Notebook

Notebook

Wet lab

<Experiments Day 1: Cell culture>

Members learnt about the fundamental concepts of cell culture, then had the basic practice using NIH3T3 cells. Our main activities were as follows: thawing and re-culturing frozen cells, changing cell media, counting cells and measuring viability, seeding cells at the target density, and freezing cells for storage. Also, we practiced using micropipettes, counting cells using a hemocytometer, and evenly seeding cells in a 96-well plate.

Time/Date

Day 1

Participants

Andrew Suh, Peter Kang, Eunu Baek

Goal

Prepare for Cell Culture Experiment

Method

  • Cell Thawing (NIH3T3 cryovials, 37 °C water bath)
  • Subculturing / Passaging (PBS wash, Trypsin-EDTA, centrifuge, resuspension)
  • Media Change (DMEM + 10% FBS + 1% PenStrep)
  • Cell Seeding (Hemocytometer + Trypan Blue for counting, seeding into plates)
  • Cell Freezing for Stocking (90% FBS + 10% DMSO, cryovials, -80 °C, LN₂)

Material

  • Frozen NIH3T3 cells
  • DMEM + 10% FBS + 1% Penicillin-Streptomycin
  • Trypsin-EDTA
  • PBS
  • Hemocytometer + Trypan Blue
  • T-flasks, 6/24/96 well plates
  • Cryovials, DMSO
Figure 14
Figure 14

Notes

  • All procedures at 37 °C, 5% CO₂ incubator
  • Maintain aseptic technique
  • Figures recorded: Pipetting, Cell Counting, Hemocytometer, Seeding

<Experiments Day 2: Plasmid Transfection>

Time/Date

Day 2

Participants

Andrew Suh, Peter Kang, Eunu Baek

Goal

Introduce NRAS-WT and NRAS-G12D plasmids into NIH3T3 cells via Lipofection

Method

  • Cell Preparation (Seed NIH3T3 cells at 60–80% confluency in 6-well plates)
  • DNA–Lipid Complex Formation (Plasmid DNA + P3000 reagent, Lipofectamine 3000, Opti-MEM)
  • Transfection (Add complexes dropwise, swirl gently)
  • Incubation (6 h, 37 °C, 5% CO₂, then replace with fresh DMEM)
  • Verification (mCherry fluorescence check for successful transfection)
  • Post-Transfection Care (Media change, morphology monitoring)
Figure 15
Figure 15
Figure 16
Figure 16

Material

  • NIH3T3 cells (60–80% confluency)
  • Plasmids: NRAS-WT and NRAS-G12D (with mCherry reporter)
  • Lipofectamine™ 3000 + P3000 reagent + Opti-MEM
  • DMEM + 10% FBS + 1% PenStrep
  • PBS, Trypsin-EDTA, sterile tubes, pipettes, tips
  • 6-well plates, T-flasks

Notes

  • Control: Untransfected NIH3T3
  • WT Group: NRAS-WT plasmid transfection
  • Mutant Group: NRAS-G12D plasmid transfection
  • Stable pools generated with puromycin selection
  • Always keep plasmids on ice, check purity (NanoDrop A260/A280 ~1.8–2.0)
  • Figures recorded: DNA purity check (NanoDrop), fluorescence validation

<Experiments Day 3: Gene Expression Analysis>

Time/Date

Day 3

Participants

Eileen Byon, Aiden Chee, Aiden Park, Eunu Baek, Sihyun Lee

Goal

Confirm expression of NRAS-WT and NRAS-G12D plasmids at RNA and protein levels

Method

  • Fluorescence Microscopy (mCherry reporter visualization, % positive cells)
  • RNA Extraction & cDNA Synthesis (PureLink RNA kit, RocketScript RT Premix)
  • PCR Amplification (NRAS-WT, NRAS-G12D, GAPDH control)
  • Gel Electrophoresis (Agarose gel, DNA ladder, visualization under gel doc)
  • Cell Freezing for Stocking (90% FBS + 10% DMSO, cryovials, -80 °C, LN₂)
Figure 17
Figure 17
Figure 18
Figure 18

