Wet Lab
1. We reviewed literature and communicated with the PI to preliminarily determine the project content.
2. We visited the laboratory, conducted initial lab safety training and engaged in learning and training of basic operations.
3.We consulted literatures, communicated with the instructors to understand the background of droplet microfluidic high-throughput screening and learned related operations.
Human Practice
1. We held the first meeting of NNU-CHINA to collectively understand the philosophy of iGEM, the significance of the project and its potential impact, and conducted team division of labor.
2. We established official accounts on WeChat, Bilibili and Tik Tok to better promote the NNU-CHINA team's project and green environmental protection concept.
3. We conducted a questionnaire survey on "How to Carry Out Synthetic Biology Education and Popularization."
4. We engaged in offline project exchanges with the iGEM team from Hubei University to discuss challenging problems, learn together and make mutual progress.
Wet Lab
1. We communicated with the PI and instructor to finalize the project content.
2. We reviewed extensive literatures to understand the background of biopesticides and 5-ALA, as well as the current context of food production.
3. We screened highly active ALAS genes from different sources, synthesized the key enzyme of 5-ALA through the C4 pathway. And we synthesized ALAS genes from various sources, constructed expression plasmids, transformed them into E. coli BL21(DE3) chassis strains for fermentation verification and obtained a highly active chassis strain.
4. We conducted single-factor experiments to optimize the concentration and timing of IPTG addition during fermentation to achieve optimal experimental conditions. In the experiment, we found that the production of 5-ALA changed little after 24h of bacterial growth, so we suspected that there might be a situation of plasmid loss. We further verified the plasmid loss finding that the plasmid loss rate reached 45% after 24h, which affected the stable expression of ALAS gene. Through literature research, we plan to enhance stability using CRISPR-associated transposons (CASTs) system in the next steps.
Human Practice
1. We completed the design and production of the team logo, uniforms, and flag.
2. We designed a questionnaire on "Understanding of Biopesticides and Synthetic Biology Among Different Countries, Industries, and Age Groups", conducted online and offline surveys and analyzed the data.
3.We published articles on our WeChat public account to introduce the project and popularize a series of green agriculture policies.
4. We held a project discussion meeting to plan upcoming promotional activities, including team material production, interviews, science popularization lectures, and other practical events.
Wet Lab
1. According to the structural characteristics of ALAS enzyme, we used artificial intelligence prediction method to predict the key enzyme, and obtained some mutation sites that may improve the enzyme activity.
2. We performed mutations based on predicted sites, conducted sequence alignment and carried out cultivation and fermentation verification in batches.
3. We constructed plasmids related to transposition to integrate the key enzyme gene ALAS into the genome and verified the transposition results.
4. We learned the relevant operations of ARTP mutagenage, explored the best mutagenesis time of E. coli and mutagenesis the modified engineering bacteria.
5. We explored suitable oil phases, surfactants and optimal inner and outer phase flow rates to generate droplets with uniform size, good stability and low emulsification tendencies.
Human Practice
1. We participated in the CCiC conference in Suzhou to further promote the team project and gain practical insights.
2. We visited the Lingshan Grain Logistics Base in Nanjing for research, gaining a more intuitive and in-depth understanding of modern grain production and logistics.
3. We carefully produced various materials to better promote the project, showcase team creativity and present our image. These included bookmarks, brochures, posters, comics, board games, picture books and poetry collections.
4. We visited Laifeng Street Community and Yudaiyuan Community in Nanjing to introduce our team, synthetic biology and green agriculture to children in simple and engaging ways. For the elderly, we went to Qimin Road Community and Guochang Community to discuss our project and share knowledge about green agriculture and biopesticides.
5. We held an online exchange with the iGEM team from Huazhong Agricultural University to learn about each other's research topics, solve project challenges and learn collaboratively.
6. We engaged in in-depth discussions with NJTech-China-A and Ulink-SZ teams at Nanjing Tech University, exploring scientific research paths and sharing experiences and insights.
7. We visited the agricultural industrial park in Nanjing to understand crop growth conditions and the use of biopesticides.
8. We released a series of micro-lesson videos on Bilibili to promote knowledge about green agriculture and microbiology.
9. We conducted a volunteer activity picking up garbage along the Yangtze River to promote green and environmental protection concepts, and visited the Nanjing Sewage Treatment Plant.
Wet Lab
1. We used enzyme-constrained models to predict beneficial targets for 5-ALA synthesis, applied pJ23119 and strong RBS to enhance the expression of related genes. Finally, we verified which targets were beneficial for product synthesis through fermentation.
2. We calculated the required concentration of E. coli for single encapsulation in droplets using the Poisson distribution formula.
3. We used high-throughput equipment to encapsulate bacterial solutions and culture media into droplets, observing the growth of E. coli. Simultaneously we cultured the same amount of E. coli in liquid media for comparison.
Human Practice
1. We visited Jiangsu Fangcaodu Agricultural Technology Co., Ltd. for interviews to further understand the development status of green agriculture and explore the field more deeply.
2. We collaborated with our university's teaching support group to conduct online lectures on microbiology and recorded experimental video demonstrations at Xiliang Village Complete Primary School in Xinxiang City, Henan Province.
3. We organized a debate competition on "Do Genetically Modified Crops Bring More Advantages or Disadvantages?"
4. We teamed up with NJTech-China-A, OUC-Haide, SDU-CHINA, and HUBU-China to create the "Biological Glow, Becoming a Torch" science popularization podcast series.
5. We developed an online dictionary providing concise explanations of professional terms related to the project to eliminate public understanding barriers caused by technical jargon.
6. We carefully produced a promotional video to better showcase our project and key technical points.
Wet Lab
1. We cultivated E. coli at appropriate temperatures. When they reached the logarithmic phase in droplets, we used OD measurements to select droplets with higher bacterial counts.
2. We broke the selected droplets, plated them on agar plates and conducted fermentation verification of the products after single colonies grew.
3. We used GraphPad Prism to process experimental data and refined all scripts.
Human Practice
1. We set up a booth on green agriculture and synthetic biology at the college's orientation site to educate new students and parents.
2. We hosted a laboratory open day, inviting nearby high school students for a tour.
3. We created an audio picture book series tailored for young children and collaborated with other teams to inspire kids through vivid and interesting stories.
