iGEM 2023 — Team ouc-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 %}
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![notebook](https://static.igem.wiki/teams/4711/wiki/fengmianzhaopian/notebook.jpg)

Notebook

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**CONTENT**

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[**January-March**](#January)

[**April-June**](#April)

[**July-August**](#July)

[**September-October**](#September)

[**Wetlab notebook**](#Wetlab)

![]()

![witch](https://static.igem.wiki/teams/4711/wiki/pet/witcher-move2.gif)

![小桌宠](https://static.igem.wiki/teams/4711/wiki/pet/linked-yeast2.jpg)

# Jan uary

---

### In January, our team of 9 officially formed and began preliminary preparations for the iGEM 2023 season. We had weekly online discussions to ultimately determine the problem we wanted to solve.

2023.1.15 During our online brainstorming session, one of our team members mentioned that her friend had mentioned the current difficulties in the production of medium-chain fatty acids, such as the generation of a large amount of acidic water pollution. This topic was particularly relevant to her as she comes from Karamay, a city known for its petroleum industry in China, and her family members work at a factory that produces medium-chain fatty acids locally. We all felt that this was a topic of great significance, so we started gathering information on this subject.

![](https://static.igem.wiki/teams/4711/wiki/hp/hp/2.png)

**Figure 1 Karamay Black Oil Mountain**

2023.1.23 During this team meeting, we discussed the information collected from various sources. HP presented her preliminary research findings, and we gained an understanding of the significant role of medium-chain fatty acids in people’s lives and the current issues in their production. We ultimately determined that our problem to solve would be the green production of medium-chain fatty acids.

2023.1.28 After reviewing various bioproduction schemes for medium-chain fatty acids, we discovered that the main limitation in their biosynthesis is the low yield. Therefore, we continued to search for literature in order to find potential solutions to this issue.

# Febr uary

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### In February, we established the team’s principal investigator (PI) and finalized the initial plan to address the green synthesis of medium-chain fatty acids. We also designed the experimental ideas for the first part of our project.

2023.2.5 Fortunately, we discovered that Professor Liu Guanglei’s team at our university had recently published a paper describing a new yeast strain called *Moesziomyces aphidis XM01*, which was obtained from the mangrove forests in Hainan. This strain produces a large amount of Mannosylerythritol lipids (MEL), which can be hydrolyzed to yield a significant quantity of medium-chain fatty acids. Excited by this finding, we reached out to Professor Liu via email to establish contact and discuss potential collaboration. Professor Liu expressed his enthusiasm for our interest in green production of medium-chain fatty acids and informed us of the current shortage of raw materials for their production. He also mentioned that the synthesis of MEL by *Moesziomyces aphidis XM01* leads to the accumulation of a substantial amount of intracellular lipid droplets. Furthermore, he expressed his willingness to act as our PI.

2.23.2.12 We had a discussion with the cafeteria manager of the Yushan Campus of Ocean University of China, where we learned that the university has a large population and there is a significant amount of food waste oil generated daily due to the large-scale food supply. Through interviews with their staff, we gained a better understanding of the methods and issues related to the treatment of kitchen waste oil.

2023.2.19 During this meeting, we had a discussion with the Principal Investigator (PI) about our ideas, proposing the concept of carbon flux reconstruction for *Moesziomyces aphidis XM01*. We suggested knocking out key genes to reduce intracellular lipid droplet synthesis.

# Mar ch

---

### In March, we started designing experimental protocols and carrying out various pre-experimental preparations.

2023.3.13 We had a discussion with Liu Zenggui, the Director of the Natural Resources Management Bureau in Yantai City, Shandong Province, to understand the current situation of reusing kitchen waste oil.

2023.3.15 We further discussed the design ideas and experimental plans with the PI. The PI expressed willingness to provide us with plasmids for gene knockout. At the same time, we also proposed the idea of using kitchen waste oil as a substrate

2023.28 We have completed the design of the plasmids for gene knockout and developed a preliminary experimental plan.

# Ap ril

---

### In April, we officially started our experiments.

2023.4.5 HP proposed that in order to increase the added value of the product and extend the industrial chain, we can produce derivative products based on medium-chain fatty acids, with the hope of generating more economic value.

2023.4.20 During this meeting, everyone exchanged ideas about producing mid-chain fatty acid derivatives. In the end, we decided to produce mid-chain α-olefins and 10-hydroxydecanoic acid.

2023.4.23 We had a discussion with Professor Zhao Yangguo because we chose waste cooking oil as our raw material. However, during the process of treating waste cooking oil, benzo[a]pyrene, a “triple threat” substance, needs to be degraded. Professor Zhao proposed that we need to optimize our approach in terms of accelerating the degradation rate and minimizing the toxicity of intermediate products. This also poses challenges for the subsequent application of our degradation process.

2023.4.25 During our discussion on the degradation of benzo[a]pyrene, we have tentatively identified a receptor for benzo[a]pyrene. It consists of two proteins, AHR (benzopyrene receptor) and ARNT (AHR nuclear translocator protein). Once AHR binds to benzo[a]pyrene, it forms a dimer with ARNT, which then initiates the expression of downstream promoter proteins.

![](https://static.igem.wiki/teams/4711/wiki/notebook/2-benzopyrene-sensor.png)

**Figure 2 Benzopyrene sensor**

2023.4.28 We discussed our ideas with the Principal Investigator (PI), and he informed us that through measuring the amount of benzo[a]pyrene in waste cooking oil, it was found to be relatively low. However, bio-sensors often cannot reach such low detection thresholds. Therefore, he suggested that we consider switching to a different sensor.

# M ay

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### In May, we started the experimental design for the later stage of the project as well as the design for the project’s surroundings.

2023.5.6 Through modeling, we discovered that the sensor indeed had difficulty detecting low levels of benzo(a)pyrene, so we have decided to replace the switch.

2023.5.10 We hope to use a riboswitch as an engineered bacteria’s suicide switch, but we have some doubts about the selection of the riboswitch and the specific implementation details. Therefore, we communicated with Professor Wang Qian from Shandong University about the design of the suicide switch. The professor provided us with many valuable suggestions, and in the end, we chose the NeuAc riboswitch.

2023.5.11 We have established the use of red light as the switch to initiate the degradation of benzo(a)pyrene.

2023.5.12 Li Peijin suggested that inclusivity can be approached from the perspective of children with autism, which is also a charity activity she frequently participates in. She proposed that we create an introductory guide to synthetic biology in the form of a picture book.

2023.5.22 We had a discussion with an associate professor from the School of Environmental Science and Engineering at the Ocean University of China. We learned about the methods for the separation and detection of polycyclic aromatic hydrocarbons (PAHs). We inquired about the severity of the hazards posed by PAHs, which further confirmed the importance of our project’s focus on harmful substances. As a result, we also laid the foundation for better industrial detection and degradation of PAHs.

