iGEM 2025 — Team korea-cx

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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](https://static.igem.wiki/teams/5925/images/note14.webp)

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](https://static.igem.wiki/teams/5925/images/note15.webp)

Figure 15

![Figure 16](https://static.igem.wiki/teams/5925/images/note16.webp)

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](https://static.igem.wiki/teams/5925/images/r-note17.avif)

Figure 17

![Figure 18](https://static.igem.wiki/teams/5925/images/note18.webp)

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](https://static.igem.wiki/teams/5925/images/note19.webp)

Figure 19

![Figure 20](https://static.igem.wiki/teams/5925/images/r-note20.avif)

Figure 20

![Figure 21](https://static.igem.wiki/teams/5925/images/note21.webp)

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](https://static.igem.wiki/teams/5925/images/note22.webp)

Figure 22

![Figure 23](https://static.igem.wiki/teams/5925/images/note23.webp)

Figure 23

![Figure 24](https://static.igem.wiki/teams/5925/images/note24.webp)

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](https://static.igem.wiki/teams/5925/images/note25.webp)

Figure 25

![Figure 26](https://static.igem.wiki/teams/5925/images/note26.webp)

Figure 26

![Figure 27](https://static.igem.wiki/teams/5925/images/note27.webp)

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.)

-

**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](https://static.igem.wiki/teams/5925/images/note1.webp)

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](https://static.igem.wiki/teams/5925/images/note2.webp)

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](https://static.igem.wiki/teams/5925/images/note3.webp)

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](https://static.igem.wiki/teams/5925/images/note4.webp)

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](https://static.igem.wiki/teams/5925/images/note5.webp)

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](https://static.igem.wiki/teams/5925/images/note6.webp)

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](https://static.igem.wiki/teams/5925/images/note7.webp)

Figure 7

![Figure 8](https://static.igem.wiki/teams/5925/images/note8.webp)

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](https://static.igem.wiki/teams/5925/images/note9.webp)

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](https://static.igem.wiki/teams/5925/images/note10.webp)

Figure 10

![Figure 11](https://static.igem.wiki/teams/5925/images/note11.webp)

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](https://static.igem.wiki/teams/5925/images/note12r.webp)

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](https://static.igem.wiki/teams/5925/images/note13.webp)

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.

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Engineering

## Engineering

### 1. Introduction

Our project aimed to engineer a robust experimental model to study the role of NRAS mutations, particularly NRAS^G12D, in cancer progression and therapy resistance. Conventional 2D culture systems fail to capture the complexity of the tumor microenvironment, and simple gene overexpression models often lack stable, long-term expression. To address these limitations, we combined **plasmid vector engineering with chip-based 3D culture technology**.

Guided by the **Design–Build–Test–Learn (DBTL) cycle**, we iteratively refined plasmid constructs, transfection strategies, and chip architecture. Through this process, we established a platform that ensures stable NRAS expression and reproducible functional analysis within a physiologically relevant 3D-like invasion environment.

### 2. Plasmid Vector Engineering

#### <Design>

![Figure 1](https://static.igem.wiki/teams/5925/images/eng1.webp)

Figure 1 (Plasmid map of NRAS^WT construct. Major features include EF1α promoter, Kozak, NRAS, Luc2, mCherry, and puromycin resistance cassette)

We designed plasmids to constitutively express either wild-type NRAS (NRAS^WT) or the oncogenic mutant NRAS^G12D. The EF1α promoter was selected due to its robust and stable activity across mammalian cells, reducing the risk of silencing compared to CMV. A Kozak sequence was included to optimize translational initiation.

To enable co-expression of reporters, we introduced self-cleaving 2A peptides (T2A/P2A) between coding sequences, allowing a polycistronic transcript to generate multiple proteins. Luc2 provided a quantitative luminescence readout of cell viability and NRAS expression, while mCherry offered a fluorescent marker for imaging and tracking transfected cells. Transcript stability was ensured with a BGH/SV40 polyA signal, and a puromycin resistance cassette facilitated selection of stable NIH3T3 lines.

For the NRAS coding region, we used the human NRAS sequence (RefSeq: NM\_002524.5). Two variants were synthesized:

* **Wild-type NRAS (NRAS^WT)**: containing the canonical glycine codon (**GGT**) at position 12.
* **Mutant NRAS (NRAS^G12D)**: carrying a single base substitution (**GGT → GAT**) at codon 12, which results in a glycine (G) to aspartic acid (D) change. This mutation is a well-known oncogenic driver in multiple cancers.

Full NRAS^WT and NRAS^G12D sequences are shown below.

**Highlighted regions:**

* Yellow = primer binding sites used for PCR verification
* Red = codon 12 (GGT → GAT) mutation site

##### NRAS^WT

```
ATGACTGAGTACAAACTGGTGGTGGTTGGAGCAGGTGGTGTTGGGAAAAGCGCACTGACAATCCAGCTAATCCAGAACCACTTTGTAGATGAATATGATCCCACCATAGAGGATTCTTACAGAAAACAAGTGGTTATAGATGGTGAAACCTGTTTGTTGGACATACTGGATACAGCTGGACAAGAAGAGTACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCTCTGTGTATTTGCCATCAATAATAGCAAGTCATTTGCGGATATTAACCTCTACAGGGAGCAGATTAAGCGAGTAAAAGACTCGGATGATGTACCTATGGTGCTAGTGGGAAACAAGTGTGATTTGCCAACAAGGACAGTTGATACAAAACAAGCCCACGAACTGGCCAAGAGTTACGGGATTCCATTCATTGAAACCTCAGCCAAGACCAGACAGGGTGTTGAAGATGCTTTTTACACACTGGTAAGAGAAATACGCCAGTACCGAATGAAAAAACTCAACAGCAGTGATGATGGGACTCAGGGTTGTATGGGATTGCCATGTGTGGTGATG
```

##### NRAS^G12D

```
ATGACAGAGTACAAACTGGTGGTGGTGGGGGCCGACGGGGTGGGGAAGAGCGCCCTGACCATTCAGCTGATCCAGAATCACTTTGTGGACGAGTACGACCCAACCATCGAGGACAGCTATAGAAAACAGGTCGTGATCGATGGTGAAACCTGTCTGCTGGACATCCTGGACACTGCAGGCCAGGAGGAGTACTCTGCCATGAGAGACCAGTACATGAGGACCGGCGAGGGATTTCTGTGCGTGTTTGCTATCAACAACTCTAAGAGTTTCGCCGATATCAATCTGTACAGAGAGCAGATCAAAAGGGTGAAGGACAGCGACGACGTGCCCATGGTGCTGGTGGGCAATAAGTGCGACCTGCCAACCCGGACAGTGGATACCAAGCAGGCCCACGAACTGGCCAAGAGTTACGGGATCCCTTTCATCGAGACCAGCGCCAAGACCAGACAGGGCGTGGAGGACGCCTTCTACACCCTGGTGAGGGAGATTCGGCAGTACCGCATGAAGAAGCTGAATAGCTCCGACGACGGAACACAGGGCTGCATGGGCCTGCCCTGTGTGGTGATG
```

By verifying these sequence constructs with targeted primers, we ensured that our plasmids faithfully model both the wild-type and oncogenic states of NRAS, providing a reliable foundation for downstream functional assays.

