iGEM 2023 — Team ibowu-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 %}

## 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)

{% endblock %}

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

# Engineering

* Home
* Project
* Engineering

## Engineering success

In iGEM 2023, iBowu\_China had deep feelings about environmental pollution. We have saw many of the shocking numbers and pictures caused by environmental problem, which has been severely affecting our own lives. Although human beings invented advanced technology and great civilization, we have neglected to protect the natural environment. Pyrethroid accumulation, as a typical example, has caused significant threat to beneficial insects in aquatic ecosystems. So, how can we solve the problem?
 This year, iBowu\_China carefully searched papers and noticed some of the natural enzymes can be used in pyrethroid degradation, which further lead us to design the new project using synthetic biology approach to biodegrade pyrethroids.

## [BBA\_K4690000](http://parts.igem.org/Part%3ABBa_K4690000)

A paper published on scientific reports mentioned the EstPS1 gene, which encodes a novel carboxylesterase of Pseudomonas synxantha PS1 isolated from oil well-produced water, showed high biodegradability towards pyrethroid pesticides.

### We used the following usual procedure to do the experiments:

1. Verification of the sequence. This part sequence we submitted was come from Pseudomonas synxantha PS1. We downloaded the sequence from NCBI and in order to be suitable for bacterial expression, we asked a biology company to undergo codon optimization and synthesize the target sequence.
2. We then constructed it into a pET28(+) plasmid and transformed the plasmids into E. coli BL21(DE3) strains.
3. After cultured on LB solid medium (Kanamycin) for 12 h, we picked a single colony into 4 ml LB medium (Kanamycin) and the mix was then shaken at 37℃ until OD600 = 0.6.
4. In order to set proper control, we divided the bacteria solution into three parts, for the first part, we added 0.5 mM IPTG and cultured in 16℃ for 20 h; the second part was added with 0.5 mM IPTG as well but cultured in 37℃ for 4 h. The last one added nothing to serve as a control.
5. When the expression process was finished, we centrifuged the bacterial solution at 12000 rpm, and the precipitation was resuspended with RIPA buffer to lysate bacteria, we also added loading buffer to heat at 96℃ for 10 min, followed by SDS-PAGE and Coomassie brilliant blue staining for expression test.
According to our SDS-PAGE result, protein EstPS1 can be expressed but precipitated in the inclusion body. In order to confirm the band we pointed out indicated our target proteins, we did an interview with our teacher, who suggested western blot assays to provide evidence, so we asked for collaboration and the following are results we got, which provided us positive results.

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690000-silver-1.png)

In the figure, the left panels are SDS-Page results obtained by us. The right panels are Western Blot results obtained by our external help (Corresponding SDS-Page runs weren’t shown).
16C-1 means the bacteria was cultured at 16 degree Celsius and the supernatant was used for the test. 16C-2 means the 16 degree Celsius culture and the precipitation was used. 37C-1 means the bacteria was cultured at 37 degree Celsius and the supernatant was used for the test. 37C-2 means the 37 degree Celsius culture and the precipitation was used.
We then wondered whether we could promote the solubility of EstPS1 through condition adjustment, so we firstly fixed the induction time and IPTG concentration, then set a series of temperature gradient. However, the SDS-PAGE result suggested that no matter what the temperature is, EstPS1 is left in the inclusion body.

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690000-silver-2.png)

The next experiment we did was to fixed the induction temperature and IPTG concentration, then set a series of time gradient. Luckily, this time the SDS-PAGE result suggested EstPS1 was expressed under 16℃, we observed little soluble bands after 3 h induction.

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690000-silver-3.png)

## [BBa\_K4690001](http://parts.igem.org/Part%3ABBa_K4690001)

Pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112, could not only degrade pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyze rho-nitrophenyl esters of various fatty acids, indicating EstP as an esterase with broad substrates.

