Notebook

Team recruitment goal: to form a diverse, interdisciplinary team whose members have different professional skills and knowledge backgrounds, who can take on different roles and tasks in the project to ensure the smooth progress of this year's iGEM project. We will publish recruitment information through school clubs and social media accounts.

1. After all team members are determined, we formulate a learning plan based on their professional background and project requirements. At a fixed time every week, team members study and discuss relevant knowledge and skills, make continuous progress, and prepare knowledge for the completion of various tasks in subsequent projects.

2. Organize team building activities every two weeks, including sharing of learning problems, time management discussions, sharing of innovative ideas, sharing of interesting life stories, etc., to promote mutual understanding and trust among members and enhance team cohesion.

Meeting topic: Project kick-off meeting

Meeting objectives: Team members get to know each other, understand the team of instructors, and clarify the iGEM competition requirements and project goals.

Meeting agenda:

Introduction of team members and instructors;

Each member briefly introduces himself to prepare for the subsequent division of labor.

Instructors introduce their research fields, professional experience, and roles and responsibilities in the project.

Emphasis the importance of the project and the goals to be achieved.

The iGEM competition requires analysis and planning for the new season, and clear goals, including the work content and expected results to be completed at each stage.

Meeting topic: Discussion meeting on the application of synthetic biology technology and global issues

Meeting objectives: Understand the current status of the application of synthetic biology technology in different industries, and team members share the global issues they are concerned about, and prepare for the inspiration of the topic.

Meeting agenda:

Members collect relevant information in advance to understand the application of synthetic biology technology in different industries. The team discusses these cases together, summarizes the application advantages and limitations of synthetic biology technology, and thinks about how to learn from and innovatively apply these technologies in the project.

And share the potential connection between the global issues they are concerned about and synthetic biology technology, and think about how to use synthetic biology technology to solve these problems.

Meeting topic: Project inspiration meeting

Meeting objectives: Based on the previous discussion, preliminarily determine the direction of the project topic.

Meeting agenda:

Team members brainstormed and sorted out several research directions closely related to humans, such as environmental protection, agriculture, food and nutrition, and proposed various possible project ideas. For example:

1. Use gene editing technology to edit key genes of crops to make them resistant to specific pests and diseases and reduce the use of chemical pesticides.

2. Use gene editing technology to transform plants or microorganisms so that they can synthesize a large amount of specific nutrients needed by the human body, providing raw materials for the development of functional foods.

After the meeting, search and review literature independently to understand feasibility and innovation.

Meeting topic: Conceptualization of the topic

Meeting objectives: Determine the research direction and key content of the topic.

Meeting agenda:

The tutor analyzed and guided, combining environmental protection, agricultural sustainability, food and nutrition, and focusing on the plant itself - plant metabolism.

Analyzed the role of plant metabolism, a physiological process of its own, in growth and development support, environmental adaptation and defense, and contribution to the ecosystem.

Task: Literature search and reading

Objective: To gain in-depth knowledge about natural plant secondary metabolites, including their production mechanism, types, characteristics, research status, and their effects and potential value on human health.

Meeting topic: Literature survey summary

Meeting objectives: Sort out the results of literature reading

Meeting agenda:

The key research results were sorted out and analyzed, and the appropriate research plant - cucumber was selected after brainstorming.

The next step plan was clarified, and team members were required to conduct further research on the plant metabolism of cucumber after the meeting, including in-depth understanding of the metabolic pathways, gene expression and types of secondary metabolites of cucumber, so as to prepare for the determination of specific research methods and experimental designs.

Meeting topic: Deepening of the topic

Meeting objectives: Further clarify the research content and methods of the topic.

Meeting agenda:

The secondary metabolites flavonoids in cucumber epidermal hairs were determined as the research object, and the key genes for flavonoid synthesis were determined.

Hypothesis: Can the key gene CsTBH and flavonoid metabolism gene (PAL) for the initiation of cucumber epidermal hairs be modified to produce more flavonoids in cucumbers by using synthetic biology?