#### <Build>

Plasmids encoding NRAS^WT and NRAS^G12D with an mCherry reporter were synthesized and introduced into NIH3T3 cells using Lipofectamine-mediated transfection. Transfection conditions followed the manufacturer’s recommended protocol (Thermo Fisher Scientific, “Transfecting Plasmid DNA into NIH3T3 Cells Using Lipofectamine® LTX Reagent”).

Stable pools were subsequently generated through puromycin selection (5 µg/ml for 2 days). This approach enriched reporter-positive cells and minimized heterogeneity for downstream assays.

To functionally model NRAS-driven phenotypes, these engineered plasmids were designed to express either wild-type or mutant NRAS^G12D upon nuclear delivery and transcription. Expression of NRAS variants allowed us to investigate proliferation, drug resistance, and migratory behavior in NIH3T3 cells, providing the foundation for downstream experiments within our lab-on-a-chip platform.

![Figure 2](https://static.igem.wiki/teams/5925/images/eng2.webp)

Figure 2 (Overview of plasmid vector transfection into NIH3T3 cells for NRAS construct delivery. Engineered plasmid vectors were introduced into NIH3T3 cells via lipid-based transfection. Once delivered into the nucleus, the plasmids undergo transcription to generate NRAS mRNA, which is subsequently translated into protein.)

#### <Test>

* **Selection with puromycin:** To confirm the functionality of the puromycin resistance cassette in our plasmid, NIH3T3 cells were subjected to puromycin (5 μg/mL) for 2 days, beginning 48 h post-transfection.
* **Reporter validation:** Brightfield and mCherry fluorescence imaging confirmed that NRAS^WT and NRAS^G12D constructs were successfully expressed in NIH3T3 cells compared with control.

![Figure 3](https://static.igem.wiki/teams/5925/images/eng3.webp)

Figure 3 (Puromycin selection of transfected NIH3T3 cells. NIH3T3 cells were transfected with plasmid and subjected to puromycin selection starting 48 h post-transfection. Brightfield images show: (left column) No puromycin treatment, (middle column) Cells treated with puromycin (5 µg/ml) for 2 days, and (right column) Cells after puromycin washout, showing only surviving resistant cells. Puromycin selection effectively eliminated non-transfected cells, leaving behind a population of resistant cells. Scale bar = 100 µm)

![Figure 4](https://static.igem.wiki/teams/5925/images/eng4.webp)

Figure 4 (Validation of plasmid expression in NIH3T3. Brightfield (top) and mCherry fluorescence (bottom) images of control (CON), NRAS^WT, and NRAS^G12D lines. Reporter fluorescence confirmed successful delivery and expression of engineered plasmids. Scale bar = 100 μm)

#### <Learn & Improve>

* 1. **Delivery optimization:** Transfection efficiency was optimized by adjusting DNA-to-reagent ratios, ensuring high viability while maintaining robust reporter expression.
* 2. **Stable pools:** Short and decisive puromycin selection (5 μg/mL for 2 days, followed by washout) generated uniform, reporter-positive populations, minimizing heterogeneity in assays.
* 3. **Lesson:** Iterative adjustment of transfection and selection enabled us to build a robust, reproducible plasmid system where EF1α-driven expression produced stable lines, reliable reporters, and phenotype-specific outcomes. These plasmid-engineered cells formed the basis for subsequent chip assays.
* 4. **Improve:** Beyond mCherry visualization, our plasmid system also contains a luciferase reporter, which can be leveraged in future assays for quantitative measurement of NRAS-driven phenotypes and drug responses. This dual-reporter strategy enhances flexibility—fluorescence allows rapid microscopy-based screening, while luminescence provides sensitive, quantitative readouts for downstream optimization.

### 3. Chip Engineering

#### <Design>

To better model tumor cell migration in a physiologically relevant context, we designed a microfluidic lab-on-a-chip (LOC) system with two compartments. Cells were seeded in the upper chamber, which was covered with a Matrigel layer to simulate the extracellular matrix barrier. The lower chamber collected migrated cells after they traversed through the Matrigel.

The connecting channels were filled with culture medium and placed on a rocking shaker, creating gentle oscillatory flow that mimicked body fluid dynamics. This setup allowed us to measure NRAS-dependent invasion under both ECM and dynamic fluid conditions.

![Figure 5](https://static.igem.wiki/teams/5925/images/eng5.webp)

Figure 5 (Schematic design of the lab-on-a-chip (LOC) device for migration assays. The chip consists of four layers: (1) a reservoir layer (8 mm holes, 4 mm thickness) for medium loading, (2) a top layer with 6 mm middle holes, 1 mm side holes, and 0.5 mm microchannels, (3) a bottom layer with 8 mm middle holes, 2 mm side holes, and 0.5 mm microchannels, and (4) a glass slide base for imaging)

#### <Build>

![Figure 6](https://static.igem.wiki/teams/5925/images/eng6.webp)

Figure 6 (Scheme of Lab-on-a-chip migration assay using Matrigel-embedded NIH3T3 cells.)

Chips were fabricated from PDMS via soft lithography, bonded to glass slides, and sterilized. Before seeding, the upper chamber was layered with Matrigel to form a 3D ECM barrier. NRAS^WT and NRAS^G12D NIH3T3 cells were seeded on top of this layer. The chip was supplied with medium in the lower chamber, and the entire system was placed on a rocking shaker, introducing gentle movement of the medium in the channels to simulate body fluid motion.

#### <Test>

* **Imaging:** Brightfield microscopy confirmed migrated cells collected in the lower chamber after 72 h for control, NRAS^WT, and NRAS^G12D groups. Representative images showed markedly higher migration in NRAS^G12D compared with NRAS^WT and control.

![Figure 7](https://static.igem.wiki/teams/5925/images/eng7.webp)

Figure 7 (Brightfield images of migrated cells in the LOC assay after 72 h (CON, NRAS^WT, NRAS^G12D). Both NRAS^WT and NRAS^G12D showed higher migration than control, with NRAS^G12D exhibiting the strongest infiltration.)