### We used the following usual procedure to do the experiments:

1. Verification of the sequence. This part sequence we submitted was come from Klebsiella sp. strain ZD112. We downloaded the sequence from NCBI and in order to be suitable for bacterial expression, we asked a biology company to undergo codon optimization and synthesize the target sequence.
2. We then constructed it into a pET28(+) plasmid and transformed the plasmids into E. coli BL21(DE3) strains.
3. After cultured on LB solid medium (Kanamycin) for 12 h, we picked a single colony into 4 ml LB medium (Kanamycin) and the mix was then shaken at 37℃ until OD600 = 0.6.
4. In order to set proper control, we divided the bacteria solution into three parts, for the first part, we added 0.5 mM IPTG and cultured in 16℃ for 20 h; the second part was added with 0.5 mM IPTG as well but cultured in 37℃ for 4 h. The last one added nothing to serve as a control.
5. When the expression process was finished, we centrifuged the bacterial solution at 12000 rpm, and the precipitation was resuspended with RIPA buffer to lysate bacteria, we also added loading buffer to heat at 96℃ for 10 min, followed by SDS-PAGE and Coomassie brilliant blue staining for expression test.
According to our SDS-PAGE result, protein EstP can be expressed but precipitated in the inclusion body. In order to confirm the band we pointed out indicated our target proteins, we did an interview with our teacher, who suggested western blot assays to provide evidence, so we asked for collaboration and the following are results we got, which provided us positive results.

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690001-silver-1.png)

We then wondered whether we could promote the solubility of EstP through condition adjustment, so we firstly fixed the induction time and IPTG concentration, then set a series of temperature gradient. We found although EstP also precipitated in the inclusion body, but 22℃ expression expression produced more soluble protein.

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690001-silver-2.png)

The next experiment we did was to fixed the induction temperature and IPTG concentration, then set a series of time gradient. This time we showed more soluble EstP protein were produced after 24 h induction under the temperature of 22℃.

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690001-silver-3.png)

## [BBa\_K4690002](http://parts.igem.org/Part%3ABBa_K4690002)

EstA-associated biodegradation of pyrethroids was confirmed in 2019, which used the key degrading gene estA and related carboxylesterase EstA from Bacillus cereus BCC01.

### We used the following usual procedure to do the experiments:

1. Verification of the sequence. This part sequence we submitted was come from Bacillus cereus BCC01. We downloaded the sequence from NCBI and in order to be suitable for bacterial expression, we asked a biology company to undergo codon optimization and synthesize the target sequence.
2. We then constructed it into a pET28(+) plasmid and transformed the plasmids into E. coli BL21(DE3) strains.
3. After cultured on LB solid medium (Kanamycin) for 12 h, we picked a single colony into 4 ml LB medium (Kanamycin) and the mix was then shaken at 37℃ until OD600 = 0.6.
4. In order to set proper control, we divided the bacteria solution into three parts, for the first part, we added 0.5 mM IPTG and cultured in 16℃ for 20 h; the second part was added with 0.5 mM IPTG as well but cultured in 37℃ for 4 h. The last one added nothing to serve as a control.
5. When the expression process was finished, we centrifuged the bacterial solution at 12000 rpm, and the precipitation was resuspended with RIPA buffer to lysate bacteria, we also added loading buffer to heat at 96℃ for 10 min, followed by SDS-PAGE and Coomassie brilliant blue staining for expression test.
According to our SDS-PAGE result, protein EstA showed clear bands around expected molecular weight. In order to confirm the band we pointed out indicated our target proteins, we did an interview with our teacher, who suggested western blot assays to provide evidence, so we asked for collaboration and the following are results we got, which provided us positive results.

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690002-silver-1.png)

We then wondered whether we could promote the expression of EstA through condition adjustment, so we firstly fixed the induction time and IPTG concentration, then set a series of temperature gradient. We found 20℃ lead to the highest EstA expression level.

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690002-silver-2.png)

he next experiment we did was to fixed the induction temperature and IPTG concentration, then set a series of time gradient. This time we showed EstA expressed pretty well under 20℃ and the induction time seems not influence the expression level.