2023.5.28 Ma Wenqi, Qiao Wang, and Liu Dun have each designed different versions of the picture book character, a little witch, as well as the image of our engineered bacteria. After discussions, we have finalized the project name and the final version of the picture book character’s image.

![](https://static.igem.wiki/teams/4711/wiki/notebook/notebook-chuanchuan.png)

![](https://static.igem.wiki/teams/4711/wiki/notebook/notebook-xiaomonu1.jpg)
![](https://static.igem.wiki/teams/4711/wiki/notebook/3-project-peripheral-products-2.png)

![](https://static.igem.wiki/teams/4711/wiki/notebook/3-project-peripheral-products-3.png)
![](https://static.igem.wiki/teams/4711/wiki/notebook/notebook-shaoping1.jpg)

**Figure 3 Project peripheral products**

# Ju ne

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### This is the stage of refining and improving the project. We placed great emphasis on communication and exchange during June. Through discussions with the team from Shandong University and our teachers, we made improvements and refinements to the design of our project.

2023.6.3 The team had a lively online exchange with the Shandong University iGEM team. We discussed our respective project designs, human practices ideas, and other topics. We also provided each other with valuable suggestions and feedback during the discussion.

2023.6.4 We had a discussion with advisor Carla about our project and received valuable advice from her.

2023.6.9 Ma Wenqi designed and refined our team uniforms. In the end, we chose light blue uniforms and printed our adorable engineered bacteria character on the back of the uniforms.

Based on our findings, we believe that the proportions of oleic acid, linoleic acid, and palmitic acid in the carbon sources determine the proportions of C10 and C8 acids in MEL. Therefore, we aim to utilize modeling techniques to predict product composition based on the choice of carbon source.

From the simulation results, we can see that the growth of the bacteria on the reagent is mainly in fermenter 1, and the bacteria in fermenter 2 will die quickly after consuming the substrate due to the lack of continuous substrate input, so the significance of refluxing the supernatant to fermenter 1 needs to be further determined to see if this step can be omitted. Overall, the model can help us simulate the actual fermentation process and thus adjust the values of the inputs as well as the time of isolation to achieve a maximum MEL yield.

# Ju ly

---

### July is a crucial month for the progress of the project. In mid-July, we started our mid-term experiments and also began some human practice work.

2023.7.6Li Peijin acted as our photographer and took some amazing group photos for us. We chose the school playground as the backdrop for the photos

![](https://static.igem.wiki/teams/4711/wiki/notebook/4-team-photo.png)

**Figure 4 Team photo**

2023.7.7-2023.7.11 We went to Hainan to participate in CCiC (China College Students’ “Internet+” Innovation and Entrepreneurship Competition). The CCiC exchange meeting greatly helped the progress of our project. We showcased our project during the exchange meeting and afterwards, we collaborated with many teams to create popular science picture books. We also joined forces on our official account to popularize synthetic biology knowledge. We received valuable advice during this process.

![](https://static.igem.wiki/teams/4711/wiki/notebook/notebook-hainan1.jpeg)
![](https://static.igem.wiki/teams/4711/wiki/notebook/notebook-hainan2.png)

**Figure 5 Hainan exchange**

2023.7.14 We had a discussion with Professor Qi and clarified the issues in the process and mechanical aspects of our project. We also discussed the equipment needed for the design of flow simulation.

2023.7.17We set up a booth at the Taitung Night Market to distribute surveys and provide popular science information about synthetic biology.

![](https://static.igem.wiki/teams/4711/wiki/notebook/notebook-set-up-stalls-to-popularize-science.jpg)

**Figure 6 Set up stalls to popularize science and exchange ideas with the masses**

2023.7.19We had a discussion with advisor Wang Ziwei about modeling.

# Aug ust

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### Similar to July, we conducted several experiments, mainly focusing on our engineered bacteria’s suicide switch. We also designed our hardware and carried out some human practice work.

2023.8.6 We had a discussion with Professor Li, who mentioned that designing devices for multiphase heterogeneous systems poses certain challenges and difficulties. This conversation inspired us to make improvements in our subsequent work. During the exchange, Professor Li provided guidance on optimizing design plans for practical production. We also received feedback on the design plans and made improvements based on the issues encountered during the actual production process.

2023.8.16 We had a discussion with our Principal Investigator (PI) and learned about the device issues our engineered bacteria encountered during fermentation. Specifically, the mixing and separation effects were not satisfactory due to poor oil-water mixing and separation. Our PI expressed the desire for us to focus on hardware-related designs to address these challenges.

2023.8.16-2023.8.26 Made a team promotional video

2023.8.24 We had a discussion with the head of the Institute of Microbiology at the Chinese Academy of Sciences. Through this exchange, we learned about the challenges and difficulties in separating the products after fermentation. We also understood the importance of controlling the state of the products during fermentation to optimize the fermentation process. Additionally, we had the opportunity to learn about sample handling, sectioning techniques, and the usage of instruments such as transmission electron microscopy and gas chromatography, through the guidance of our project team.

2023.8.23 We collaborated with OUC-Haide to conduct popular science activities on synthetic biology for high school students.

2023.8.24 We went to an autism children’s institution to guide autistic children in coloring our picture book

# Sept ember

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### In September, we entered the final stage of our project, completing the finishing operations of the experiments and starting to create our wiki.

2023.9.6 Through communication with Shanghai Jingyaoshi Biological Technology Co., Ltd., we learned that the convenient detection of medium-chain fatty acids poses a challenge. This also presents a challenge for measuring the content of our products. It has also inspired us to continue improving the detection of medium-chain fatty acids in the future.

2023.9.13 Carla helps translate our picture book.

2023.9.15 We took individual photos of the filming team.

2023.9.18 Translation: Through communication with the manager of Karamay Jiangrun Chemical Materials Co., Ltd., we received feedback that in order to further commercialize and industrialize our products, it is necessary to clarify their market feasibility and conduct comprehensive market research. This enabled us to better engage in industrial production. Additionally, we have found potential partner companies interested in collaborating with our product.

# Octo ber

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### In October, we embarked on intensive wiki creation and the production of a presentation video, preparing for the final event.

10.12 We have completed the creation of the wiki

10.13-10.19 We have created the project presentation PPT and recorded the presentation video.

# Wetlab notebook

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{% 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 %} -->
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![Engineering Success](https://static.igem.wiki/teams/4711/wiki/fengmianzhaopian/4d5c2335213219f79e5b1a25696d33d.jpg)

Engineering Success

**CONTENT**

[**Overview**](#Overview)

[**1.MEL synthesis**

**system**](#Part1. MEL synthesis system)

[**2.synthesis of**

**medium Alpha-olefin**](#PART2. synthesis of medium Alpha-olefin)

[**3.synthesis of**

**10-hydroxycapric acid**](#PART3. synthesis of 10-hydroxycapric acid)

[**4.Kill switch**](#PART4. Kill switch)

[**5.Benzopyrene**

**degradation systemm**](#PART5. Benzopyrene degradation system)

[**6.Model**](#Model)

[**7.References**](#References)

![]()

![witch](https://static.igem.wiki/teams/4711/wiki/pet/witcher-move2.gif)

![小桌宠](https://static.igem.wiki/teams/4711/wiki/pet/linked-yeast2.jpg)

# Over view

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Synthetic biology encompasses the creation and assembly of new biological components, mechanisms, and structures, as well as the reconfiguration of natural biological systems for practical applications. Similar to other engineering disciplines, synthetic biologists adhere to the iterative DBTL cycle (design, build, test, learn) to effectively design innovative biological systems.