* **Functional assays:** Migrated cells were counted across multiple fields. Normalized to control (CON = 100%), NRAS^WT showed ~150–250%, and NRAS^G12D ~250–450% migration.
* **Dynamic conditions:** rocking shaking supported nutrient exchange and physiological relevance.
* **Quantification:** Both NRAS^WT and NRAS^G12D cells showed significantly increased migration compared with control, with NRAS^G12D showing the highest infiltration capacity (p < 0.01).

![Table 1](https://static.igem.wiki/teams/5925/images/eng-table-1.webp)

Table 1 (Cells (CON, NRAS^WT, and NRAS^G12D) were seeded at 1×10^5 cells on Matrigel-coated LOC chambers. After 72 h, migrated cells in the lower compartment were counted from multiple microscopic fields and expressed as mean cell number ± SD. Data were normalized to the control group (CON = 100%). Both NRAS^WT and NRAS^G12D groups showed significantly higher migration compared to control, with NRAS^G12D exhibiting the strongest infiltration capacity (p < 0.05, \*p < 0.01 vs. CON))

#### <Learn & Improve>

During the chip-based migration experiments, several technical challenges emerged:

1. **1. Matrigel barrier thickness**
   * ○ When the Matrigel layer was too thick, very few cells were able to infiltrate.
   * ○ When it was too thin, the barrier collapsed and failed to mimic a realistic ECM.
   * ○ Iterative adjustment led us to an intermediate thickness that allowed differential migration between groups while maintaining barrier stability for 72 h.
2. 2. **Rocking shaker speed**
   * ○ Excessive shaking disrupted the Matrigel surface, causing irregular migration patterns.
   * ○ Insufficient shaking reduced nutrient exchange, which decreased cell viability.
   * ○ By calibrating the orbital speed, we reproduced physiologically relevant body fluid dynamics while keeping the ECM barrier intact.
3. 3. **Imaging and quantification**
   * ○ Early counts varied due to inconsistent imaging fields.
   * ○ Standardizing the imaging area and normalizing migrated cell numbers to the control (CON = 100%) improved reproducibility across replicates.

Through optimization of ECM thickness, fluid dynamics, and imaging methods, we established a reproducible LOC invasion platform. This system consistently revealed that NRAS^G12D cells have markedly stronger migration capacity than NRAS^WT, faithfully reflecting oncogenic phenotypes in a 3D-like, dynamic environment.

### 4. Integration of Plasmid & Chip Platforms

The major innovation of our project lies in combining engineered NRAS plasmid vectors with a Matrigel-based LOC migration model. Stable NRAS^WT and NRAS^G12D lines were seeded at 1×10^5 cells per chip and migration was quantified after 72 h under rocking shaking.

This integration allowed us to:

* Directly compare wild-type vs mutant NRAS phenotypes within the same microenvironment.
* Quantify migration capacity using normalized brightfield counts.
* Leverage reporter signals (mCherry, luciferase) to validate plasmid expression and support parallel functional assays.
* Enable drug response evaluation under physiologically relevant ECM + fluid conditions.

By merging plasmid engineering with chip-based invasion assays, we created a robust platform to study oncogenic NRAS-driven phenotypes in a reproducible and translatable format.

### 5. Conclusion

By iteratively applying DBTL cycles, we engineered a composite platform that integrates plasmid vector design with microfluidic chip technology. This approach enabled the establishment of a stable, reproducible, and physiologically relevant system for investigating NRAS-driven oncogenesis.

Our findings demonstrate that precise genetic circuit design, combined with optimized chip engineering, can yield a scalable model for cancer biology and drug evaluation. This work underscores the potential of synthetic biology to bridge molecular-level engineering with tissue-level disease modeling, providing a versatile framework for future therapeutic discovery and translational research.

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

Experiments

## Experiments

### Experiments Day 1: Cell Culture

#### <Part 1. Basic Theory of Cell Culture>

##### Experimental Principle

* **Definition**: Cell culture is the process of growing live cells in a controlled, artificial environment outside the body.
* **Purpose**: To study cell behavior, drug responses, and genetic changes.
* **Cell Types**: Immortalized cell lines such as NIH3T3 are commonly used.
* **Immortalized Cells**: These are cells engineered to divide indefinitely by overcoming senescence, often using viral genes, oncogenes, or telomerase.

##### Essential Components

1. **Media**: Provides nutrients
2. **FBS**: Serum supplying growth factors (10% v/v).
3. **Penicillin-Streptomycin**: Prevents bacterial contamination (1% v/v).
4. **Trypsin-EDTA**: Enzyme for detaching adherent cells.
5. **PBS**: For washing cells.

#### <Part 2. Cell Culture Procedures>

##### Materials Needed

* Frozen NIH3T3 cells in cryovials
* DMEM + 10% FBS + 1% PenStrep
* Trypsin-EDTA
* PBS
* Hemocytometer & Trypan Blue
* 6/24/96 well plates, T-flasks

##### Procedure

1. **1. Cell Thawing**
   * Thaw cryovial in 37 °C water bath (2 min).
   * Dilute into warm media, centrifuge (1350 rpm, 3 min).
   * Resuspend in fresh media, transfer to T-flask, incubate at 37 °C, 5% CO₂.
2. **2. Subculturing (Passaging)**
   * Remove old media, wash with PBS.
   * Add trypsin, incubate 1–3 min until cells detach.
   * Neutralize with media, collect cells, centrifuge, resuspend, and split.
3. **3. Media Change**
   * Replace old media with fresh pre-warmed DMEM.
4. **4. Cell Seeding**
   * Count cells using hemocytometer + Trypan Blue.
   * Calculate volume for target density.
   * Seed into plates or chips for downstream assays.
5. **5. Cell Freezing (Stocking)**
   * Harvest cells, centrifuge.
   * Resuspend in freezing media (90% FBS + 10% DMSO).
   * Aliquot into cryovials, freeze at -80 °C, transfer to LN₂ for long-term storage.