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690002-silver-3.png)

## [BBa\_K4690003](http://parts.igem.org/Part%3ABBa_K4690003)

PytH, isolated from Sphingobiumsp. strain JZ-1, encoding a pyrethroid-hydrolyzing carboxylesterase, which was able to transform p-nitrophenyl esters of short-chain fatty acids and a wide range of pyrethroid pesticides.

### We used the following usual procedure to do the experiments:

1. Verification of the sequence. This part sequence we submitted was come from Sphingobiumsp. strain JZ-1. We downloaded the sequence from NCBI and in order to be suitable for bacterial expression, we asked a biology company to undergo codon optimization and synthesize the target sequence.
2. We then constructed it into a pET28(+) plasmid and transformed the plasmids into E. coli BL21(DE3) strains.
3. After cultured on LB solid medium (Kanamycin) for 12 h, we picked a single colony into 4 ml LB medium (Kanamycin) and the mix was then shaken at 37℃ until OD600 = 0.6.
4. In order to set proper control, we divided the bacteria solution into three parts, for the first part, we added 0.5 mM IPTG and cultured in 16℃ for 20 h; the second part was added with 0.5 mM IPTG as well but cultured in 37℃ for 4 h. The last one added nothing to serve as a control.
5. When the expression process was finished, we centrifuged the bacterial solution at 12000 rpm, and the precipitation was resuspended with RIPA buffer to lysate bacteria, we also added loading buffer to heat at 96℃ for 10 min, followed by SDS-PAGE and Coomassie brilliant blue staining for expression test.
According to our SDS-PAGE result, protein PytH showed clear bands around expected molecular weight. In order to confirm the band we pointed out indicated our target proteins, we did an interview with our teacher, who suggested western blot assays to provide evidence, so we asked for collaboration and the following are results we got, which provided us positive results.

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690003-silver-1.png)

We then wondered whether we could promote the expression of PytH through condition adjustment, so we firstly fixed the induction time and IPTG concentration, then set a series of temperature gradient. We found 16℃ lead to the highest PytH expression level. As the temperature arises, PytH comes to the inclusion body, so it prefers lower temperature.

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690003-silver-2.png)

The next experiment we did was to fixed the induction temperature and IPTG concentration, then set a series of time gradient. This time we showed the highest PytH expression level was achieved with 18 h or 20 h induction under the temperature of 16℃.

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690003-silver-4.png)

{% endblock %}

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

# Experiments

* Home
* Project
* Experiments

Video: Protein Solution Injecting into Cold Coagulation Bath

### Molecular Cloning

【1】PCR Primers were designed by snapgene software and synthesized at BGI Company. PCR was performed by following the manuals of PrimeSTAR Max Premix (2X) (Taraka, R045) or 2 X EasyTaq PCR SuperMix (+ dye) (Transgene, M256). The former was used for construction and the latter was used for identification.
【2】Gel extraction
DNA fragment were separated by agarose gel electrophoresis and then recycled by TIANgeo Midi Purification Kit (TIANGEN, DP209-02) or AxyPrep DNA gel Extraction Kit (AXYGEN, AP-GX-250).
【3】Gibson
Gibson was performed by following the manual of pEASY- Basic Seamless Cloning and Assembly Kit (Transgene, CU201).
【4】Endonuclease digestion and ligation
The endonucleases and T4 ligase were bought from NEB Company and the experiments were performed by following instructions.
【5】Transformation
Transformation was performed by following the manuals of Trans5α Chemically Competent Cell (Transgene, CD201) or Trans1-T1 Phage Resistant Chemically Competent Cell (Transgene, CD501).
【6】Plasmid extraction
Bacteria were cultured in lysogeny broth (LB) at 37℃ overnight and plasmid extraction was performed by following the manuals of TIANprep Mini Plasmid Kit (TIANGEN, DP103-02) or AxyPrep Plasmid Miniprep Kit (AXYGEN, AP-MN-P-250).
【7】Sequencing
Sanger sequencing was done by BGI Company and snapgene software is used to align the sequences.