We utilized a newly discovered fungus, *M. aphidis XM01*, from mangroves to regulate its metabolic pathways for producing a large quantity of MEL. Additionally, we expressed corresponding enzymes to catalyze the synthesis of medium-chain α-olefins and 10-hydroxydecanoic acid. To address the issue of waste oil disposal, we designed a light-controlled degradation circuit to remove benzo[a]pyrene from the waste oil. Finally, we designed a NeuAc riboswitch to prevent gene leakage and enhance engineering security.

# MEL synthesis system

---

Design:

The marine yeast, *Moesziomyces aphidis XM01*, produces a substantial amount of intracellular lipids during MEL production. By identifying and knocking out key genes involved in intracellular lipid synthesis in the XM01 strain, the synthesis of MEL can be enhanced. Previous studies have shown that fungal intracellular lipid synthesis is closely related to genes such as DGA1, LRO1, and ARE1. Complete knockout of LRO1 results in the inability to generate intracellular lipids. With the help of our PI and advisor, Zhang Chao, we have identified the sequences of DGA1 and ARE1 genes in *Moesziomyces aphidis XM01*. Therefore, we have decided to knock out the DGA1 and ARE1 genes to observe whether it can reduce the formation of intracellular lipid droplets.

![](https://static.igem.wiki/teams/4711/wiki/design/oil-to-mels.png)

**Figure 1 XM01 Fatty Acid Metabolism Diagram**

Build:

We chose to construct double knockout plasmids for the DGA1 and ARE1 genes. Using the genomic DNA of the XM01 strain as a template, we amplified the DGA1-5’arm and DGA1-3’arm, as well as the ARE1-5’arm and ARE1-3’arm fragments of the XM01 strain. The correctly sequenced and digested fragments were then ligated to the FL4A-HTP-loxp vector provided by the PI using T4 DNA ligase. Finally, using the FL4A-NAT-loxp-ARE1(DGA1) plasmid as a template, we performed PCR amplification with corresponding primers to obtain linearized knockout fragments. Since the probability of simultaneous double gene knockout is relatively low, we chose to perform the ARE1 gene knockout on the basis of DGA1 knockout.

![](https://static.igem.wiki/teams/4711/wiki/engineering/tongyuanxulie.png)

**Figure 2 Gene double knockout diagram**

Test:

First, we verified the successful double gene knockout through DNA gel electrophoresis. We compared the growth curves of the XM01 strain with the ΔDGA1ΔARE1 strain using OD600 measurements and found that gene knockout had little impact on the growth of the XM01 strain. To qualitatively measure the changes in intracellular lipid droplets and MEL production, we used gas-liquid chromatography to determine the MEL production ability and intracellular lipid content of the original XM01 strain and the ΔDGA1ΔARE1 strain. Additionally, to visually observe the reduction of intracellular lipid droplets, we stained the intracellular lipids with Nile Red fluorescence staining and observed them under a fluorescence microscope. It was observed that there was a significant reduction in intracellular lipid droplets after the double gene knockout.

Learn:

The ΔDGA1ΔARE1 strain showed no significant change in growth rate compared to the wild-type strain, and there was a significant reduction in intracellular lipid droplet production. We have successfully achieved our goal.

# Synthesis of medium Alpha-olefin

---

Design:

To synthesize medium-chain α-olefins, we employed the OleTJE P450 enzyme from Jeotgalicoccus sp. ATCC 8456 to catalyze the single-step decarboxylation of fatty acids, which is a relatively simple approach in the biosynthesis of α-olefins. It directly utilizes free fatty acids as substrates to produce α-olefins in a straightforward and controllable manner. Additionally, to obtain a large amount of protein, we chose Escherichia coli BL21 as the host organism.

Build:

The gene fragment of OleTJE P450 was directly synthesized by a gene synthesis company and then ligated to the PET28a vector using restriction enzyme sites NcoI and XhoI. To facilitate protein purification, a 6×His tag was added at the C-terminus of the protein

![](https://static.igem.wiki/teams/4711/wiki/design/8-2.png)

Test:

After culturing the cells successfully transformed with the plasmid for a certain period of time, the cells were lysed, and the cell lysate was purified using Ni-NTA resin to collect the protein. Gel electrophoresis was performed to verify the presence of OleTJE P450 enzyme. Since the medium-chain fatty acid substrate produced by MEL has a chain length of C8-12, we first determined whether the purified P450 fatty acid decarboxylase OleTJE had the ability to catalyze medium-chain fatty acid substrates by testing different substrates including C8:0, C9:0, C10:0, C11:0, C12:0, C12:1, and C14:0. Finally, using the medium-chain fatty acids obtained in our experiments as substrates, the actual conversion rate of α-olefins was determined by GC-MS analysis. The amount of each fatty acid in the fatty acid composition was used as the denominator, corresponding to the measured amount of α-olefins obtained. The actual conversion rate of α-olefins for each medium-chain fatty acid was calculated.

Learn:

The results showed that fatty acid chain lengths between 10 and 12 were more easily catalyzed by the P450 fatty acid decarboxylase. The conversion rates for C8, C10, and C12 were 73.58%, 57.86%, and 75.67%, respectively. The higher substrate conversion rates demonstrate that using medium-chain fatty acids as substrates for the synthesis of medium-chain α-olefins is a promising and sustainable biosynthetic strategy. However, in the future, protein mutations can be explored to achieve higher specificity and conversion rates for C8-C12 substrates.

# Synthesis of 10-hydroxycapric acid

---

Design:

In order to achieve a high yield of 10-hydroxydecanoic acid and generate a high concentration of alkane hydroxylase, we chose the BL21 strain as our expression host. For the ω-hydroxylase system, we selected the alkBGT alkane hydroxylase system, which consists of three enzymes, AlkB, AlkG, and AlkT, derived from the alk operon of Pseudomonas oleovorans GPo1. In this system, AlkB is a membrane-bound monooxygenase, which is a key component of the alkane hydroxylase system. AlkG and AlkT are electron transfer proteins involved in the oxidation of NADH and electron transfer in the system. Studies have suggested that intracellular NADPH in Escherichia coli may not be sufficient for sustained biosynthesis of 10-hydroxydecanoic acid. Therefore, an efficient coenzyme regeneration system can be designed to improve biosynthetic efficiency. We chose glucose dehydrogenase (GDH) from Bacillus subtilis as the driving enzyme for the NADPH cofactor recycling cycle. To further enhance the catalytic capacity of the system, we used the SpyCatcher-SpyTag system to design an intracellular self-assembling multienzyme complex, assembling the cofactor GDH and AlkT to enable more efficient electron transfer. This allows for the formation of an ordered cofactor transfer channel between the two enzymes, thereby achieving efficient biosynthesis.