#### <Part 3. Practice Skills>

1. **Pipetting Practice**
   * Use micropipettes (P10, P20, P200, P1000).
   * Accuracy check with water-weighing (100 µL = 0.100 g, ±0.005 g).
   ![Figure 1](https://static.igem.wiki/teams/5925/images/r-exp1.avif)

   Figure 1 (Pipetting Practice done by Korea-CX member)
2. **Cell Counting with Trypan Blue**
   * Mix cells and Trypan Blue 1:1.
   ![Figure 2](https://static.igem.wiki/teams/5925/images/exp2.webp)

   Figure 2 (Korea-CX member mixing cells with Trypan Blue)

   * Load into hemocytometer (10 µL).
   ![Figure 3](https://static.igem.wiki/teams/5925/images/exp3.webp)

   Figure 3 (Cells are loaded into hemocytometer and shown on the screen)

   * Count 4 squares, calculate concentration and viability.
   ![Figure 4](https://static.igem.wiki/teams/5925/images/exp4.webp)

   Figure 4 (Korea-CX member counting cells inside the 4 squares)
3. **Seeding in 96-well Plate**
   * Determine seeding density (e.g., 2 × 10⁴ cells/well).
   * Calculate suspension volume, prepare master mix.
   * Dispense evenly, avoid bubbles, incubate at 37 °C, 5% CO₂.
   ![Figure 5](https://static.igem.wiki/teams/5925/images/exp5.webp)

   Figure 5 (Korea-CX member seeding cells in the 96-well plate)

   ![Figure 6](https://static.igem.wiki/teams/5925/images/exp6.webp)

   Figure 6 (Korea-CX member seeding cells in the 96-well plate)

   ![Figure 7](https://static.igem.wiki/teams/5925/images/exp7.webp)

   Figure 7 (96-well plate fully loaded with cells)

### Experiments Day 2: Plasmid Transfection

#### <Part 1. Basic Theory of Plasmid Transfection>

##### Experimental Principle

* **Definition**: Plasmid transfection is the process of introducing foreign DNA into eukaryotic cells using non-viral methods (e.g., lipofection or electroporation).
* **Purpose**: To induce gene expression of wild-type or mutant NRAS (WT vs G12D) and visualize successful delivery via mCherry fluorescence.
* **Application**: This enables downstream analysis of cell viability, migration, and drug sensitivity in genetically modified cells.
* **Plasmid Preparation**: Plasmids encoding NRAS^WT and NRAS^G12D with an mCherry reporter were synthesized and directly introduced into NIH/3T3 cells via Lipofectamine-mediated transfection. Stable pools were generated by puromycin selection.

##### Key Concepts

* **NRAS Mutation (G12D)**: A point mutation leading to constitutive activation of NRAS signaling.
* **Experimental Groups**:
  + **Control** : Untransfected NIH3T3 cells
  + **Wild-type group** : NIH3T3 cells transfected with NRAS-WT plasmid (with mCherry reporter)
  + **Mutant group** : NIH3T3 transfected with NRAS-G12D plasmid (with mCherry reporter)
* **Fluorescence Check** : Successful transfection is verified by mCherry fluorescence.
* **Transfection Methods**:
  + Lipofection: DNA-lipid complexes merge with cell membranes.

#### <Part 2. Transfection Procedures>

##### Materials Needed

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

##### Procedure (Lipofection Method)

1. **1. Cell Preparation**
   * Seed NIH3T3 cells in 6-well plates (2–3 × 10⁵ cells/well).
   * Ensure ~70% confluency on the day of transfection.
2. **2. DNA–Lipid Complex Formation**
   * Tube A: Dilute 2.5 µg plasmid DNA + 5 µL P3000 reagent in 125 µL Opti-MEM.
   * Tube B: Dilute 7.5 µL Lipofectamine 3000 in 125 µL Opti-MEM.
   * Combine Tube A and B, mix gently, incubate 10–15 min at room temperature.
3. **3. Transfection**
   * Add mixture dropwise to each well containing 2 mL fresh medium.
   * Gently swirl plate to distribute complexes evenly.
4. **4. Incubation**
   * Incubate 6 h at 37 °C, 5% CO₂.
   * Replace with fresh complete media.
   * Maintain for 24–48 h before downstream assays.

#### <Part 3. Practice Skills>

1. **1. Plasmid Handling**
   * Always keep plasmids on ice during setup.
   * Measure DNA purity with NanoDrop (A260/A280 ~1.8–2.0).
   ![Figure 8](https://static.igem.wiki/teams/5925/images/r-exp8.avif)

   Figure 8 (Korea-CX member loading a drop of liquid onto Nano Drop)
2. **2. Transfection Controls**
   * Replace media to reduce cytotoxicity.
   * Monitor cells daily for morphology changes.
3. **3. Post-Transfection Care**
   * Replace media to reduce cytotoxicity.
   * Monitor cells daily for morphology changes.

### Experiments Day 3: Gene Expression Analysis

#### <Part 1. Basic Theory of Gene Expression Analysis>

##### Experimental Principle

* **Definition**: Gene expression analysis measures whether transfected plasmids are expressed at RNA and protein levels.
* **Purpose**: To confirm successful delivery of NRAS-WT and NRAS-G12D plasmids in NIH3T3 cells.
* **Techniques**:
  + Fluorescence Microscopy for mCherry reporter expression.
  + RT-PCR to detect NRAS transcripts.
  + Gel Electrophoresis to visualize amplified products.

##### Key Concepts

* **mCherry Reporter**: Red fluorescence indicates successful plasmid uptake.
* **RNA → cDNA → PCR**: Stepwise workflow for validating transgene expression.
* **Internal Control (GAPDH)**: Ensures accuracy by normalizing expression levels.

#### <Part 2. Experimental Procedures>

##### Materials Needed

* NIH3T3 cells transfected with plasmids (Exp. 2)
* 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

##### A. Fluorescence Microscopy

1. Wash cells with PBS, add fresh media.
2. Focus under brightfield, switch to mCherry filter.
3. Capture images at multiple fields; record % positive cells.

##### B. RNA Extraction & cDNA Synthesis

1. Harvest 1 × 10⁶ cells, lyse with buffer + β-mercaptoethanol.
2. Bind RNA to column, wash, elute in RNase-free water (50 µL).
3. Quantify RNA with NanoDrop (260/280 ~1.8–2.1).
4. Use 500 ng RNA for cDNA synthesis (50 °C, 30 min → 95 °C, 5 min).

![Figure 9](https://static.igem.wiki/teams/5925/images/exp9.webp)

Figure 9 (Korea-CX member loading RNA extraction into a micro tube)

##### C. PCR Amplification

* Reaction (20 µL): cDNA template, GAPDH primer (internal control), NRAS-WT or NRAS-G12D primer, premix, water.
* PCR program:
  + Initial denaturation : 95 °C 1 min
  + Cycling : 95 °C 15 s, 55 °C 15 s, 72 °C 30 s) × 35 cycles
  + Final extension : 72 °C 7 min.
  + Hold: 4 °C
  + ![Figure 10](https://static.igem.wiki/teams/5925/images/exp10.webp)

    Figure 10 (A PCR machine used for the amplification process)

##### D. Gel Electrophoresis

1. 1. Prepare 2% agarose gel with nucleic acid stain.
2. 2. Load 15 µL PCR product + 4 µL ladder.
3. 3. Run at 130 V for 30 min.
4. 4. Visualize under gel doc; confirm expected band sizes.