Material

  • NIH3T3 cells (from Exp. 2 plasmid transfection)
  • Fluorescence microscope with mCherry/Texas Red filter
  • Invitrogen PureLink RNA Mini Kit
  • Bioneer RocketScript RT Premix (cDNA synthesis)
  • PCR premix, GAPDH & NRAS primers
  • NanoDrop spectrophotometer
  • Agarose gel, TAE buffer, DNA ladder, Gel doc system

Notes

  • Reporter: mCherry confirms transfection
  • Internal Control: GAPDH normalizes expression
  • RNA integrity checked via NanoDrop (A260/280 = 1.8–2.1)
  • Include no-template control (NTC) in PCR
  • Figures recorded: RNA extraction, PCR machine, Gel electrophoresis
  • Practice skills: Avoid photobleaching, maintain RNase-free setup, compare WT vs G12D band intensity

<Experiments Day 4: CCK-8 Drug Response Assay with MEK inhibitor U0126>

Time/Date

Day 4

Participants

Eileen Byon, Aiden Chee, Aiden Park, Eunu Baek, Sihyun Lee

Goal

Evaluate cytotoxic effects of MEK inhibitor U0126 on NIH3T3 cells using CCK-8 assay

Method

  • Cell Seeding: 1 × 10⁴ cells/100 µL per well, 96-well plate, 24 h incubation
  • Drug Dilution & Treatment: Serial dilutions of U0126 (40–2.5 µM), triplicates, DMSO vehicle control
  • CCK-8 Addition: Add 20 µL reagent, incubate 2 h, protect from light
  • Absorbance Measurement: Plate reader at 450 nm, triplicate readings, normalized to controls
Figure 19
Figure 19
Figure 20
Figure 20
Figure 21
Figure 21

Material

  • NIH3T3 cells
  • U0126 stock solution (10 mM in DMSO)
  • CCK-8 kit (WST-8 reagent)
  • 96-well plate
  • DMEM + 10% FBS + 1% Pen/Strep
  • Plate reader (450 nm)
  • Sterile pipettes, tips, conical tubes

Notes

  • Principle: WST-8 reduced by dehydrogenases → orange formazan dye, absorbance ∝ viability
  • U0126 MW = 380.4 g/mol, stock prep: 10 mM (3.804 mg in 1 mL DMSO)
  • Controls: Blank wells (media + CCK-8, no cells), DMSO vehicle-only = 100% viability
  • Practice Skills: Serial dilution, bubble-free pipetting, run triplicates for reliability
  • Figures recorded: Serial dilution, plate seeding, CCK-8 loading, absorbance reading

<Experiments Day 5:Lab-On-A-chip Fabrication>

Time/Date

Day 5

Participants

Eileen Byon, Aiden Chee, Eunice Kang, Elliot Hoyoung Nam, Aiden Park, Soo Young Pee, Aiden Seunghwan Chee, Minjun Hwang

Goal

Fabricate a PDMS-based Lab-on-a-Chip for cell seeding, migration assays, and drug testing

Method

  • PDMS Preparation: Mix base:curing agent (10:1), pour into molds, de-gas, cure at 60 °C (3–4 h or overnight)
  • Layer Cutting & Punching: Remove cured PDMS, punch reservoirs/chambers/inlets (1–8 mm)
  • Plasma Bonding: Plasma-treat PDMS layers + glass slide, align and bond (Layer 3 → Layer 2 → Membrane → Layer 1 → Glass)
  • Chip Washing & Conditioning: Sequential wash (70% EtOH → PBS → DMEM), check flow uniformity and leakage

Material

  • PDMS base + curing agent (10:1)
  • Silicon molds for each layer
  • Biopsy punches (1, 2, 6, 8 mm)
  • Plasma surface treatment system
  • Glass slides
  • Transwell porous membrane insert
  • 70% EtOH, PBS, DMEM
Figure 22
Figure 22
Figure 23
Figure 23
Figure 24
Figure 24

Notes

  • Design Overview: Layer 1 (Reservoir), Layer 2 (Chamber Top), Layer 3 (Chamber Bottom + Channels + Membrane), Layer 4 (Glass slide)
  • Advantages of PDMS: Transparent, biocompatible, permeable, moldable
  • Practice Skills: Avoid bubbles in PDMS, consistent curing, membrane alignment, plasma bonding (O₂ 45 sccm, RF 90 W, 1 min)
  • Figures recorded: PDMS mixing, mold pouring, layer trimming, plasma bonding, final washed chips