Build:

Due to the large number of proteins in the gene circuit, we chose to use two plasmids, PET28a and PCDFDuet-1, with different replication origins for plasmid co-transformation. They both contain the T7 promoter, which is a strong promoter that can effectively express the enzymes we desire. Therefore, we cloned the target DNA into the PET28a and PCDFDuet plasmids and constructed two vectors according to our plan. The first vector, PET28a+alkBGT+SpyCatcher, was designed to carry alkBGT and SpyCatcher, while the second vector, PCDFDuet-1+GDH+SpyTag, was designed to carry GDH and SpyTag.

![](https://static.igem.wiki/teams/4711/wiki/hardware/plasmid/1-pet28a-alkbgt-spycatcher.png)
![](https://static.igem.wiki/teams/4711/wiki/hardware/plasmid/2-pcdfduet-1-gdh-spytag.png)

Test:

First, the expression of proteins was detected by SDS-PAGE, and the gas-liquid chromatograms of the generation of 10-hydroxydecanoic acid after catalysis by three strains, alkBGT, alkBGT+GDH, and alkBGT+GDH+SpyCatcher+SpyTag, were compared to determine whether the yield of 10-hydroxydecanoic acid was successfully increased.

Learn:

It was found that under the catalysis of 0.2mM IPTG, the expression levels of the proteins were low, and there were significant differences in expression levels among the proteins, with the fusion protein alkT+SpyCatcher having the highest expression level. No peak of the target product was observed after gas extraction and GC analysis, suggesting that methylation was not performed and the boiling point of the product was high, resulting in unsuccessful results.

Improve:

Different IPTG concentrations (0.2mM, 0.5mM, 1mM) were set for induction to observe the results of SDS-PAGE and determine the optimal induction concentration for protein expression. At the same time, the samples were methylated before HPLC analysis. The parameters were also modified: the initial injection temperature was changed from 250°C, holding at 50°C for 1 min, and then increasing at a rate of 15°C/min to 250°C, holding for 10 min to an injector temperature of 250°C and a detector temperature of 300°C. The initial temperature was set at 80°C for 2 min and then increased at a rate of 5°C/min to 250°C, holding for 10 min.

Learn:

It was found that the protein induction was most effective under the 0.5mM condition. However, the target product still did not appear. In the future, we will try cell disruption for HPLC analysis, as it is speculated that the low product yield combined with failure to transport it out of the cells may have resulted in the inability to detect the product. We also consider redesigning the RBS to achieve similar expression levels of the proteins, as this is also a factor affecting catalysis.

# Kill switch

---

Design:

In order to prevent leakage, a suicide switch was designed based on the NeuAc riboswitch. The sequence reported by Schyung Cho, obtained through in vitro screening, was used. The switch consists of an RNA aptamer and a hammerhead ribozyme. The principle of its action is that NeuAc binding to the RNA aptamer causes a conformational change in the entire switch, leading to self-cleavage of the hammerhead ribozyme, creating a gap. Then, with the action of the RecJ nuclease in Escherichia coli cells, the downstream genes are cleaved, reducing the abundance of mRNA expression of the downstream genes. We chose ΦX174 as the lysing protein downstream to achieve the suicide of Escherichia coli.

Build:

We chose to have the company directly synthesize the fragment and then connect it to the plasmid PET28a.

![](https://static.igem.wiki/teams/4711/wiki/hardware/plasmid/3-pet28a-riboswith-174e.png)

Test:

We integrated the plasmid successfully and validated its expression. Subsequently, varying concentrations of N-acetylneuraminic acid (NeuAc) ranging from 0g/L to 8g/L were introduced into the culture medium. The optical density (OD) values of E. coli BL21 were monitored over time to ascertain the optimal NeuAc concentration necessary for the bacteria's normal viability.

Learn:

The results showed that when the culture medium contained 10g/L NeuAc, Escherichia coli could maintain normal growth status. However, once removed from the environment, it would quickly die, which took about 1-2 hours.

# Benzopyrene degradation system

---

Design:

In order to achieve controllability of degradation, we designed a red light switch to control the expression of benzo[a]pyrene-degrading enzyme. Among them, phyA is a functional photosensitive pigment receptor, composed of a lipoprotein and a photosensitive pigment-binding protein (chromophore). Since the chromophore cannot be synthesized in yeast, a similar compound called phycocyanobilin (PCB), purified from cyanobacteria, was added to the culture medium. PCB is easily absorbed by yeast cells and binds to the photosensitive pigment lipoprotein, forming a photosensitive pigment photoreceptor. Under red light (λmax = 660 nm) or far-red light (λmax = 730 nm), PhyA can reversibly change its conformation, allowing it to bind to FHY1 and through a linker, bind PhyA to BD (DNA-binding domain) and PHY1 to AD (transcription activation domain). By utilizing the principle of yeast double hybrid, the target gene can be expressed under the induction of red light.

Build:

We chose to directly synthesize the two fusion proteins of the red light system and then connect them to the existing yeast eukaryotic expression plasmid with the PGK promoter and terminator (provided by PI).

![](https://static.igem.wiki/teams/4711/wiki/engineering/5-2.png)
![](https://static.igem.wiki/teams/4711/wiki/engineering/6-2.png)
![](https://static.igem.wiki/teams/4711/wiki/engineering/7-2.png)

Test:

Due to time constraints, we only separately validated the function of the benzo[a]pyrene-degrading enzyme UPO1, and did not integrate it with the red light system to achieve overall control. However, we have used modeling to explore the conditions for future experiments. We plotted the relationship between the time required for the cP concentration to reach its maximum and the time required for the cP concentration growth rate to reach its maximum with the red light intensity RL. This helps in controlling the separation time in semi-continuous fermentation and selecting the light intensity in wet experiments, narrowing down the range of light intensity selection. The results showed that the expression level and expression rate of the target protein have a strong marginal effect on the time required to reach the maximum as the light intensity increases. When the light intensity reaches 50 umol/m^2/nm, it has almost reached the optimal state. Therefore, we chose a light intensity range of 50-60 umol/m^2/nm for subsequent testing.

![](https://static.igem.wiki/teams/4711/wiki/engineering/7-time-for-max-cp-by-rl-value.png)

Learn:

Upo1 has good benzo[a]pyrene degradation ability, and we have selected a light intensity range of 50-60 umol/m^2/nm for subsequent testing of the red light system.

# Mo del

---

## **● Sensing system**

Design:

In order to control the fermentation, we designed a receptor for initiating fermentation in engineered bacteria, and initially we intended to use a benzo(a)pyrene receptor to perform this function, so we modeled the benzo(a)pyrene receptor to test whether it could detect benzo(a)pyrene in kitchen waste oil and activate downstream gene expression.

