Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

IGEM daily work July 4,2024: 1. We went to the lab. The pDawn plasmid arrived! Janna, Eison and Jack planned some educational videos for our Gold Medal category. July 3,2024: 1. We went to the lab. Mr. Huang let us know that he will send the pDawn plasmid today. July 2,2024: 1. We went to the lab. 2. After lunch, we had a 20-minute meeting. And then do our work. July 1,2024 1. Martina focuses on the daily work. 2. Micheal and Carson work on the PPT of introduction of our team. June 27,2024: 1. June 26,2024: 1. June 25,2024: 1. June 24,2024: 1. June 20,2024: 1. June 19,2024: 1. June 18,2024: 1. June 17,2024: Mons 1. IGEM team went to the lab together. We divided into three parts and each part has a different job. Part, one needs to. Part two needs to. Part three needs to be completed the. June 12,2024：Wed. (Caron and Alin not here today) 1. June 11, 2024: Tues. (Carson, Jessia Wu, Micheal, Jack, and Eson not here.) 1.Louis went to the lab by himself. 2.Janna and Alin still work on our box. They use a hot melt adhesive stick fan in the opening. 3.Violet still draws. 4.Allen works on the research. June 8,2024: Sat. (Dragon Boat Festivals） 1.Prof Jin came to ask the PDawn plasmid, he can give us some information about that. June 6,2024: Thur. 1.Mr. Gert explained to us our IGEM project and how to proceed carefully. It took us about 20 mins. [two papers about it]. 2.Janna, Alin, and Eson started to put a fan into the box. 3.Jack, Jessia W and Violet worked on the video. 4. Louis, Jessia Wu, Violet, and Martina went to the lab this morning and came back before lunch time. First, they put yellow trash bags on all the rubbish bins in the lab. Next, they put the experimental apparatus in order, so that we can get space for our materials. Finally, they learned how to clean the Electroporation. And then they practiced measuring the concentration value of the DsDNA by using Nanodrop. June 5,2024: Wed. 1.Mr. Gert collected the written consent of the celebration of IGEM in Pairs, France. 2.Some students need to sign in again. June 4,2024: Tues. 1. A list of what you did. 2. Jerry starts to build the page of the Wiki about our team on the computer. June 3,2024: Mons. 1. Box and video. May 31,2024：Fri. 1. We went to the lab as a team together and we divided into three different groups. Janna, Martina, and Carson: rinse the glass bottles. Louis, Violet, and Allen: learn how to make agar. Jack, Jessica W and Eson: subpackage and sterilize the tip cone of the pipettor.



May 30,2024: 1. Box group tried to install the timing mechanism. 2. Micheal Chen helped our video group. May 29,2024: 1. Box group started to test their box. They sticked the blue and red lights and put the yeast inside. 2. Micheal Chen still helped video group. May 28,2024: 1. The hardware team worked on their prototype. 2. The video group works on concept art and script. 3. Micheal Chen came to help video group. May 27 ,2024: 1. The hardware team worked on their prototype. May 24,2024: 1. Louis, Jack, Mr. Gert, Mr. Sam and Ms. Hou went to Guangming Science City to communicate with Dr. Lou about the light gene expression system that UCAS IGEM 2018 as used in their project 2. .