![Figure 11](https://static.igem.wiki/teams/5925/images/exp11.webp)

Figure 11 (Korea-CX member loading product into slots of agarose gel)

![Figure 12](https://static.igem.wiki/teams/5925/images/exp12.webp)

Figure 12 (Agarose gel filled with samples)

![Figure 13](https://static.igem.wiki/teams/5925/images/exp13.webp)

Figure 13 (Samples going through Agaros gel electrophoresis)

#### <Part 3. Practice Skills>

##### Materials Needed

* NIH3T3 cells transfected with plasmids (Exp. 2)
* 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

* **Microscopy**
  + Avoid photobleaching by minimizing light exposure.
  + Use the same exposure time for all groups.
* **RNA Work**
  + Always use RNase-free consumables.
  + Keep samples on ice during prep.
* **PCR & Gel**
  + Include no-template control (NTC) to check contamination.
  + Compare band intensity between WT and G12D for expression lev

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

#### <Part 1. Basic Theory of CCK-8 Assay>

##### Experimental Principle

* **Definition**: The CCK-8 assay is a colorimetric method to determine cell viability based on metabolic activity.
* **Purpose**: To evaluate the cytotoxic effect of the MEK inhibitor U0126 on cultured cells by measuring dose-dependent viability.
* **Mechanism**: The CCK-8 reagent contains WST-8, a tetrazolium salt reduced by dehydrogenases in metabolically active cells to form a soluble orange formazan dye.

The intensity of color (absorbance at 450 nm) is directly proportional to the number of viable cells.

##### About U0126

* U0126 is a selective inhibitor of MEK1 and MEK2, components of the MAPK/ERK signaling pathway. It is widely used in research to block ERK phosphorylation and study cell proliferation, survival, and differentiation.
* **U0126 Concentration Calculation**
  U0126 MW = 380.4 g/mol

```
Basic formula:
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑀) = 𝑚𝑎𝑠𝑠 (𝑔) / 𝑀𝑊 (𝑔/𝑚𝑜𝑙) × 𝑣𝑜𝑙𝑢𝑚𝑒 (𝐿)

Example:
To prepare a 10 mM stock solution in DMSO:

* Desired final volume: 1 mL* Target concentration: 10 mM
  * Required mass of U0126:

                  10𝑚𝑀×380.4𝑔/𝑚𝑜𝑙×0.001𝐿 = 3.804𝑚𝑔
```

#### <Part 2. Experimental Procedures>

##### Materials Needed

* NIH3T3 cells
* U0126 stock solution (10 mM in DMSO)
* CCK-8 solution (Cell Counting Kit-8)
* 96-well plate
* DMEM with 10% FBS + 1% Pen/Strep
* Plate reader (450 nm)
* Sterile tips, pipettes, conical tubes

##### Procedure

1. **1. Cell Seeding**
   * Prepare cell suspension (1 × 10⁴ cells/100 µL/well).
   * Seed into a 96-well plate (100 µL/well).
   * Incubate 24 h at 37 °C, 5% CO₂ before drug treatment.
2. **2. Drug Dilution & Treatment**
   * Prepare serial dilutions of U0126 in medium from 10 mM stock.
   * Example dilution series (final concentrations, µM): 40, 20, 10, 5, 2.5
   * Add 100 µL of each dilution to triplicate wells.
   * Include control wells with vehicles (DMSO only).
   * Incubate cells for 24 h.
   ![Figure 14](https://static.igem.wiki/teams/5925/images/exp14.webp)

   Figure 14 (Korea-CX member conducting serial dilution of the stock and medium)

   ![Figure 15](https://static.igem.wiki/teams/5925/images/exp15.webp)

   Figure 15 (Korea-CX member seeding samples, finished with dilution, into a 96-well plate)
3. **CCK-8 Addition**
   * Add 20 µL CCK-8 reagent to each well (final volume 120 µL).
   * Gently tap the plate to mix.
   * Incubate 2 h at 37 °C (protect from light).
   ![Figure 16](https://static.igem.wiki/teams/5925/images/exp16.webp)

   Figure 16 (Samples of Cells with CCK-8 reagent loaded in a 96-well plate)
4. **Absorbance Measurement**
   * Wipe bottom of plate with Kimwipe + ethanol.
   * Measure absorbance at 450 nm using a microplate reader.
   * Record triplicate readings for each condition.
   ![Figure 17](https://static.igem.wiki/teams/5925/images/exp17.webp)

   Figure 17 (Measured absorbance of the 96-well plate at 450nm using microplate reader )

#### <Part 3. Practice Skills>

1. **Serial Dilution & Pipetting**
   * Practice preparing serial dilutions using Trypan Blue or dye before working with U0126.
   * Always mix thoroughly before transferring to the next tube.
2. **Assay Controls**
   * Include blank wells (media + CCK-8, no cells) for background correction.
   * Use vehicle-only wells (DMSO without drug) as 100% viability control.
3. **Data Reliability**
   * Run samples in triplicates for each condition.
   * Avoid bubbles in wells, as they interfere with absorbance readings.
   * Normalize data against control wells.

   ```
   𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) = 𝐴𝑏𝑠.𝑠𝑎𝑚𝑝𝑙𝑒 / 𝐴𝑏𝑠.𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100
   ```

### Experiments Day 5: Lab-on-a-Chip (LOC) Fabrication

#### <Part 1. Basic Theory of Lab-on-a-Chip (LOC)>

##### Experimental Principle

* **Definition:** A Lab-on-a-Chip (LOC) is a microfluidic platform integrating laboratory functions into a millimeter–centimeter-sized device.
* **Purpose**: To fabricate a PDMS-based LOC that allows cell seeding, migration assays, and drug testing in controlled microenvironments.
* **Advantages**:
  + Minimal reagent/sample consumption
  + Real-time monitoring under microscopy
  + Mimics physiological structures (tissue, vasculature, tumor)
  + Enables flow control and chemical gradient formation

##### Why PDMS?

* Transparent → ideal for microscopy
* Biocompatible and gas permeable
* Easy to mold (soft lithography)
* Hydrophobic (temporarily hydrophilic after plasma treatment for bonding)

##### Chip Design Overview

* Layer 1 (Reservoir): Media exchange & drug delivery (5 × 8 mm circles, 4 mm thick).
* Layer 2 (Chamber Top): Cell seeding chamber (6 mm central well, aligned with porous membrane).
* Layer 3 (Chamber Bottom + Channels): Collection chamber with microchannels, mimicking vasculature.
* Porous Membrane: Inserted between layers to simulate biological barriers.
* Layer 4 (Glass Slide): Bottom support, allowing imaging.