Build:

We modeled the benzo(a)pyrene receptor to simulate its opening at a certain benzo(a)pyrene concentration, and since the actual concentration of benzo(a)pyrene in kitchen waste oil is very low, we used this to set the initial conditions to verify whether it can function or not.

Test:

We explored the relationship between the binding rate at specific benzo(a)pyrene concentrations as a function of its KA value, n value.

![](https://static.igem.wiki/teams/4711/wiki/model/5-relation-p-versus-n-and-k.png)

Learn:

It is concluded that if we want the binding rate P to reach more than 0.5, we need to mutate the binding site of benzo(a)pyrene to make its KA close to the order of magnitude of 10^-7, and this is almost impossible. We therefore decided to replace the switch.

Improve:

So we considered using a red light system to control the activation of the XM01 strain at specific times.

![](https://static.igem.wiki/teams/4711/wiki/model/8-simulation-of-red-light-switching-dynamics-chat.png)

Learn:

The results show that the changes of each substance under different red light intensities are as expected, indicating that the model itself can well describe the kinetic process of red light switching.

Improve:

During the actual experiment, we found that the yeast culture would attenuate the red light. Therefore, we took this factor into consideration and redesigned the model to obtain the optimal red light intensity for application in the final experiment.

![](https://static.igem.wiki/teams/4711/wiki/222.png)

Learn:

The fermentation tank we used is a cylindrical vessel with a radius of approximately 10cm and a height of approximately 40cm. Considering the actual fermentation process, most of the fermentation culture is located in the upper layer, while the lower layer is mainly occupied by the product MEL. After continuous adjustment of the number and positioning of the lights, we finally determined that wrapping 8 light bulbs with an intensity of 180 umol/m^2/nm at distances of 4cm, 12cm, and 20cm from the top of the tank would ensure that the upper 2/3 region within the fermentation tank reaches a light intensity of 50 umol/m^2/nm.

## **● Fusion protein System**

Design:

We first performed kinetic simulations of the reaction system and found that increasing the electron transfer efficiency can effectively increase the rate of product production in the early stages without changing the relevant enzyme activities. It can therefore be determined that the construction of fusion proteins can indeed increase the production of medium-chain fatty acids. In order to further provide the catalytic ability of the system, we intended to construct a fusion protein of AlkT and GDH to increase the efficiency of electron transfer.

Build:

Because alkT is a key enzyme in the production of electrons, its activity is critical to the success or failure of the entire experiment. To verify that alkT with the addition of the SpyCatcher-SpyTag system performs its original function, we molecularly docked alkT with its substrate, NADH, and performed the same test on the original alkT and various alkT-GDH connected with linkers to compare and analyze their substrate-binding abilities.

Test:

We first simulated the change in product expression over time as the electron transfer efficiency constant grew from 0.5 to 2 to observe the effect of electron transfer efficiency on product expression.

![](https://static.igem.wiki/teams/4711/wiki/model/20-enzymatic-kinetic-analysis-chat.png)

To validate the structure of the fusion proteins, we performed structure prediction on fusion proteins constructed by each of the linker and spytag-spyteacher systems.

![](https://static.igem.wiki/teams/4711/wiki/model/25-alkt-spycatcher-spytag-gdh.png)

To ensure that the monomer of the fusion protein is still active, we performed molecular docking using its substrate and judged the catalytic efficiency of the fusion protein from the docking data.

![](https://static.igem.wiki/teams/4711/wiki/model/29-validation-of-alkt-activity3.png)

Learn:

Through structure prediction and screening, we finally decided to construct an intracellular self-assembling multi-enzyme complex designed to assemble cofactors GDH and AIKT using the SpyCatcher-SpyTag system.

Improve:

Since the simulated structures show that SpyTag and SpyCatcher will interact with the AlkT and GDH proteins causing them to move closer to them, this may cause the SpyTag-SpyCatcher system to not work properly or have an effect on the structure and activity of the protein itself. Considering that the linkage site of the SpyTag-SpyCatcher system is ASP on SpyTag and Lys on SpyCatcher, we decided to substitute glycine for some of the amino acids.

![](https://static.igem.wiki/teams/4711/wiki/777.png)
![](https://static.igem.wiki/teams/4711/wiki/888.png)

Learn:

Structural simulations were performed after replacing amino acids in the relevant sites, and SpyTag no longer interacted with the protein itself. This method can be used as a direction to optimize the performance of fusion proteins in the future, and the specific binding efficiency and enzyme activity changes need to be determined by subsequent experiments.

## **● imulation of MEL fermentation process**

Design:

In order to better understand and improve the fermentation process of engineered bacteria, we intend to simulate the production of intracellular oil and MEL during fermentation of engineered bacteria.

Build:

We used MATLAB to model the fermentation part of the engineered bacteria, and established a fermentation model for the growth and production of MEL by the engineered bacteria. The relevant parameters were obtained experimentally.

Test:

We tested changes in the concentration of engineered bacteria, MEL concentration, and substrate concentration over time under a specific initial condition.

![](https://static.igem.wiki/teams/4711/wiki/444.png)

Learn:

The results give a good response to the growth of the engineered bacteria and the fermentation of MEL, but we intend to improve the model considering that intracellular lipids are also produced during the fermentation process and that we will be using a semi-continuous fermentation for the fermentation.

Improve:

We added to the model a relevant equation for measuring intracellular lipid production and made the fermentation process consistent with a semi-continuous fermentation: a product separation step was added.

![](https://static.igem.wiki/teams/4711/wiki/model/42-simulation-of-mel-fermentation-process-graph1.png)

Learn:

In the end, we obtained a model that can be applied under semi-continuous fermentation, which can respond to the growth of engineered bacteria, the production of MEL, and the production of intracellular oil, and the results initially met the expectations, which can help us to adjust the relevant fermentation parameters during the fermentation process in order to achieve optimization.

# Refer ences

---

[1] Yu G, Wang X, Zhang C, et al. Efficient production of mannosylerythritol lipids by a marine yeast Moesziomyces aphidis XM01 and their application as self-assembly nanomicelles [J]. Marine Life Science & Technology, 2022, 4(3) : 373-383.

[2] Oelkers P, Cromley D, Padamsee M, et al. The DGA1 gene determines a second triglyceride synthetic pathway in yeast [J]. Journal of Biological Chemistry, 2002, 277(11) : 8877-8881.

[3] Cho, S, et al, In vitro selection of sialic acid specific RNA aptamer and itsapplication to the rapid sensing of sialic acid modified sugars. Biotechnol Bioeng,2013.110(3): p.905-13.

[4] Sorokina, O., Kapus, A., Terecskei, K. et al. A switchable light-input, light-output system modelled and constructed in yeast. J Biol Eng 3, 15 (2009).

[5] Hochrein L, Machens F, Messerschmidt K, Mueller-Roeber B. PhiReX: a programmable and red light-regulated protein expression switch for yeast. Nucleic Acids Res. 2017 Sep 6;45(15):9193-9205.