May 22,2024: 1. Today we do the second step of bioinformatics—MEGA. (a) Press the “Align” tab. (b) Edit alignment. (c) Create a new alignment. (d) DNA (e) Insert sequences from FASTA files. (f) Align selected sequences with Muscle. (g) Align DNA. (h) Check alignment for any gaps where sequences do not match up. (i) Do manual editing by comparing your sequences with the original Chromas file. (j) Export alignment for further use as a FASTA file. May 21,2024: 1. Today we do the first step of Bioinformatics—Chromas (a) Open the AB1 files. (b) Work though the sequence and delete the bases that you cannot trust. (c) Save the new version in a new folder. (d) Export the files as FASTA files. May 20,2024: 1. The video group started to draw art for the video. 2. Micheal, Martina, and Carson looked for other participants who near us. May 14,2024: 1. The video team brainstormed their ideas and script. 2. The hardware team researched their initial materials: 3.
May 15, 2024: 1. Allen and Michael finished their PPT of the process of Vanillin synthetic. 2. The video team work on the script for the promotional video. May 6, 2024: 1. We decided that we would like to design our plasmid so that the expression of vanalin can be controlled by the presence of blue light and the expression of menthol can be activated by the presence of red light. We decided to start with the expression of limonene as the first step in the menthol pathway. 3. The hardware team designed the first prototype of our hardware that will switch between blue and red light. April 22,2024: 1. Put the DNA into the gel electrophoresis. We decided to focus on Vanillin and Menthol Synthesis 2. We divided our tasks between a group in charge of the promotional video, a group in charge of the hardware and a group in charge of the wet lab. April 17,2024: 1. We divided the previous IGEM wikis among us and searched through them for projects that worked on aromatherapy. . Carson—2004 and 2023 Allen—2009 and 2018 Micheal—2005 and 2022 Eisson—2010 and 2017 Janna—2006 and 2021 Martina—2011 and 2016 Alin—2007 and 2020 Violet—2012 and 2015 Jerry—2008 and 2019 Jessica—2013 and 2014 April 16,2024: PCR reaction: 1. Template DNA—–4 ul. 2. Loumole rbcLaf—–2 ul. 3. Loumole rbcLarev—2 ul. 4. 2 times Taq Master Mix—25 ul. 5. ddHzo————-17 ul. April 15,2024: 1. We had our first meeting with our IGEM mentor, and she shared her experience of participating in the IGEM competition. 2. Finish the last step of the Plant Genomic DNA (10) Transfer the adsorption column CB3 into a clean centrifugal tube, drop 50-200 ul elution fine rinse TE into the middle part of the adsorption film, place at room temperature for 2-5 minutes, centrifuge at 12,000 RPM（~13,400xg）for 2 minutes, and collect the solution into the centrifugal tube. April 11,2024： 1. We continued with the DNA extraction: We started the fifth to ninth step: (5) The mixed liquid was transferred to adsorption column CB3, centrifuged at 12,000 rpm (-13,400xg) for 30 sec, and the waste liquid was discarded. (6) Add 500 U buffer GD into adsorption column CB3 (check whether anhydrous ethanol has been added before using the spectrum), centrifuge at 12,000 rpm (-13,400xg) for 30 sec, pour away the waste liquid, and put adsorption column CB3 into the collection tube. (7) Add 600 ul bleaching solution PW (check whether anhydrous ethanol has been added before use) to adsorption column CB3, centrifuge it at 12,000 rpm (~13,40xg) for 30 sec, pour away the waste solution, and put adsorption CB3 into the collection tube. （8）Repeat step 7 (9) The adsorption column CB3 was out back into the collection tube, centrifuged at 12,000 rpm (~3,400xg) for 2 minutes, and the waste liquid was dumped. The adsorption column CB3 was placed at room temperature for several minutes to thoroughly dry the residual bleaching solution in the adsorption material. April 10, 2024: 1. We continued with the DNA extractions. Some people had to repeat the first step. 2. Other people started the second to forth steps: （2）The ground powder was quickly transferred to a centrifuge tube with 700 ul 5℃ top thermal buffer GP1 (before the experiment, hydrophobic ethanol was added to the preheated GP1 to make its final concentration 0.1%) ，and then the centrifuge tube was placed in a 65℃-water bath for 20 minutes. During the water bath, the centrifuge tube was reversed to mix the sample several times. (3) Mix thoroughly, centrifuge at 12,000 rpm (~13,400xg) for 5 minutes. (4) Carefully transfer the upper water phase obtained in the previous step into a new centrifugal tube, add 700 U buffer GP2, and mix thoroughly. April 9, 2024: 1. The remaining plants have been delivered to school, such as eucalyptus, halcyon, Mint, citronelle, and rosemary. 2. We smell the fragrance. Mr. Gert explained how we can extract the Plant Genomic DNA 3. We started the first step of the Plant Genomic DNA extraction: （1）take about 100 mg of fresh tissue or 30 mg of dry tissue and grind it thoroughly. 4. Martina—rosemary Jerry—agilawood Alin ——halcyon Jessia—agilawood Carson—-chrysanthemum Micheal-parsley Louis——eucalyptus Jack—–citronelle Allen——-agilawood Eson—–mint April 8, 2024: 1. Miss Aoife gave presented a workshop about Aromatherapy to us. April 7, 2024: 1. Most of the plants that we need for our project have been delivered, such as chrysanthemum and agilawood. 2. We packed tips and Eppendorf’ and autoclaved them for future use. 3. Check the Escherichia coli that we striped out. April 2,2024: 1. Louis taught us how to stripe out bacteria on a gel.

March 28,2024: 1. We search the name of genes involved in terpene synthesis in our chosen plants. March 27,2024: 1. We decided what plants we need for our project. March 22,2024: 1. Our games started on the third floor. The students from grade 1 to grade 12 came to have fun. March 18- March 21,2024: 1. Plan our event. For example, site selection, make the posters and stick them on school or on the stairs, and purchase required materials. March 16,2024: 1. We have a webinar about the IGEM software online at 10 p.m. March 15,2024: 1. We went to the lab for the first time first as a team and talked about our team’s project with Prof. Shi Chen and met Dr. Hou and her team who will help us!