![Figure 18](https://static.igem.wiki/teams/5925/images/exp18.webp)

Figure 18 (Step by step design of the lab-on-a-chip)

#### <Part 2. Experimental Procedures>

##### Materials Needed

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

1. **1. PDMS Preparation**
   * Mix PDMS base:curing agent (10:1).
   * Pour into molds; de-gas under vacuum.
   * Cure at 60 °C for 3–4 h (overnight preferred).
   ![Figure 19](https://static.igem.wiki/teams/5925/images/exp19.webp)

   Figure 19 (Korea-CX member mixing PDMS base with curing agent)

   ![Figure 20](https://static.igem.wiki/teams/5925/images/exp20.webp)

   Figure 20 (Korea-CX member pouring mixed base into the molds)
2. **2. Layer Cutting & Punching**
   * Remove cured PDMS layers.
   * Punch reservoirs, chambers, inlets/outlets (sizes: 1–8 mm).
   ![Figure 21](https://static.igem.wiki/teams/5925/images/exp21.webp)

   Figure 21 (Korea-CX member unnecessary parts of PDMS layers)
3. **3. Plasma Bonding**
   * Cut membrane from transwell insert.
   * Plasma-treat each PDMS layer + glass slide.
   * Align and bond in order: Layer 3 → Layer 2 → Membrane → Layer 1 → Glass slide.
   ![Figure 22](https://static.igem.wiki/teams/5925/images/exp22.webp)

   Figure 22 (Each PDMS layer being plasma treated)

   ![Figure 23](https://static.igem.wiki/teams/5925/images/exp23.webp)

   Figure 23 (Machine observed from outside when plasma bonding)
4. **4. Chip Washing & Conditioning**
   * Leave overnight for hydrophobic recovery.
   * Wash sequentially: 70% EtOH → PBS → DMEM.
   * Check flow uniformity and absence of leakage.
   ![Figure 24](https://static.igem.wiki/teams/5925/images/r-exp24.avif)

   Figure 24 (Chips after being all washed and conditioned)

#### <Part 3. Practice Skills>

* Soft Lithography Handling
  + Avoid bubbles during PDMS pouring/de-gassing.
  + Use consistent curing times for reproducible thickness.
* Membrane Alignment
  + Handle membrane with forceps, avoid folding/tearing.
  + Ensure central alignment with chambers.
* Plasma Surface Treatment
  + Settings used: O₂ gas 45 sccm, RF power 90 W, 1 min.
  + Immediately bond after treatment (hydrophilicity is temporary).

### Experiments Day 6: 3D Bone Marrow Microenvironment & Migration Assay

#### <Part 1. Basic Theory>

##### Experimental Principle

* **Definition**: Construction of a 3D bone marrow-like microenvironment in LOC to study cancer cell migration.
* **Purpose**: To mimic ECM and stromal interactions using Matrigel and track cancer cell behavior under realistic conditions.

##### Background

* **Bone Marrow Niche**: Provides signals (stromal cells, ECM proteins, cytokines) that regulate hematopoietic and malignant cell behavior.
* **Matrigel**: Basement membrane extract rich in laminin/collagen IV, forming ECM scaffolds that support 3D growth.
* **LOC + Matrigel**: Combines fluidic control with ECM simulation → closer to in vivo.

Note: NIH3T3 fibroblasts were used as a model system instead of human multiple myeloma cells, in compliance with iGEM safety guidelines.

#### <Part 2. Experimental Procedures>

##### Materials Needed

* LOC device (from Exp. 5)
* Matrigel (thawed on ice, 4 mg/mL final conc.)
* NIH3T3
* DMEM + 10% FBS + 1% Pen/Strep
* PBS, 70% EtOH
* CO₂ incubator
* Ice bucket, micropipettes with pre-cooled tips, sterile dishes, timer

1. **1. Chip Preparation**
   * Wipe LOC surface with 70% EtOH.
   * Wash channels: EtOH → PBS → medium (repeat twice).
   * Fill channels with medium; condition for 30 min.
2. **2. Matrigel Injection**
   * Thaw Matrigel on ice.
   * Load with pre-cooled tips.
   * Slowly inject into the ECM chamber (avoid bubbles).
   * Incubate 37 °C, 30–45 min until polymerized.
3. **3. Cell Preparation & Seeding**
   * Harvest NIH3T3 cells; resuspend at 1 × 10⁵ cells/mL.
   * Seed 100 µL of suspension above the Matrigel layer.
   * Fill reservoirs with medium.
   ![Figure 25](https://static.igem.wiki/teams/5925/images/exp25.webp)

   Figure 25 (Korea-CX member Seeding Matrigel onto the chip)
4. **4. Incubation & Observation**
   * Incubate LOC in 37 °C, 5% CO₂ for 24–72 h.
   * Monitor cell morphology and migration under fluorescence or brightfield microscopy.
   * To mimic dynamic body-fluid flow, the LOC was placed on a low-speed rocking shaker inside the incubator. This gentle shaking maintained continuous medium circulation and shear stress similar to physiological conditions.
   ![Figure 26](https://static.igem.wiki/teams/5925/images/r-exp26.avif)

   Figure 26 (LOC placed on the rocking shaker inside the incubator)

#### <Part 3. Practice Skills>

* **1. Matrigel Handling**
  + Always keep on ice; pre-cool tips/tubes.
  + Avoid repeated freeze–thaw; aliquot before use.
* **2. Injection Technique**
  + Avoid bubble formation during Matrigel filling.
  + Ensure uniform ECM distribution for reproducible migration results.
* **3. Imaging & Analysis**
  + Capture time-lapse images for migration tracking.
  + Use fluorescence (mCherry) to distinguish transfected cells.

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Results

## Results

### Research Goal & Overview

The primary goal of our project was to determine how the oncogenic mutation NRAS^G12D influences cell behavior compared to wild-type NRAS (NRAS^WT) and untransfected control cells. Since NRAS mutations are frequently implicated in cancer progression, especially in blood and solid tumors, we aimed to establish an in vitro platform that allows quantitative and reproducible comparison of mutation-driven phenotypes.

Our experimental workflow was designed to assess three major aspects of NRAS function:

1. Expression validation – confirming successful delivery and transcription of NRAS^WT and NRAS^G12D.
2. Cell viability and drug response – evaluating how the mutation alters survival under MEK inhibition.
3. Cell migration – comparing the motility of WT and mutant cells in both 2D and 3D environments.

By combining standard assays (RT-PCR, CCK-8, scratch assay) with advanced approaches (Lab-on-a-Chip migration), we built a stepwise pipeline from basic confirmation to functional characterization. This overview demonstrates not only the specific impact of NRAS^G12D but also establishes a general workflow that other iGEM teams can apply to study mutation-driven cellular behaviors.