<Experiments Day 6: 3D Bone Marrow Microenvironment Migration Assay>

Time/Date

Day 6

Participants

Eileen Byon, Aiden Chee, Eunice Kang, Elliot Hoyoung Nam, Aiden Park, Soo Young Pee, Aiden Seunghwan Chee, Minjun Hwang

Goal

Recreate a 3D bone marrow-like microenvironment in LOC with Matrigel to study NIH3T3 migration

Method

  • Chip Preparation: Wash LOC (EtOH → PBS → medium), pre-condition 30 min
  • Matrigel Injection: Thaw on ice, inject into ECM chamber with pre-cooled tips, incubate 37 °C (30–45 min polymerization)
  • Cell Seeding: Prepare NIH3T3 (1 × 10⁵ cells/mL), seed 100 µL suspension above Matrigel, fill reservoirs with medium
  • Incubation & Observation: Place LOC in 37 °C, 5% CO₂, 24–72 h; observe under fluorescence/brightfield; use rocking shaker to mimic dynamic flow
Figure 25
Figure 25
Figure 26
Figure 26
Figure 27
Figure 27

Material

  • LOC device (from Day 5)
  • Matrigel (final conc. 4 mg/mL, thawed on ice)
  • NIH3T3 cells
  • DMEM + 10% FBS + 1% Pen/Strep
  • PBS, 70% EtOH
  • CO₂ incubator, rocking shaker
  • Ice bucket, sterile micropipettes/tips, dishes

Notes

  • Principle: LOC + Matrigel simulates ECM and stromal environment → closer to in vivo
  • Safety: NIH3T3 fibroblasts used instead of myeloma cells (iGEM guidelines)
  • Practice Skills: Keep Matrigel on ice, avoid bubbles, ensure uniform ECM fill
  • Imaging: Capture time-lapse, use fluorescence (mCherry) for tracking
  • Figures recorded: Matrigel seeding, LOC incubation with shaker

Meetings

<Team Korea_CX Meetings>

Date : 03/17/2025
Event: 1st Whole Team Meeting(Virtual)
Agenda:
Members introduced themselves for the first time. They were also shared with the history and accomplishments of the Korea-HS team and iGEM. Instructors shared the characteristics of the iGEM competition, simple concepts of synthetic biology, and the overall plan for the competition (Grand Jamboree, etc.)

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Date: 3/30/2025
Event: Kick-off meeting
Agenda:

Members shared their ideas about team operation, including communication methods, Google Drive, and the availability form. Members also had an icebreaking activity, sharing their majors and areas of interest. They were then assigned to the groups they were interested in: wet lab, inclusivity, education, experiments, and wiki/deliverables.

Members made additional agreements on some policies, such as equal contribution and strikes, and went through the medal criteria.

Figure 1
Figure 1

Date: 04/06/2025
Event: 3rd Whole team meeting – lecture and elections
Agenda: Members had a lecture on “Lab-on-a-Chip for Modeling Genetic Mutation-Driven Multiple Myeloma Progression,” covering topics such as the definition of blood cancer and its three types, the pathological characteristics of multiple myeloma, its mutations, current treatments, and future prospects. The election for the whole team leader, co-leader, and sub-team leaders also took place.

Figure 2
Figure 2

Date: 04/13/2025
Event: 4th Whole team meeting
Agenda: Members created their iGEM accounts and went through the iGEM policies. They were then assigned to subteams and made specific future plans.

Date: 04/20/2025
Event: 5th Whole team meeting – competition updates and sub-team progress
Agenda: Subteams shared their plans related to the competition. The experiment team discussed simple ideas and the direction of their experiments. Members listened to the lecture “Modern Illustrative Introduction to Genetics,” which covered replication, transcription, and translation of DNA to protein, using NRAS G12D as an example.