### Expression and Measurements

1. Transform the plasmids into E. coli BL21(DE3)
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 4ml LB medium with kanamycin. Add 1mM IPTG to all experimental groups as needed. Incubate at 37 degree Celsius in a shaker overnight.
3. Fluorescence. Acquire 1-3 ml bacteria culture after overnight inoculation. Took fluorescence picture under UV illumination.
4. SDS-PAGE.
4.1 Take 1ml of bacterial solution, centrifuge at 12000g for 1min at 4℃, discard the supernatant, add 100ul of pre-cooled RIPA lysate (high), vortex to mix, settle for 5min, centrifuge at 4℃, 12000g for 10min, and take the supernatant which contains the protein extract. After protein quantification by BCA method using commercial test box, adjust the concentration of the protein solution to 1mg/ml. Take 4 parts of the protein solution and 1 part of 5X protein loading buffer and mix it in a boiling water bath for 5 minutes, centrifuge at 12000g at 4°C for 10 minutes, take the supernatant, and store on ice. Use precast gel purchased from commercial company (Transgen, Precast Tris-Glycine Gel) for protein electrophoresis.
4.2 Add marker at 5μl, and add sample on all other lanes with 10ul sample on each lane. Use 100V for electrophoresis. After the electrophoresis, take out the gel, put it in the staining box, add Coomassie Brilliant Blue dye solution to about 3mm below the gel surface, shake on a horizontal shaker, dye for 30min at room temperature, discard the dye solution, wash off the floating color with distilled water, and add decolorization liquid, decolorize on a horizontal shaker until the result is in good condition for taking pictures.

{% endblock %}

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

# Results

* Home
* Project
* Results

## Overview

The ecological environment is an important basis for human survival and development, but under the influence of human activities, the ecological environment has suffered serious damage. This year, iBowu\_China focused on addressing the environmental concern of pyrethroid accumulation in aquatic ecosystems.
Pyrethroids are a class of pesticides that, despite their effectiveness in pest control, pose a significant threat to non-target organisms, including beneficial insects like bees. Our project designed a synthetic biology approach to biodegrade pyrethroids, thereby reducing their toxicity in aquatic ecosystems, protecting biodiversity, promoting ecosystem health, and contributing to the broader goals of environmental sustainability.

### Find the five target enzymes sequence to degrade pyrethroids

Pyrethroid insecticides are reported to be neurotoxic to both insects and mammals, to find more enzymes have functions in pyrethroids degradation, we searched in previous iGEM projects and published papers, the following are some of the targets we found:
1) iGEM Warwick 2022 team presented a project, namely Pyre-Pyrethroid Remediation, involved the design of an integrated system to test and degrade build-up of λ-cyhalothrin (a type of pyrethroid) in the environment. The enzyme they used is our first target: Pyre1. [Pyre1]
2) A paper published on scientific reports mentioned the EstPS1 gene, which encodes a novel carboxylesterase of Pseudomonas synxantha PS1 isolated from oil well-produced water, showed high biodegradability towards pyrethroid pesticides, so we chose EstPS1 as our second target. [EstPS1]
3) Pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112, which is our third target, could not only degrade pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyze rho-nitrophenyl esters of various fatty acids, indicating EstP as an esterase with broad substrates. [EstP]
4) EstA-associated biodegradation of pyrethroids was confirmed in 2019, which used the key degrading gene estA and related carboxylesterase EstA from Bacillus cereus BCC01. We selected EstA as the forth target. [EstA]
5) A fifth novel esterase gene, pytH, isolated from Sphingobiumsp. strain JZ-1, encoding a pyrethroid-hydrolyzing carboxylesterase, which was able to transform p-nitrophenyl esters of short-chain fatty acids and a wide range of pyrethroid pesticides. [PytH]
 **the above five enzymes have been reported to degrade pyrethroids, their research is still insufficient. We do not know whether these enzymes can be expressed in more basic chassis organisms, whether there are more suitable expression conditions, and whether their expression amounts meet the needs of water purification. Therefore, we launched this year’s project. We chose to use E.coli, the most common strain in synthetic biology research, as our carrier. E.coli can meet our needs in terms of safety as a common strain living in the gut of humans and animals, so can E.coli express these five enzymes?**