[6] Athenstaedt K. Yali0e32769g (dga1) and yali0e16797g (lro1) encode major triacylglycerol synthases of the oleaginous yeast yarrowia lipolytica [J]. BBA Molecular and Cell Biology of Lipids, 2011, 1811(10) : 587-596.

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![Experiments](https://static.igem.wiki/teams/4711/wiki/fengmianzhaopian/experiment.jpg)

Experiments

![小桌宠](https://static.igem.wiki/teams/4711/wiki/pet/linked-yeast2.jpg)

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# Protocols and Experiments

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![Results](https://static.igem.wiki/teams/4711/wiki/fengmianzhaopian/results-docx.jpg)

Results

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**CONTENT**

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[**1.Part1**](#Part1: MEL synthesis system)

[**2.Part2**](#Part2: Medium chain fatty acid derivatives)

[**3.Part3**](#Part3: Benzo[a]pyrene degradation system)

[**4.Part4**](#Part4: Kill switch)

[**5.References**](#References)

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![witch](https://static.igem.wiki/teams/4711/wiki/pet/witcher-move2.gif)

![小桌宠](https://static.igem.wiki/teams/4711/wiki/pet/linked-yeast2.jpg)

# Over view

---

The solution for addressing the issues related to pollution from medium-chain fatty acid production and the inefficient utilization of kitchen waste oil can be divided into three parts: optimization of medium-chain fatty acid synthesis, synthesis of derivatives of medium-chain fatty acids, and benzo[a]pyrene degradation. We conducted experiments in a sequential manner to validate these components. Additionally, we demonstrated the feasibility of a suicide switch and identified current shortcomings in suicide switch technology. We proposed a more suitable, controllable, and simplified suicide switch that aligns better with the requirements.

# Part1: MEL synthesis system

---

Due to the coexistence of different fatty acid pathways within the XM01 strain, the formation of MEL leads to the accumulation of intracellular lipids. Whether it is the synthesis of intracellular lipids or MEL, energy, fatty acid precursors, and acetyl-CoA are essential for these metabolic processes. The synthesis of intracellular lipids will inevitably hinder the extensive production of MEL to a certain extent.

![](https://static.igem.wiki/teams/4711/wiki/proof-of-concept/dga1-hh.png)
![](https://static.igem.wiki/teams/4711/wiki/proof-of-concept/are1-lh.png)

**Figure 1 Schematic diagram of double gene knockout plasmid construction.**

We chosed to construct double knockout plasmids for the DGA1 and ARE1 genes. Using the genomic DNA of the XM01 strain as a template, we amplified the DGA1-5’arm and DGA1-3’arm, as well as the ARE1-5’arm and ARE1-3’arm fragments of the XM01 strain. The correctly sequenced and digested fragments were then ligated to the FL4A-HTP-loxp vector provided by the PI using T4 DNA ligase. Finally, using the FL4A-NAT-loxp-ARE1(DGA1) plasmid as a template, we performed PCR amplification with corresponding primers to obtain linearized knockout fragments. Since the probability of simultaneous double gene knockout is relatively low, we chose to perform the ARE1 gene knockout on the basis of DGA1 knockout.

![](https://static.igem.wiki/teams/4711/wiki/results/pcr-verification-of-double-gene-knockout.png)

**Figure 2 PCR verification of double gene knockout.**

After inoculating the ΔDGA1ΔARE1 strain and the original XM01 strain into 5 mL of seed liquid culture medium and incubating them at 28°C with shaking at 180 rpm for 48 hours, they were transferred to MEL fermentation medium for 7 days of oscillatory cultivation. Changes in MEL production capacity and intracellular lipid content of both the original XM01 strain and the ΔDGA1ΔARE1 strain were measured. The results are shown in the graph below.

Under the same culture conditions, the ΔDGA1ΔARE1 strain exhibited a significantly increased MEL production capacity compared to the original XM01 strain. The production of MEL increased from 62.8 g/L to 90.6 g/L, demonstrating that the double knockout of ARE1 and DGA1 greatly enhanced the strain's ability to produce MEL. At the same time, the intracellular lipid content decreased from 68% to 12.5%.

![](https://static.igem.wiki/teams/4711/wiki/results/r4.png)

**Figure 3 MEL production, biomass, and intracellular oil weight per gram of biomass in the ΔDGA1ΔARE1 strain**

In addition, we conducted measurements to determine whether the double knockout of the DGA1 and ARE1 genes would have an impact on the cell's growth rate. As shown in the graph below, the results indicate that, compared to the original XM01 strain, the growth rate of the ΔDGA1ΔARE1 strain was not affected by the knockout of the DGA1 and ARE1 genes.

![](https://static.igem.wiki/teams/4711/wiki/results/r5.png)

**Figure 4 Growth rate changes in the ΔDGA1ΔARE1 strain**

The cells cultured for seven days from both the ΔDGA1ΔARE1 strain and the original XM01 strain were subjected to staining of intracellular lipids using the Nile Red fluorescent staining method. Subsequently, they were observed under a fluorescence microscope. In comparison to the original strain, which exhibited a long chain of golden-yellow lipid droplets inside the cells, the knockout of ARE1 and DGA1 genes clearly had a significant impact on intracellular lipid synthesis.

![](https://static.igem.wiki/teams/4711/wiki/results/r6.png)

**Figure 5 Staining images of intracellular lipids in the original XM01 strain and the ΔDGA1ΔARE1 strain.**

To investigate the relationship between the composition of carbon sources (natural plant oils) and the resulting products, specifically the production of MEL (mannosylerythritol lipids), you used soybean oil, peanut oil, rapeseed oil, corn oil, sunflower oil, and safflower oil as carbon sources for fermentation in shake flasks. We collected samples and performed methylation on both the carbon sources (natural plant oils) and the corresponding generated MEL. Subsequently, we conducted gas chromatography/mass spectrometry (GC/MS) analysis to analyze the fatty acid composition of the carbon sources and the fatty acid composition of the produced MEL. The fatty acid compositions of the carbon sources and the corresponding MEL are shown in the figure below.

![](https://static.igem.wiki/teams/4711/wiki/results/r7.png)
![](https://static.igem.wiki/teams/4711/wiki/results/r8.png)

**Figure 6 Fatty acid composition of natural plant oil and MEL**

The results of the correlation analysis have demonstrated that the proportion of fatty acid components in the substrate determines the changes in the composition of medium-chain fatty acids in MEL synthesis. The higher the content of a particular component in the substrate, the more it will be reflected in the corresponding component in the produced MEL. Understanding this pattern allows us to produce a wide range of medium-chain fatty acids in large quantities, enabling customized production of medium-chain fatty acids.