Figure 3
Figure 3

Date: 05/18/2025
Event: 6th Whole Team Meeting – Post-AP restart + midterm planning
Agenda: After the break due to the AP exams, members had a check-up session for each subteam. They shared ideas they had developed independently during the break: the education team organized previous data and turned them into a presentation; the inclusivity team planned interview and outreach activities; the experiments team prepared for the summer wet lab (experiment procedures, material lists, etc.); and the wiki team worked on website structuring and design sharing. Members agreed to have the first mid-term check in June.

Date: 05/25/2025
Event: 7th Whole team meeting - tumor microenvironment lecture and midterm planning
Agenda: Members attended a lecture by Dr. Min Hee Park, titled “Understanding the Tumor Microenvironment.” The lecture covered the limitations of currently used anticancer drugs, the definition and role of TEE, differences between 2D, 3D, and organ-on-a-chip models, and the microenvironment of bone marrow in multiple myeloma.

Figure 4
Figure 4

Date: 06/01/2025
Event: 8th Whole Team meeting – lab-on-a-chip lecture and experimental design
Agenda: Members attended the professor’s lecture, which covered the concept of lab-on-a-chip, microfluidics, the process of making a PDMS chip and using CAD, as well as its advantages and limitations.

Figure 5
Figure 5

Date: 06/08/2025
Event: 9th Whole Team meeting – lab-on-a-chip lecture and experimental design
Agenda: Members attended the professor’s second lecture on the concept of lab-on-a-chip. The lecture included topics such as organ-on-a-chip, a case study of Crohn’s disease-on-a-chip, and building a multiple myeloma (MM) chip. The model included: Top for MM cells (with/without mutation); Middle for Matrigel or collagen gel representing a bone marrow-like ECM environment; Bottom for Collection zone to measure migrated cells. Members hypothesized that mutated MM cells might migrate further and faster

Figure 6
Figure 6

Date: 06/15/2025
Event: 10th Whole Team Meeting - Wiki Discussion Workshop
Agenda: Members were divided into two groups and had a discussion on “What makes good writing?” After the group discussion, they shared what they found important. Some of the key qualities that typically define good writing include: Clear and concise expression, Logical categorization, Well-structured and organized sentences, Simple yet detailed explanations of the topic, Clarity throughout the writing, and specific examples (facts, graphs, statistics) to support the main idea or thesis

Figure 7
Figure 7
Figure 8
Figure 8

Date: 06/22/2025
Event: 11th Whole Team meeting – Conceptual overview of Cell experiments
Agenda: Members attended the professor’s lecture, learning about the importance of experiments—turning ideas into visible results—cell culture, gene modulation, and other topics. Our conclusion was that each experiment provides one piece of the puzzle, and together, they tell the full story. Members also planned an in-person meeting.

Figure 9
Figure 9

Date: 06/29/2025
Event: 12th Whole Team meeting – in-person meeting(award strategy and wiki documentation
Agenda: Members gathered in person and set an internal calendar for iGEM 2025, including both the internal calendar and the competition calendar. They conducted a subteam process check (HP, Education, Experiments, Wiki/Documentation) and discussed award strategies. They also discussed why some iGEM teams fail, citing reasons such as a lack of understanding of the project and issues with copyright/citation. ⇒ The teams considered how to ensure the success of their project by emphasizing urgency, creativity, and quantitative proof. Lastly, the members got separated into each subteams, conducting outside activities for human practices.

Date: 07/06/2025

Event: 13th Whole Team Meeting – LOC Development & Cell Migration (Lecture 6)
Agenda: Members attended a lecture by Joo Young Sim, M.S., on lab-on-a-chip (LOC) development and cell migration, focusing especially on vector design and cell groups. Additionally, members set up the experiment schedule and determined the lab location. The workflow included: cell culturing to an on-chip migration assay. Subteams were divided into three groups: Group 1(Building lab-on-a-chip and migration assays), Group 2(Modulating genes), and Group 3(Working on cell culture and vector optimization)

Figure 10
Figure 10
Figure 11
Figure 11

Date: 08/09/2025
Event: 14th Whole Team meeting - 2nd In-person Meeting
Agenda: Members gathered offline to discuss the directions of our project. They made a mindmap for 2025 iGEM Project of Korea-CX by writing down all the activities and intentions on each branch. Next, they worked on Human Practices by searching for community centers to visit. Other than that, the members took time to make a promotion video.

