Notebook
Jan
uary
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.
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
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
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.
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
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.
Figure 3 Project peripheral products
Ju
ne
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
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
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.
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.
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
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
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
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