we need to make that VqmAphage could be expressed in *E.coli*. By SDS-PAGE analysis we identified the expression of VqmAphage.

![](https://static.igem.wiki/teams/4645/wiki/quorum-sensing/vqma-sds.jpg)

**Figure 9. SDS-PAGE analysis of VqmAphage.**

We inserted eCFP reporter gene behind qitp promoter (**Figure 10**) to verify whether qtip promoter could work as expected in *E. coli* BL21(DE3). However, bacteria that have transformed this plasmid never shown any fluoresce, even though the sequence didn’t mutate. Later, we found that the sequence of qtip promoter contains more than one initiation codon. This may lead to the losing of efficiency of ECFP. So, we tried to delete sequence between eCFP and the last initiation codon and between eCFP and the penult one. Sad to say, we were not able to make this part work finally.

![](https://static.igem.wiki/teams/4645/wiki/quorum-sensing/cha-pqtip.png)

**Figure 10. The circuit to verify whether qtip promoter could work as expected.**

So, we implemented the alternative plan: AHL quorum-sensing system. However, compared to the originally designed system, it lacks the same level of specificity. Thanks ShanghaiTech-China for offering BBa\_K4115039, BBa\_K4115040 to us. So, in the construction of Not-Gate, we use AHL quorum-sensing system to realize regulation.

# NOT-Gate

When the number of engineered bacteria is insufficient, bacteria will express Hsa-CsgA, which is in the downstream of TetA promoter. With the growth of bacteria, AHL accumulates. Then LuxR will combine to Lux promoter and activate transcription of TetR, NeuA and NeuB. TetR will represses the expression of Hsa-CsgA.

![](https://static.igem.wiki/teams/4645/wiki/quorum-sensing/not-gate/not-gate-comp.png)

**Figure 11. The mechanism of NOT-Gate.**

To verify whether NOt-Gate could work as expected, we built this circuit (**Figure 12**).

![](https://static.igem.wiki/teams/4645/wiki/quorum-sensing/not-gate/not-gate-veri.png)

**Figure 12. The circuit for functional verification of Not-Gate.**

Blank *E.coli* BL21(DE3), engineered *E.coli* BL21(DE3) with this biobrick were cultured in microplate reader 37°C, 220 rpm for 10 hours and detected OD600, fluorescence intensity (458 nm excitation light, 489 nm emission light) every 10 minutes.

![](https://static.igem.wiki/teams/4645/wiki/quorum-sensing/luxr-tetr-chara.jpg)

**Figure 13. Changes of fluorescence intensity / OD600 in blank *E.coli* BL21(DE3), engineered *E.coli* BL21(DE3) with this biobrick over time.**

The result shows that in the early growth stage, transformed bacteria expressed Amcyan fluorescent protein continually. When the group density reached the threshold value, the expression of Amcyan fluorescent protein began to be blocked up.

This means that the Not-Gate we built worked successfully.

Sterilization

We learned that *Streptococcus mutans* has been recognized as one of the principle causative agents of dental caries [10]. The purpose of this module is to prevent cariogenic *S. mutans* from utilizing the biofilms formed by engineered bacteria on tooth.

We use the chimeric lysin (ClyR), which is the first lysin that demonstrates activity against the dominant dental caries-causing pathogen [11]. The circuit diagram of the module is presented below.

![](https://static.igem.wiki/teams/4645/wiki/project/design/ss-circuit.png)

**Figure 14. The outline of genetic circuit.**

The *E.coli* BL21(DE3) strain containing plasmid pET28a-ClyR, originated from Hubei Jianxia Laboratory. *E. coli* strain was cultured to an optical density (OD600) of ~0.6, induced by adding IPTG to 0.2 mM, and allowed to grow overnight at 16℃. Purification was performed following the instructions of Ni2+-affinity chromatography. To allow ClyR to be secreted out, the protein using the OmpA signal peptide was purified following the same procedure. The following is the purification result of the two proteins.