Figure 12
Figure 12

Date: 08/17/2025
Event: 15th Whole Team Meeting – progress updates & wiki finalization
Agenda: Members watched the promotion video and shared their opinions on some limitations that could be improved. They also reviewed the wiki page formatting and documentation process.

Figure 13
Figure 13

Date: 08/24/2025
Event: 16th Whole Team Meeting – progress updates & wiki finalization
Agenda: Our team shared subteam updates, set timelines for August–September, and reviewed Wiki and safety deadlines. We also confirmed that all Wiki pages must be finalized by October 6, before the Freeze. Immediate follow-up meetings were scheduled for HP, Experiments, and Wiki/Documentation.

Date: 09/07/25
Event: 17th Whole Team Meeting
Agenda: The team conducted a progress check on sub-teams. We also discussed what we will be doing in each subteam from now on. Next we did a progress check on wiki pages. We went over the criteria for wiki format once again.

Date: 09/14/25
Event: 18th Whole Team Meeting
Agenda: Members made a document in which they added the list of items that we need in our Team Booth, and started to discuss and design what our Team booth will look like in the Grand Jamboree.

<Education Meetings>

Date: 03/30/2025
Event: Education Introduction Meeting
Agenda: Our team held the first education sub-team meeting to introduce the education prize and the criterias. We reviewed examples of activities and projects other teams did in the past, went over the overall timeline of the activities and the responsibilities of each member.

Date: 04/06/2025
Event: Timeline Decision & Feedback Session
Agenda: We went over the internal calendars to determine the exact dates and deadlines of the tasks and activities. We also reviewed the past educational outreach activities from previous teams and assessed their effectiveness. Then, we discussed what worked well and what could be improved, ultimately to apply them to our own activities.

Date: 05/18/2025
Event: Outreach Plans Development Session
Agenda: We introduced the two new members of our team the tasks and activities we would have to do in Education. Our team started brainstorming possible outreach activities and organizations that we would need to contact. We reviewed each others’ ideas and provided feedback in order to implement and develop better ideas and strategies.

Date: 06/30/2025
Event: Podcast Work Session #1
Agenda: We selected the podcast as one of our main outreach strategies. We planned out three episodes describing the connection between Multiple Myeloma and Lab-on-a-chip. We also started writing the script for episode 1, which is dedicated to explaining the MM and its symptoms.

Date: 07/13/2025
Event: Podcast Work Session #2
Agenda: We continued working on our podcasts that will be posted on our team instagram. We filmed episode 1 of our podcast while getting started on writing the script for episode 2 and finishing the research for episode 3.

Date: 07/27/2025
Event: Podcast Work Session #3
Agenda: We continued working on our podcasts. We finished editing episode 1 of our podcast about Multiple Myeloma and the Lab-on-a-chip technology. We filmed the episode 2 parts while finishing the script of the final episode.

Date: 08/10/2025

Event: Podcast Work Session #4
Agenda: We finished working on the podcast, as we already had filmed episode 3 part as homework and focused on editing the both episodes. We were eventually able to finish and share the finalized version of all 3 episodes to the whole team.

<Wiki/Deliverables Meetings>

Date: 04/12/2025
Event: Wiki/Deliverables Introduction Meeting
Agenda: Our team held the first education sub-team meeting to go over the brief timeline of the Wiki/Deliverables tasks and the medal criterias related to our job. As homework, we assigned each of the members to come up with their own logo and t-shirt design ideas for our team.

Date: 06/06/2025
Event: Website Design Session
Agenda: We brainstormed various ideas about our website design. We discussed the color scheme, icons/logos, fonts, and how all of the elements connect with our project. We came up with multiple samples and gave feedback to each of them, ultimately combining various ideas to get the best design we could think of.

Date: 07/10/2025
Event: T-shirt & Hoodie Design
Agenda: We finalized our logo design as the one containing the pink ribbon representing multiple myeloma, along with t-shirt and hoodie designs. We sent the finalized designs to the maker so he could produce it based on the designs we suggested.