Conclusion:
In summary, by knocking out the ARE1 and DGA1 genes, we significantly reduced the formation of intracellular lipid droplets during MEL synthesis, which facilitated both the synthesis and secretion of MEL. Under the experimental conditions of shake flask fermentation, we achieved a MEL yield of 90.6g/L, with the intracellular lipid content reduced to 12.5%. This successful transformation of the original XM01 strain demonstrates the effectiveness of our approach. Additionally, we have uncovered the potential for customized production of medium-chain fatty acids.

# Part2: Medium chain fatty acid derivatives

---

Synthesis of α-olefins

The recombinant expression vector, provided by the Institute of Bioenergy and Process Research, Chinese Academy of Sciences, was introduced into Escherichia coli BL21 cells for seed culture in LB liquid medium. Subsequently, these cells were inoculated into LB liquid medium supplemented with glycerol and a rare salt solution for further cultivation. When the culture reached a certain optical density (OD), isopropyl-β-D-thiogalactoside (IPTG) and δ-aminolevulinic acid were added to induce the expression of the P450 enzyme. After a specific period of cell cultivation, the cells were lysed, and the cell lysate was purified using Ni-NTA resin. The protein was collected, and gel electrophoresis was performed to verify the presence of the OleTJE P450 enzyme.

It was observed that the protein secreted by Escherichia coli BL21 cells, after purification and processing, exhibited a single and clear band on the gel electrophoresis, with a size ranging from 45 kDa to 60 kDa. The OleTJE P450 enzyme used in this study has a size of 50.53 kDa, which aligns well with the position of the band shown on the protein gel.

![](https://static.igem.wiki/teams/4711/wiki/results/a-gene-circuit-b-sds-page-result-image.png)

**Figure 7 (A) Gene circuit (B) SDS-PAGE result image**

Due to the production of medium-chain fatty acids by MEL, with substrate chain lengths ranging from C8-12, we initially determined the in vitro activity of the P450 fatty acid decarboxylase OleTJE through a recombinant in vitro reaction. We conducted experiments using various fatty acids, including C8:0, C9:0, C10:0, C11:0, C12:0, C12:1, and C14:0, as substrates, with the addition of 0.2 μM of the P450 fatty acid decarboxylase OleTJE. The reaction was carried out for two hours, and by measuring the changes in substrate content, we determined whether the purified P450 fatty acid decarboxylase OleTJE possesses the ability to catalyze medium-chain fatty acid substrates in vitro. As a control group, we used C14:0 palmitic acid substrate to confirm the protein activity of P450 fatty acid decarboxylase OleTJE.

![](https://static.igem.wiki/teams/4711/wiki/results/r10.png)

**Figure 8 Conversion rate of fatty acid substrates catalyzed by P450 enzyme.**

Using the MEL obtained from the experiments described in this study, we prepared medium-chain fatty acids as substrates for the reaction. The reaction system included 0.5 μM of the enzyme, 200 μM of medium-chain fatty acids, and 500 μM of H2O2. The reaction was carried out at 28°C for 2 hours. The products obtained from the reaction were analyzed using GC-MS. Based on the GC-MS analysis results, the actual conversion rates of α-olefins were calculated. For each type of fatty acid in the fatty acid composition, the corresponding content of α-olefins obtained from the measurements was used as the numerator, with the content of each fatty acid type as the denominator, to calculate the actual conversion rate of α-olefins for each medium-chain fatty acid.

![](https://static.igem.wiki/teams/4711/wiki/results/r11.png)

**Figure 9 Conversion yield of α-olefins.**

Synthesis of 10- hydroxydecanoic acid

In order to enhance the efficiency of preparing 10-hydroxydecanoic acid, we introduced the cofactor GDH (Glucose Dehydrogenase) to provide more NADH. This was achieved by designing a SpyCatcher-SpyTag system to construct a dual-enzyme complex. Due to the relatively large quantities of both alkBGT and the cofactor GDH, expressing them in the same plasmid can often be challenging. Therefore, we considered the approach of dual-plasmid transformation and selected two plasmids, PET28a and pCDFuet-1, each with different origins of replication.

Firstly, the verification of the alkBGT system was conducted. Colony PCR results showed the appearance of the target band at around 4000 bp, indicating successful plasmid integration into Escherichia coli. SDS-PAGE analysis revealed the successful expression of the AlkB, AlkG, and AlkT proteins. Additionally, by setting a gradient of IPTG concentrations, it was observed that the protein expression was most efficient when induced with 0.5 mM IPTG, yielding the highest protein expression levels.In order to better visualize the expression intensity of proteins at different IPTG induction concentrations, we used software(GelAnalyzer) to analyze the results of SDS-PAGE and obtained a data graph of the relative protein expression intensity.

![](https://static.igem.wiki/teams/4711/wiki/results/figure-10.png)

**Figure 10 (A) Gene circuit (B) Colony PCR (C) SDS-PAGE M: Protein Marker 1: 0mM IPTG 2: 0.2mM IPTG 3: 0.5mM IPTG 4: 1mM IPTG. The protein bands from top to bottom are alkT (56KDa), alkB (45KDa), and alkG (18KDa).**

![](https://static.igem.wiki/teams/4711/wiki/design/35210630e3590abd1d7c9c85ebd3aaf4.png)

**Figure 11 Expression intensity of proteins at different induction concentrations**

Next, the validation of the alkBGT+GDH+SpyTag+SpyCatcher system was conducted. Colony PCR results showed the appearance of target bands at approximately 4000 bp and 1000 bp, indicating successful plasmid integration into Escherichia coli. SDS-PAGE analysis revealed successful expression of the AlkB, AlkG, AlkT, and GDH proteins. By setting a gradient of IPTG concentrations, it was observed that the protein expression was most efficient when induced with 0.2 mM IPTG, yielding the highest protein expression levels.In order to better visualize the expression intensity of proteins at different IPTG induction concentrations, we used software to analyze the results of SDS-PAGE and obtained a data graph of the relative protein expression intensity.However, the obtained data graph is inconsistent with the gel electrophoresis results due to inconsistent light distribution during the image capturing process, leading to errors in the software analysis results. Therefore, when using relevant software(GelAnalyzer) to process gel electrophoresis results, it is important to pay attention to the distribution of light sources during image capturing.

Moreover, a distinct band was observed at around 90 KDa, indicating successful linkage of alkT and GDH through SpyTag and SpyCatcher. As a result, the band corresponding to GDH was lighter compared to the previous one due to partial migration to the 90 KDa position as a result of binding with GDH.