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/clyr/clyr5.png)

**Figure 15. SDS-PAGE analysis of ClyR and OmpA-ClyR.**

Next, to determine the dose dependence and time dependence of ClyR's lytic activity, *S. mutans* UA159 was resuspended to an optical density (OD600) of ~0.8 using PBS buffer. In a 96-well plate, we sequentially added 35 μL of ClyR solution at different concentrations and 165 μl of bacterial suspension. The OD600 was continuously measured using a Synergy H1 microplate reader under constant shaking at 37℃ for 1 hour. The experimental results are shown in the figure below.

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/clyr/clyr2.jpg)

**Figure 16. Variation curve of OD of *S. mutans* UA159 treated with different ClyR concentrations.**

As shown in the above figure, ClyR demonstrates a certain level of antibacterial activity, which becomes increasingly significant as its concentration increases.

In order to dynamically regulate the expression of ClyR in response to environmental changes, we aimed to introduce the quorum sensing two-component system from *S. mutans* into the engineered bacteria. When the concentration of CSP produced by *S. mutans* reaches a certain threshold, it activates the *nlmC* promoter, thereby initiating downstream ClyR expression [12].

Suicide

To ensure the safety of engineered bacteria, we designed a suicide module to kill the bacterial strains both in the external environment and in a cat's digestive tract, avoiding potential leakage of engineered bacteria.

The suicide circuit contains three main gene pathways: temperature-sensitive pathway, pH-sensitive pathway, and toxin-antitoxin pathway.

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/suicide-model.jpg)

**Figure 17. Suicide circuit design diagram.**

# Temperature-sensitive suicide

The temperature-sensitive pathway can detect temperature changes. When engineered bacteria leak out into the external environment, the temperature-sensitive pathway takes effect, expressing the toxin protein RelE while expressing CI protein to shut down the expression of the antitoxin protein MntA. The circuit design is shown in the diagram. We performed targeted validation of the key elements contained in the module.

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/temperature1.jpg)

**Figure 18. Temperature-sensitive suicide.**

## 1. Functional validation of RNA thermosensor

To validate the function of the RNA thermosensor, we used two methods to experimentally verify it. In Method 1, we placed the Amcyan protein after the RNA thermosensor in the PUC57 plasmid and expressed the plasmid in *E.coli* DH5α. After culturing for 4 hours continuously at 36°C, 26°C and 16°C respectively, we detect the relative fluorescence intensity.

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/rna2-1.jpg)

**Figure 19. Analysis of Differential Relative Fluorescence Intensity under Four Hours of Different Temperature Induction Conditions.**

In order to examine the possible effects of RNA leakage at 36°C on toxin protein expression and bacterial growth, In Method 2, We referred to the verification method of iGEM21\_HZAU-China [BBa\_K3733043](http://parts.igem.org/Part%3ABBa_K3733043) to verify the RNA thermosensor. For more information you can see here: [BBa\_K3733011](http://parts.igem.org/Part%3ABBa_K3733011).

The experimental results are shown in **Figure 20 and Figure 21**.

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/rna2-2.jpg)

**Figure 20. Bacterial OD600 over time.**

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/img-20231007-1520221.jpg)

**Figure 21. Phenotypic result comparison chart.**

Both verification methods successfully validated that the RNA thermosensor can function and express proteins at 26°C or lower temperatures. However, the experimental results also showed that there was some leakage from the RNA thermosensor at 36°C, which may lead to some adverse effects.

## 2. Functional validation of RelE toxin protein

In our experiment, we constructed a plasmid vector with RelE toxin protein connected after the lactose promoter and introduced it into *E.coli* BL21(DE3). IPTG was used to induce expression and OD600 was detected continuously for 4 hours. Samples were taken every 30 minutes, diluted and plated to determine viable cell counts.

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/rele2-1.jpg)

**Figure 22. Bacterial OD600 over time.**

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/rele2-2-ab.jpg)

**Figure 23. Viable cell counts over time.**

Since RelE is a weak toxin protein, as shown in **Figure 22**, the OD600 detection showed obvious inhibition of bacterial growth after induced expression, which delayed the log phase, but the bacteria could still grow in the later phase. The viable cell count method also produced significant results. As shown in **Figure 23**, viable cell counts decreased rapidly within one hour after induction, exhibiting significant growth inhibition, which demonstrated the bacteriostatic effect of this toxin.