### Expression of the five target enzymes

The first step in our experiments is to do a preliminary test of enzyme expression, to do this, we connected with the company to construct those enzymes into pET28a(+) plasmids. After which, the plasmids were transfected into E.coli BL21(DE3) strains and used IPTG as induction. The preliminary expression conditions were set as 1) induction for 20 h under 16℃; 2) induction for 4 h under 37℃.
The following are the results:

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690002-silver-1.png)

SDS-Page Results of EstA

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690003-silver-1.png)

SDS-Page Results of PytH

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690000-silver-1.png)

SDS-Page Results of EstPS1

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690001-silver-1.png)

SDS-Page Results of EstP

As we observed through SDS-PAGE, protein EstA and PytH showed clear bands around expected molecular weight, protein EstPS1 and EstP can be expressed but precipitated in the inclusion body, however, protein Pyre1 was not expressed.

**In order to confirm those bands we pointed out indicated our target proteins, we did an interview with Prof. Geng from Minzu University of China, who suggested western blot assays to provide evidence, so we asked for collaboration and received her kind support in providing the Western Blot runs to confirm our findings. The WB in the above plots clearly provided us positive results.**

### Find the best expression temperature of EstA, PytH, EstPS1 and EstP

After we confirmed the expression of protein EstA, PytH, EstPS1 and EstP, we wanted to find the optimized expression conditions for these proteins.
**We have communicated with the person in charge of the sewage treatment company and learned that the current temperature of the sewage treatment tank is generally 16℃ in winter and 37℃ in summer. Therefore, we hope to find the most suitable temperature in the range of 16-37℃.**

So we firstly fixed the induction time and IPTG concentration, then set a series of temperature gradient. The SDS-PAGE result suggested:
1) 20℃ lead to the highest EstA expression level.
2) 16℃ lead to the highest PytH expression level. As the temperature arises, PytH comes to the inclusion body, so it prefers lower temperature.
3) Although EstP also precipitated in the inclusion body, but 22℃ expression expression produced more soluble protein.
4) No matter what the temperature is, EstPS1 is left in the inclusion body.

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690002-silver-2.png)

EstA Temperature Condition Tests

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690003-silver-2.png)

PytH Temperature Conditions Tests

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690001-silver-2.png)

EstP Temperature Conditions Tests

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690000-silver-2.png)

EstPS1 Temperature Conditions Tests

### Find the best induction time of EstA, PytH, EstPS1 and EstP

**After solve the problem of temperature, we would like to know how long is it appropriate for sewage to circulate in the treatment tank? The worker in the sewage treatment company said that according to his experience, 16 h is more appropriate in winter and 4 h is major indicated in summer. So the next experiment we did was to fixed the induction temperature and IPTG concentration, then set a series of time gradient around 16 h or 4 h. The SDS-PAGE result suggested:**

1) EstA expressed pretty well under 20℃ and the induction time seems not influence the expression level..
2) The highest PytH expression level was achieved with 18 h or 20 h induction under the temperature of 16℃. .
3) More soluble EstP protein were produced after 24 h induction under the temperature of 22℃. .
4) EstPS1 was expressed under 16℃, we observed little soluble bands after 3 h induction..