March 6,2024: 1. We learned how to do gel electrophoresis. We used coloured candy 😊 March 5,2024: 1. We learned how to use pipettes with Mr. Gert and Louis ’s help. March 4,2024: 1. We brainstormed our first fund raising event. For the event we will hold a games day with a cafe. The following people are in charge of different booths: Martina —pouring water. Jessic and Violet —Cafe with milk tea and pancakes Janna and Alin—Ring toss Micheal and Carson—Math game Louis —Crack the golden egg game and Hand of Fire Allen—Arm wrestling
February 27-March 1, 2024: 1. Focus on the questionnaire. We let grade 5 to grade 12 do the questionnaire. February 21,2024: 1. The first meeting after holiday. We started to make our online survey. January 10,2024: 1. Have the meeting with the IGEM guide. We met the people at 6,30 p.m. and we used about 30 minutes to learn something that is important to a new team just like us. For example, we know the normal time plan now. January 5-8,2024: 1. We discussed what topic we want to research. We decided to do a survey among our peers to find out what problems they might have. December 20-27,2023: 1. We recruited our team. We are 12 students in our team.

Methods followed in some of the experiments that we learned:

  1. Plasmid Extraction (Kit)
  • Transfer 1ml of grown LB medium into 1.5ml ependorf
  • Centrifuge at 10000×g for 60sec
  • Remove LB and leave bacteria precipitates
  • Repeat until all bacteria precipitate is collexted at bottom of ependorf
  • Add 250ul of Solution I/RNase A, mix thoroughly
  • Add 250ul of Solution II, rotate gently to obtain a clear lysate
  • Add 350ul of Solution III, immediately invert and mix thoroughly to prevent localized precipitation
  • Centrifuge at 13000×g for 10min
  • Insert filter column into 2ml collection tube
  • Transfer cleared supertant into filter column
  • Centrifuge at 13000×g for 60sec
  • Discard Filtrate
  • Add 500ul HBC buffer
  • Centrifuge at 13000×g for 60sec.
  • Discard Filtrate
  • Add 700ul DNA Wash Buffer
  • Centrifuge at 13000×g for 60sec.
  • Discard Filtrate
  • Add 700ul DNA Wash Buffer
  • Centrifuge at 13000×g for 60sec.
  • Dry filter column
  • Insert filter colum into 1.5ml ependorf
  • Add 50ul ddH2O
  • Rest at room temperature for 1min
  • Centrifuge at 13000×g for 60sec
  • Store at -20°C
  1. Gibson assembly (Kit)
  • 20ul system
  • 10ul 2x clone mix
  • 0.5ul plasmid template
  • ddH2O up to 20ul
  • Pipet to mix
  • PCR 50°C 50min
  1. Chemical translation
  • Thaw potent cells on ice
  • Add sequence into potent cell in open bench
  • Rest on ice for 30min
  • Heat in 42°C for 90sec
  • Rest on ice for 2min
  • Revive in non-antibiotic liquid medium for 40min
  • Plate bacteria onto culture medium with corresponding antibiotics
  • Grow in incubator
  1. DNA barcoding

  1. Primers for rbcL Gene Amplification: Forward primer (rbcL-af): 5’-ATGTCACCACAAACAGAGACTAAAGC-3’ -Reverse primer (rbcL-ar): 5’-GTAAAATCAAGTCCACCRCG-3’

  2. DNA Extraction:

    • Extract genomic DNA from the plant sample using a plant DNA extraction kit.
    • Check the quality and concentration of the extracted DNA using a Nanodrop spectrophotometer or gel electrophoresis.

  3. Prepare PCR Master Mix:

    • In a PCR tube, add the following reagents:
      • 7 µL of plant DNA template
      • 25 µL Master Mix solution (contains PCR buffer, dNTPs and Taq Polymerase)
      • 0.5 µL of 10 µM rbcL-af forward primer
      • 0.5 µL of 10 µM rbcL-ar reverse primer
      • Nuclease-free water to make the final volume up to 50 µL.

  4. PCR Cycling Conditions: Program the thermocycler to run the following cycles:

    • Initial denaturation: 94°C for 3 minutes (to denature the DNA)
    • Denaturation: 94°C for 30 seconds (melts the DNA strands)
    • Annealing: 55°C for 30 seconds (allows primers to bind to the rbcL gene)
    • Extension: 72°C for 1 minute (Taq polymerase extends the DNA strand)
    • Repeat for 35 cycles to ensure sufficient amplification.
    • Final extension: 72°C for 5 minutes (to complete extension of all products).

  5. PCR Verification via Gel Electrophoresis:

    • Prepare a 1.0% agarose gel with ethidium bromide or another DNA stain.
    • Load 5 µL of PCR product into the gel along with a DNA ladder.
    • Run electrophoresis at 90–100 volts for 45 minutes.
    • Visualize the gel under UV light. The expected band size for the rbcL gene fragment is around 600–700 bp, depending on the plant species.

  6. Sequencing (for Barcoding):

  • The PCR product was sent to GENEWIZ for sequencing.
  • Use the same primers (rbcL-af and rbcL-ar) for sequencing.
  1. Data Analysis:
    • Sequences were checked and analyzed with Chromas and MEGA. The obtained rbcL sequences were compared to databases in GenBank and Barcode of Life Database (BOLD) to identify the plant species.