# Acid-sensitive suicide and Toxin-antitoxin system

The pH-sensitive circuit can detect pH changes when engineered bacteria enter a cat's digestive tract and take effect, expressing CI protein to inhibit the expression of antitoxin protein MntA. At the same time, it expresses the two-component system RstAB. RstB can sense the decrease in pH to phosphorylate RstA, and RstA can increase the activity of the acid-induced promoter Pasr [13], thus quickly increasing the expression of CI protein.

The toxin protein HepT and antitoxin protein MntA form a toxin-antitoxin pathway, where the expression of MntA protein is inhibited by CI protein.The circuit design is shown in the diagram below. We performed targeted validation of the key elements contained in the module.

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/ph.jpg)

**Figure 24. Acid-sensitive suicide and Toxin-antitoxin system.**

## 1. Charaterization of asr promoter with fluorescence intensity measurement

To validate the promoter activity and its activation range in response to pH, we connected Amcyan protein after this promoter and introduced it into *E. coli* BL21(DE3). For more information you can see here: BBa K1231000. The experimental results are shown below.

The experimental results are shown in the **Figure 27. and Figure 28**

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/pasr2-23.jpg)

**Figure 25. Relative fluorescence intensity over time under different pH induction.**

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/pasr2-1.jpg)

**Figure 26. Differential analysis of relative fluorescence intensity after 4 hours of induction under different pH.**

As shown in **Figure 25**, Pasr has almost no expression under pH 7-8 conditions, but begins low-dose expression at pH 6 and gradually increases as the pH decreases, reaching a peak at pH = 5, then maintaining a relatively low expression level as the pH continues to decline. As shown in **Figure 26**, we selected the final values for differential analysis, which showed extremely significant differences between pH 6, pH 5 and the control group. This experiment validated that this promoter has almost no expression under pH 7-8 conditions, consistent with our project design expectations. Pasr can be used to activate the suicide circuit in the gastric environment of cats to cause engineered bacteria death, thus avoiding potential hazards from leakage.

## 2. Functional validation of HepT toxin protein

We referred to the method of verifying Hept by iGEM21\_HZAU-China BBa\_K3733010. To verify the cytotoxicity of HepT, we connected the HepT toxin protein after the lactose promoter and transferred it to *E. coli* BL21(DE3) using pET-28a(+) vector.

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/hept1-1.jpg)

**Figure 27. Bacterial OD600 over time**

As shown in **Figure 27**, the toxin protein HepT has a very obvious bactericidal effect, and this effect is dose-dependent, correlating with the induction concentration of IPTG within a certain range.

## 3. HepT-MntA toxin-antitoxin-CI-PR System

We constructed a circuit to validate the function of the toxin-antitoxin system (HepT/MntA) and the CI-PR system.We constructed the CI protein downstream of the LacI promoter, and constitutively expressed HepT, and connected the antitoxin protein MntA downstream of a promoter repressed by the CI protein, using the pet28a plasmid transformed into *E. coli* BL21(DE3).

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/lacimnta2.jpg)

**Figure 28.Circuit design**

Since the toxin protein HepT is highly toxic, and a strong promoter J23119 and strong RBS B0034 were used, if MntA cannot function, the bacteria transformed with this plasmid should grow slowly or even not survive. But as shown in Figure 29., the bacteria grew well on the plates.We also cultured the *E. coli* strain to OD600 = 0.4-0.6, induced with 1.0mM IPTG, and allowed continuous growth for 4 hours at 37°C. In 96-well plates, by comparing the OD600 between the experimental group and control group using the Synergy H1 enzyme-labeled tester, it displayed the function of the antitoxin protein MntA. The experimental results are shown in Figure 30..

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/cimnta.jpg)

**Figure 29.Bacterial culture plates**

![](https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/mnta.jpg)

**Figure 30.Bacterial OD600 over time**

Through analysis of the experimental results, we can find that MntA plays an important role in maintaining the survival of bacteria with constitutive expression of HepT. We also successfully verified the availability of the CI-PR (PCI) system. We also found the bacteria in the control group grew slightly slower than normal bacteria, which may be due to low-dose leakage from the LacI promoter.

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