Meeting topic: Project division meeting

Meeting objectives: Make reasonable project division according to the research content and members' skills and expertise.

Meeting agenda:

The instructor introduces the principles of project division to ensure that each member can give full play to their strengths. Emphasize the importance and necessity of division of labor, as well as the key role of teamwork in the project.

According to the principle of division of labor, the research tasks of the project are broken down into specific subtasks, and the dry and wet team members are assigned. The work responsibilities and task objectives of each member are clarified, and effective communication and collaboration are carried out in the team WeChat group.

Preliminary experimental design

Under the guidance of our instructor, we defined the experimental objectives: to construct a co-expression vector of CsTBH and CsPAL, verify the gene interaction, and explore its effect on the synthesis of flavonoids in cucumber epidermal trichomes.

The CsTBH and CsPAL genes were obtained through gene cloning, and related vectors were constructed. The gene interaction was verified by yeast single hybridization, and the constructed vector was transformed into cucumber plants using Agrobacterium-mediated transformation. Finally, the experimental results were verified by phenotypic observation and flavonoid content determination (liquid chromatography-mass spectrometry and fluorescence staining). It is expected that the expected results will be achieved in terms of gene cloning and vector construction, gene interaction verification, plant transformation phenotype and flavonoid content determination.

Note: General laboratory safety knowledge includes rules and regulations, personal protective equipment, use of laboratory instruments and equipment, biosafety knowledge including biohazard identification, operating specifications, and reagent classification and storage and use. Master operating procedures and emergency measures to ensure your own safety and the laboratory environment.

RNA was extracted from cucumber plants and reverse transcribed into cDNA using reverse transcriptase.

Specific primers were designed based on the known CsTBH and CsPAL gene sequences, and PCR amplification was performed using cDNA as a template to obtain the target gene fragments.

The PCR products were ligated to the TOPO vector and transformed into Escherichia coli. Colony PCR and sequencing were used to verify the consistency of the cloned CsTBH and CsPAL genes with those in the cucumber genome database to ensure that the selected cucumber materials had not mutated in the natural state.

Construct PCY-CsTBH and PCY-CsPAL overexpression vectors and CsTBH-PLacZi vector (for activating CsPAL promoter) respectively.

Verify the constructed vectors by colony PCR and sequencing to ensure that the target gene fragment is correctly inserted into the vector.

Extract the plasmid DNA of the constructed CsTBH-PLacZi vector and related control vectors.

Transform the plasmid DNA into yeast competent cells (such as EGY48) and perform operations according to specific transformation methods, including adding Carrier DNA, PEG/LiAc and other reagents, water bathing, centrifugation and other steps, and finally spread the transformed cells on SD plates for culture.

Prepare color development medium, inoculate yeast cells grown on SD plates into the color development medium, and culture at a specific temperature (such as 29°C) for a specific time (such as 48-96h).

Observe the color development of yeast cells. If yeast cells containing CsTBH-PLacZi vector appear blue in the color development medium, while control cells do not show color, it proves that CsTBH can bind to CsPAL promoter and there is an interaction between the two.

The constructed PCY-CsTBH and PCY-CsPAL overexpression vectors were transformed into Agrobacterium (GV3101) and transformed by freeze-thaw method or electroporation method.

The Agrobacterium containing the overexpression vector was infected into cucumber cotyledons, and transgenic cucumber plants were obtained through tissue culture steps such as co-cultivation, induction of adventitious buds, and rooting.

Cucumber plants are cultivated in artificial greenhouses, and environmental conditions such as light, temperature, and water are artificially controlled to monitor plant growth in real time and improve the survival rate of genetically modified plants.

The transgenic cucumber plants and wild-type cucumber plants were observed and their fruit thorn morphology was compared. If the transgenic plants had many small black thorns on their fruit thorns, it might be related to the production of flavonoids, which needs further verification.

The results of LC-MS analysis showed that the flavonoid content in the fruits of transgenic cucumber plants was significantly higher than that in wild-type plants, and the fluorescence staining results assisted in proving the changes in flavonoid content.