Date: 08/07/2025
Event: PV work session
Agenda: We wrote down scripts for our promotion video as well as planning scenes and cuts of the video itself. After recording everything, we managed to match the video we created based on our plans with our voice recordings, and make some edits to increase the quality. Later after the meeting, Aiden Chee was able to finalize and submit the PV.

<Human Practices / Inclusivity Meetings>

Date: 03/30/25
Event: 1st HP Meeting
Agenda: Our team held the first meeting to introduce Human Practices (HP) and the Inclusivity Award. We reviewed past award-winning examples, discussed how to integrate inclusivity activities into our project, and created brief planning of our surveys, interviews, and awareness campaigns.

Date: 04/12/25
Event: 2nd HP Meeting

Agenda: Our team analyzed past Inclusivity Award projects from Jiashu Shanghai and Thailand RIS, discussing each team’s strengths and weaknesses to refine our own inclusivity strategy. We also began crafting our online awareness survey.

Date: 05/17/25
Event: 3rd HP Meeting
Agenda: Our team focused on promoting the online survey to reach over 60 responses. We also held a brainstorming session to select our Inclusivity topic, concluding that in Korea, conservative attitudes toward synthetic biology limit public acceptance. We decided to address this challenge by promoting awareness through public campaigns and community seminars.

Date: 05/31/25
Event: 4th HP Meeting
Agenda: Our team reviewed survey summaries and began planning stakeholder interviews for broader understanding in synthetic biology. Members were assigned to contact relevant stakeholders–such as teachers, professors, or researchers–to gather insights on public perceptions of gene editing and multiple myeloma.

Date: 07/10/25
Event: 5th HP Meeting
Agenda: Our team reviewed the iGEM Judging Handbook and discussed strategies for the Inclusivity and Integrated Human Practices components. We brainstormed ideas for campaigns focused on supporting the elderly as our target minority group.

Interview / Outreach Meetings

Date: 06/07/25
Interviewee:
Medical Doctor Min Gu Kang
Philosophy Teacher Eun Jung Heo
Doctor Sung Ung Kang
Fortuga Bio CEO Sungjoon Yoon
Bio Teacher Lenny Musungu
Professor Minkyung Joo
Bio Teacher Minha Oh
Doctor Baek Daehyun
Bio Ethics Teacher Jae Eun Shim
Director from the International Myeloma Foundation
Bio Teacher Jae Jin Lim
Philosophy Major Alponse Munyentwari
Bio Teacher David Dai
Biology Student Charlotte Godfrey
Participant: Peter Kang, Eunice Kang, Aiden Park, Aiden Lee, Minjun Hwang
Agenda: Member got to discuss

Date: 07/09/25
Interviewee: Professor Wonil Choi
Participants: Aiden Park, Eunu Baek
Agenda: Members learned about the development of tumor therapies and techniques related to it. They got a chance to ask professor questions about multiple myeloma and get a validation on their project related to the current situations of cancer treatments.

Date: 07/12/25
Interviewee: CEO of Decodecell (RNA Sequencing Company)
Participants: Eunu Baek, Peter Kang
Agenda: Members learned about next generation sequencing, technology related to fragments of DNA. By asking the interviewee questions, they could get to know ways of conducting single-cell RNA sequencing on multiple myeloma samples. They also got advice for their project, such as being careful with designing the control group and having a well defined purpose.

Date: 07/19/25
Interviewee: Professor Seungmin Kim
Participants: Aiden Lee, Aiden Chee, Sihyun Lee
Agenda: Members went on a bio chip lab tour and learned about organoid and bio chip. They got meaningful insights from the professor related to bio chips and our project.

Date: 09/26/2025
Event: Children Center Visit
Agenda: We visited the local child care center at Gyeongki-do to share our project and knowledge with the children as well as introducing them to the concept of synthetic biology through various activities. After a brief presentation, we had sessions where the children learned terms related to cancer and treatment, and drew pictures related to the ideas of our project.

Date: 09/26/2025
Event: Nursing Home Visit
Agenda: We visited the local nursing home for elders during the Chuseok break. We passed on the pamphlets we made to inform the elders about our project and basic knowledge of synthetic biology. We went through a brief presentation and quickly moved on to the puzzle matching activities we prepared, imitating some of the dementia prevention activities. After the activities we received feedback from each of the elders before leaving.