### <Result 1: Transfection and Fluorescence Microscopy>

#### [1-1. Description]

To validate successful introduction of NRAS constructs into NIH3T3 cells, we performed Lipofectamine 3000-mediated transfection with plasmids containing NRAS^WT and NRAS^G12D, each linked to an mCherry fluorescent reporter.After 48 hours of transfection, cells were subjected to puromycin selection at 5 µg/ml for 2 days, followed by observation under brightfield and fluorescence microscopy.

In control cells, no fluorescence signal was detected, whereas both NRAS^WT and NRAS^G12D groups showed strong red fluorescence signals consistent with mCherry expression. The transfected cells also exhibited typical fibroblast-like morphology under brightfield conditions, indicating good viability and absence of morphological stress.

![Figure 1](https://static.igem.wiki/teams/5925/images/res1.webp)

Figure 1. Confirmation of mCherry fluorescence in NIH3T3 cells following plasmid transfection. NIH3T3 cells were transfected with the mCherry-expressing plasmid using a lipid-based transfection reagent. 48h post-transfection, cells were subjected to puromycin selection (5 μg/ml) for 2days, after which surviving cells were imaged under fluorescence microscopy. Representative bright-field images (top) and fluorescence images (bottom) are shown. A strong red fluorescence signal corresponding to mCherry expression was detected in the transfected cells, whereas untransfected control cells exhibited no detectable fluorescence. These results confirm successful plasmid uptake and expression of the mCherry reporter gene. Scale bars, 100 μm.

#### [1-2. Interpretation]

These results confirm that NRAS^WT and NRAS^G12D plasmids were successfully delivered and expressed in NIH3T3 cells. The consistent fluorescence across multiple fields supports reproducibility and indicates that the mCherry reporter is a reliable marker of transfection efficiency. This provides a validated platform for downstream experiments, including gene expression verification, drug response, and migration assays.

### <Result 2: Quantitative Measurement of Transfection Efficiency>

#### [2-1. Description]

To quantitatively validate transfection efficiency beyond microscopy, we measured mCherry fluorescence intensity using a microplate reader at 48 h post-transfection, followed by 2 days of puromycin selection (5 µg/ml). NIH3T3 cells transfected with NRAS^WT or NRAS^G12D plasmids showed significantly higher fluorescence compared to control cells. A baseline fluorescence signal was observed in the control group, likely due to cellular autofluorescence. Statistical analysis (unpaired Student’s t-test) confirmed that both NRAS^WT and NRAS^G12D groups were significantly different from controls (\*p < 0.05, \*\*p < 0.01). The fluorescence intensity between WT and G12D groups showed comparable levels, suggesting that the mutation did not alter transfection efficiency at this stage.

![Table 1](https://static.igem.wiki/teams/5925/images/res-table-1.webp)

Table 1. Quantification of mCherry fluorescence in NIH3T3 cells following plasmid transfection. Fluorescence intensity was measured 48 h post-transfection, followed by 2 days of puromycin selection (5 µg/ml) using a microplate reader. Data are presented as mean ± SD (n = 3). Baseline fluorescence was detected in control cells, likely due to cellular autofluorescence. Statistical significance was determined using two-tailed unpaired Student’s t-tests. \*p < 0.05, \*\*p < 0.01 vs. control.

#### [2-2. Interpretation]

This quantitative analysis complements the fluorescence microscopy results in Result 1. Together, they confirm that both NRAS^WT and NRAS^G12D plasmids were successfully transfected and expressed in NIH3T3 cells. The reproducibility of the fluorescence signal across biological replicates demonstrates the robustness of our transfection protocol. Importantly, fluorescence intensity should not be interpreted as a direct measure of transfection efficiency, but rather as a confirmation of successful transgene expression. Furthermore, because our designed vectors include a luciferase assay module, more rigorous quantitative analysis of transfection efficiency can be performed using luciferase-based measurements.

### <Result 3: Verification of NRAS Expression by RT-PCR>

#### [3-1. Description]

To confirm whether the NRAS^WT and NRAS^G12D plasmids were transcribed after successful transfection, we performed reverse transcription PCR (RT-PCR) followed by agarose gel electrophoresis. GAPDH was used as an internal control to normalize expression levels.

As shown in Figure 2, GAPDH bands were consistently detected in all groups, confirming equal RNA input and successful cDNA synthesis. In control (untransfected) cells, weak endogenous NRAS bands were observed, reflecting the basal expression of the native NRAS gene in NIH3T3 cells. In contrast, cells transfected with NRAS^WT or NRAS^G12D plasmids displayed much stronger NRAS-specific bands (~120–150 bp), confirming robust exogenous expression in addition to the endogenous signal.

![Figure 2](https://static.igem.wiki/teams/5925/images/res2.webp)

Figure 2. Amplified NRAS DNA products by RT-PCR are visualized using agarose gel electrophoresis.

* Lane 1: DNA size marker (100–150 bp).
* Lane 2–4: Control (empty vector, GAPDH, NRAS^WT, NRAS^G12D).
* Lane 5–7: NRAS^WT-transfected cells (GAPDH, NRAS^WT, NRAS^G12D).
* Lane 8–10: NRAS^G12D-transfected cells (GAPDH, NRAS^WT, NRAS^G12D).
* GAPDH was amplified as an internal control.

Endogenous NRAS expression is detectable in control cells, reflecting the presence of the native NRAS gene in NIH3T3 cells. In transfected groups, exogenous NRAS^WT or NRAS^G12D plasmid expression led to stronger NRAS-specific bands, confirming successful transfection.

#### [3-2. Interpretation]

These results validate that both NRAS^WT and NRAS^G12D constructs are not only transfected but also actively transcribed in NIH3T3 cells. The absence of NRAS amplification in controls confirms specificity, while consistent GAPDH expression ensures accuracy. Together with Results 1 and 2, this establishes a reliable expression system for downstream functional assays, including viability testing and migration studies.

### <Result 4: Effect of NRAS^G12D Mutation on Cell Growth>

#### [4-1. Description]

To investigate whether the NRAS^G12D mutation influences cell proliferation independently of drug treatment, we monitored the growth of NIH3T3 cells transfected with NRAS^WT or NRAS^G12D plasmids. Cells were seeded at equal densities and incubated for up to 48 hours. Cell numbers were quantified at each time point using the CCK-8 assay, with absorbance measured at 450 nm.

As shown in Figure 3, both groups exhibited an increase in cell numbers over time. However, NRAS^G12D cells displayed a consistently higher growth rate compared to NRAS^WT cells, indicating that the mutation promotes enhanced proliferation under normal culture conditions.