![](https://static.igem.wiki/teams/4711/wiki/results/figure-11.png)

**Figure 12 (A) Gene circuit (B) Colony PCR (C) SDS-PAGE M: Protein Marker 1: 0mM IPTG 2: 0.2mM IPTG 3: 0.5mM IPTG 4: 1mM IPTG.The protein bands from top to bottom are alkT+GDH (87KDa), alkT (56KDa), alkB (45KDa), GDH (31KDa), and alkG (18KDa).**

![](https://static.igem.wiki/teams/4711/wiki/design/191190effe6f5fafa485332ddf9c0fd3.png)

**Figure 13 Expression intensity of proteins at different induction concentrations**

Additionally, we amplified the GDH fragment without the SpyTag from the previously constructed pCDFuet-1+GDH+SpyTag plasmid using PCR. The purpose was to verify that the catalytic efficiency of the engineered bacteria without the enzyme complex is lower than that of the engineered bacteria with the enzyme complex. However, to ensure that the primer’s Tm values did not differ too much, we adjusted the length of the PCR fragment and retained a portion of the SpyTag sequence, aiming to disrupt the formation of the enzyme complex. After sequencing the PCR fragment, we reconnected it to a new pCDFuet-1 vector to construct a new expression vector. The newly constructed expression vector was then introduced into the E.coli BL21 strain, which already contained the PET28a+AlkBGT+SpyCatcher plasmid. Colony PCR confirmed the successful introduction of the dual plasmids. SDS-PAGE results indicated the presence of bands corresponding to the enzyme complex between 75 kDa and 100 kDa. This suggests that we did not remove the amino acids responsible for binding with SpyCatcher. In the future, we will PCR amplify the GDH fragment without any SpyTag and repeat the validation process.

![](https://static.igem.wiki/teams/4711/wiki/results/figure-12.png)

**Figure 14 (A) Gene circuit (B) Colony PCR (C) SDS-PAGE M: Protein Marker 1: 0mM IPTG 2: 0.5mM IPTG**

After constructing the three engineered bacteria, we conducted catalytic experiments with decanoic acid. Despite varying IPTG concentrations, induction temperatures, and extraction conditions, we were unable to obtain the desired product, 10-hydroxydecanoic acid. We speculate that this is due to unequal expression levels of the proteins in the catalytic system, which is evident from the SDS-PAGE results. It is also possible that adding too many proteins to the same plasmid has overloaded the engineered bacteria, preventing efficient expression of each protein. In the future, we will continue by redesigning the RBS to achieve similar expression levels for each catalytic protein.

# Part3: Benzo[a]pyrene degradation system

---

Due to time constraints, we were unable to verify the red light switch. However, we independently verified the degradation effect of benzo[a]pyrene.According to the graph, it can be seen that after approximately 15 days, almost all of the benzopyrene has been degraded, which can demonstrate the strong benzopyrene degradation activity of the Uop1 enzyme.

![](https://static.igem.wiki/teams/4711/wiki/results/f49c8e6965871679723ea2eb8cdaf845.png)

**Figure 15 The change in benzopyrene content**

# Part4: Kill switch

---

To prevent the leakage of foreign genes into the environment, we constructed a suicide switch as shown in the diagram below. Successful integration of the plasmid into the bacterial strain was confirmed through colony PCR, as shown in the figure below, where a target band of approximately 750 bp was obtained. After inducing with IPTG for 5-6 hours, SDS-PAGE analysis revealed clear bands in the control group, while no distinct bands were observed in the induced group. This is because the induced group expressed a lysis protein, resulting in self-destruction and the release of a hydrolytic enzyme, which subsequently degraded all other proteins.

![](https://static.igem.wiki/teams/4711/wiki/results/figure-13.png)

**Figure 15 (A) Gene circuit (B) Colony PCR (C) SDS-PAGE M: Protein Marker 1: 0mM IPTG 2: 0.2mM IPTG**

After successful plasmid integration and expression validation, different concentrations of NeuAc (N-acetylneuraminic acid) ranging from 0g/L to 8g/L were added to the culture medium. The OD (Optical Density) values of E. coli BL21 were measured over time to determine the optimal NeuAc concentration required to ensure the normal survival of the bacteria. In the graph, "0h" represents the time point at which IPTG induction was added.

It can be observed that the control group and E. coli BL21 without IPTG induction exhibited typical sigmoidal growth curves. In contrast, the OD values of E. coli BL21 in the experimental group decreased as the NeuAc concentration decreased. This phenomenon occurred because at lower NeuAc concentrations, fewer NeuAc molecules bound to the riboswitch. As a result, the ribozyme was unable to perform self-cleavage, and the downstream target gene remained hidden, avoiding degradation by the ReJ enzyme. Consequently, the lysis protein ΦX174 was highly expressed, leading to the death of E. coli.

This demonstrates the successful functionality of our NeuAc riboswitch. Through modeling and data analysis, it was determined that E. coli would undergo lysis and die when removed from the culture environment if NeuAc was added at a concentration of 10g/L in the culture medium.

![](https://static.igem.wiki/teams/4711/wiki/results/r20.png)

**Figure 17 The change in OD value at different NeuAc concentrations(0.2mM IPTG)**

![](https://static.igem.wiki/teams/4711/wiki/results/r21.png)

**Figure 18 The change in OD value at different NeuAc concentrations(0.5mM TPTG)**

It can be observed that in the end, the control group had lower OD values compared to the induced group. This is due to the fact that we used a 96-well plate for detection, and the smaller growth space in the wells allowed the inhibited growth of the induced bacterial strains to remain relatively stable for a longer period. Consequently, there would be a higher cell count in the induced group compared to the control group in the later stages. However, the actual effect should be assessed based on the OD values in the earlier part of the growth curve.

Additionally, we compared the differences in OD values under different IPTG concentrations and found that the suicide effect was less effective under 0.5 mM IPTG induction compared to 0.2 mM IPTG. This suggests that the reason behind this is likely due to excessive protein expression, which can lead to the formation of inclusion bodies, thereby reducing the activity of the proteins themselves. As a result, the lysis effect is compromised.

# Refer ences

---

[1] He, Q., Metabolic engineering of Escherichia coli for the synthesis of ω-hydroxy fatty acids from medium-chain free fatty acids. (Doctoral dissertation, East China University of Science and Technology, 2019). DOI: 10.27148/d.cnki.ghagu.2019.000094.
[2] Wang, J., Fang, K., Wang, R., et al. A genetically engineered bacterium and its application in the preparation of 10-hydroxy-2-decenoic acid. CN202211249301.9. (2023-07-24).
[3] Liu, L., Yin, L., Huang, F., et al. Construction of intracellular self-assembling multienzyme complexes using SpyTag/SpyCatcher for efficient biosynthesis. Chinese Journal of Biotechnology, 38(7), 75-82. (2018). DOI: 10.13523/j.cb.20180710.
[4] Sorokina, O., Kapus, A., Terecskei, K., et al. A switchable light-input, light-output system modelled and constructed in yeast. Journal of Biological Engineering, 3, 15. (2009). DOI: 10.1186/1754-1611-3-15.
[5] He, Q., Bennett, G. N., San, K. Y., Wu, H. Biosynthesis of medium-chain ω-hydroxy fatty acids by AlkBGT of Pseudomonas putida GPo1 with native FadL in engineered Escherichia coli. Frontiers in Bioengineering and Biotechnology, 7, 273. (2019).
[6] Wang, J. Evolution of N-acetylneuraminic acid-recombinant Escherichia coli based on riboswitch screening system. (Master’s thesis, Shandong University, 2016).

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