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690002-silver-3.png)

EstA Induction Time Tests

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690003-silver-4.png)

PytH Induction Time Tests

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690001-silver-3.png)

EstP Induction Time Tests

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4690000-silver-3.png)

EstPS1 Induction Time Tests

### The improvement of Pyre sequence

The last part of our project is to solve the problem of Pyre expression. In the preliminary test, we downloaded the sequence from Warwick 2022 team, however, it did not work. **We then carefully reviewed the whole project and related papers, we finally found out the key issue lies in the promoter and RBS part.** The sequence Warwick uploaded contains promoter and RBS, we then destructed the original promoter and RBS on pET28a(+), which may lead to the incompatibility for E.coli BL21(DE3). So we isolated Pyre to construct a new plasmid named pET28a(+)-Pyre, which showed the expression band of Pyre protein, indicating the successful improvement of the existing part.

![](https://static.igem.wiki/teams/4690/wiki/registry/bba-k4210004-bronze-2.png)

### Test the degradation ability of the five target enzymes

After successfully expressing five types of pyrethroid degradation enzymes, we induced the expression of these proteins again under the optimal conditions obtained in the previous experiments. We then mixed the 2 ml bacterial solution with 0.5 ml of specified concentration pyrethroid solution and subjected it to 4 hours of rotational mixing. To conveniently and quickly detect the pyrethroid content within the system, we used commercially available test strips. As shown in the figure below, compared to the control group, all five experimental groups exhibited the ability to degrade pyrethroids. Among them, the degradation effect of enzyme EstA was the most significant, followed by enzyme PytH, and then Pyre1, while the degradation effects of enzyme EstPS1 and EstP were similarly weak, i.e., poor degradation effect. One red line on the top indicates positive test outcome, meaning the test sample contains pyrethroid. Two red lines indicate negative test outcome, meaning no (or less) pyrethroid in the test sample.

![](https://static.igem.wiki/teams/4690/wiki/team/registry/bba-k4690002-res4.jpg)

### Future plan of pyrethroids degradion Implementation

We still need to consider more aspects if we are to put our project into practice.
Firstly, how to check the activity of pyrethroids degrading enzymes? We found that this experiment can be performed using a solution of p-nitrophenol acetate. If there is esterase activity, the solution will change from colorless to yellow. Then we can measure the activity of five enzymes in order to obtain more accurate experimental conditions through calculation.
Secondly, how can our reactions be stopped? If pyrethroids is not currently present in the sewage, or if the sewage treatment plant is facing special circumstances such as holidays, we need to stop the engineered E. coli from working. After searching for related publication, we noticed antibiotic signal-based CRISPR-Cas9-based kill switches (CRISPRks) and temperature signal-based T-switch systems, and if we can integrate suicide systems into the E. coli genome in the future, then we will have the potential for more precise and efficient human control.

#### ==== Reference ====

[PytH] Zuo Z, Gong T, Che Y, Liu R, Xu P, Jiang H, Qiao C, Song C, Yang C. Engineering Pseudomonas putida KT2440 for simultaneous degradation of organophosphates and pyrethroids and its application in bioremediation of soil. Biodegradation. 2015 Jun;26(3):223-33.
[EstA] Hu W, Lu Q, Zhong G, Hu M, Yi X. Biodegradation of Pyrethroids by a Hydrolyzing Carboxylesterase EstA from Bacillus cereus BCC01. Applied Sciences. 2019; 9(3):477.
[EstPS1] Cai, X., Wang, W., Lin, L. et al. Autotransporter domain-dependent enzymatic analysis of a novel extremely thermostable carboxylesterase with high biodegradability towards pyrethroid pesticides. Sci Rep 7, 3461 (2017).
[EstP] Pei C. Wu, Yu H. Liu, Zhuo Y. Wang, Xiao Y. Zhang, He Li, Wei Q. Liang, Na Luo, Ji M. Hu, Jia Q. Lu, Tian G. Luan, and Li X. Cao. Journal of Agricultural and Food Chemistry 2006 54 (3), 836-842
[Pyre1] DING, J., LIU, Y., GAO, Y., ZHANG, C., WANG, Y., XU, B., YANG, Y., WU, Q. & HUANG, Z. 2022. Biodegradation of λ-cyhalothrin through cell surface display of bacterial carboxylesterase. Chemosphere, 289, 133130.

{% endblock %}