![Figure 3](https://static.igem.wiki/teams/5925/images/res3.webp)

Figure 3. Cell proliferation analysis of CON, WT and G12D groups. Cell proliferation was measured at 0, 24, and 48 h after seeding using the Cell Counting Kit-8 (CCK-8). All groups showed time-dependent increases in proliferation; however, G12D cells exhibited significantly enhanced growth compared with both CON and WT, whereas WT cells displayed the lowest proliferation. Statistical significance was determined using two-tailed unpaired Student’s t-tests (\*\*\*p < 0.001).

#### [4-2. Interpretation]

These results demonstrate that the NRAS^G12D mutation alone is sufficient to accelerate cell growth, even without external stimulation or drug treatment. This finding aligns with previous knowledge that oncogenic NRAS mutations drive uncontrolled cell proliferation. Establishing this baseline difference in proliferation is essential for interpreting subsequent experiments, such as drug response and migration assays.

### <Result 5: Migration Assay (Scratch Wound Healing Test)>

#### [5-1. Description]

To evaluate the effect of NRAS^G12D mutation on cell migration, a scratch wound healing assay was performed using NIH3T3 cells transfected with NRAS^WT or NRAS^G12D. A uniform gap (“scratch”) was introduced in confluent cell monolayers, and wound closure was monitored for up to 72 hours.

As shown in Figure 4, control cells exhibited limited closure of the scratch, whereas both NRAS^WT and NRAS^G12D groups showed progressive closure over time. Notably, NRAS^G12D cells closed the wound gap more rapidly than NRAS^WT cells, suggesting that the mutation enhances migratory capacity.

![Figure 4](https://static.igem.wiki/teams/5925/images/res4-1.webp)

Figure 4. 2D cell migration (1) Representative images of wound healing assays at 0, 24, 48, and 72 h (scale bar = 100 μm). At later time points, NRAS^WT and NRAS^G12D groups showed faster wound closure compared to the control.

![Figure 4](https://static.igem.wiki/teams/5925/images/res4-2.webp)

Figure 4. 2D cell migration (2) Quantification of wound closure (%) based on three independent fields per group (n=3). At 72 h, NRAS^WT and NRAS^G12D exhibited significantly higher wound closure compared to the control (p < 0.001).

#### [5-2. Interpretation]

These findings indicate that the NRAS^G12D mutation significantly promotes fibroblast migration compared to NRAS^WT. This is consistent with the known role of oncogenic NRAS mutations in activating downstream MAPK signaling, which enhances cytoskeletal rearrangements and motility. By establishing that NRAS^G12D increases both proliferation (Result 4) and migration (Result 5), we provide strong evidence that this mutation drives aggressive cellular behaviors, which are hallmarks of cancer progression.

### <Result 6: Lab-on-a-Chip migration>

#### [6-1. Description]

To investigate how the NRAS^G12D mutation alters invasive potential in a 3D extracellular matrix–like context, we employed a PDMS-based lab-on-a-chip (LOC) system filled with Matrigel. NIH3T3 cells (1×10^5) from control (CON), NRAS^WT, and NRAS^G12D groups were seeded into the upper chamber. After 72 h, cells that penetrated the Matrigel and reached the lower compartment were imaged and quantified. Representative images (Figure 5) showed few migrated cells in the control group, a moderate increase in the NRAS^WT group, and the highest number in the NRAS^G12D group. Quantification (Table 2) confirmed significantly greater migration in NRAS^WT and NRAS^G12D compared to control, with NRAS^G12D displaying the strongest infiltration capacity.

![Figure 5](https://static.igem.wiki/teams/5925/images/res5-1.webp)

Figure 5. 3D cell migration in LOC assay (1) Final lab-on-a-chip (LOC) device fabricated for 3D migration studies.

![Figure 5](https://static.igem.wiki/teams/5925/images/res5-2.webp)

Figure 5. 3D cell migration in LOC assay (2) Representative microscopic images of migrated cells collected in the lower compartment after 72 h for control (CON), NRAS^WT, and NRAS^G12D groups. A greater number of migrated cells was observed in NRAS^WT and NRAS^G12D compared with control, with NRAS^G12D showing the highest infiltration.

![Table 2](https://static.igem.wiki/teams/5925/images/res-table-2.webp)

Table 2. Quantification of 3D migration in LOC assay. Cells (CON, NRAS^WT, and NRAS^G12D) were seeded at 1×10^5 cells on Matrigel-coated LOC chambers. After 72 h, migrated cells in the lower compartment were counted from multiple microscopic fields and expressed as mean cell number ± SD. Data were normalized to the control group (CON = 100%). Both NRAS^WT and NRAS^G12D groups showed significantly higher migration compared to control, with NRAS^G12D exhibiting the strongest infiltration capacity (p < 0.05, \*p < 0.01 vs. CON).

#### [6-2. Interpretation]

The LOC assay demonstrates that the NRAS^G12D mutation markedly enhances migration in a 3D environment that better mimics the bone marrow extracellular matrix. While NRAS^WT cells exhibited some increase in motility relative to control, the G12D mutation further amplified this effect, consistent with its oncogenic role in promoting invasion. These findings complement the 2D scratch assay, showing consistent trends across both platforms. Importantly, the LOC system provided a physiologically relevant readout of cell infiltration through a tissue-like barrier, underscoring its utility as an alternative to animal models for studying mutation-driven cancer progression.

### <Result 7: Drug Response Assay (U0126 Treatment, CCK-8 Viability Test)>

#### [7-1. Description]

We compared cell viability of three groups—Control (untransfected), NRAS^WT, and NRAS^G12D—with or without the MEK inhibitor U0126 (72 h). Viability was measured by CCK-8 at 450 nm and normalized to each group’s untreated condition.

![Figure 6](https://static.igem.wiki/teams/5925/images/res6.webp)

Figure 6. Drug response to the MEK inhibitor U0126. Cells were treated with U0126 for 72 h, and viability was measured by CCK-8 assay (mean ± SD, n=4). U0126 treatment reduced viability in the control (~20%) and NRAS^WT (~5%) groups, whereas NRAS^G12D cells showed increased viability (~5–7%). One-way ANOVA revealed significant differences among groups (F(2, 9) = 131.27, p < 0.0001), with Tukey’s test confirming significance for all pairwise comparisons.

#### [7-2. Interpretation]

The data indicate differential sensitivity to MEK inhibition:

* Control cells show the expected growth suppression with U0126.
* NRAS^WT cells appear drug-sensitive, with viability reduced both at baseline (likely expression burden) and further under U0126.
* NRAS^G12D cells display a paradoxical, slight increase in viability with U0126, consistent with tolerance/resilience to MEK inhibition. This may reflect feedback rewiring of RAS–MAPK signaling or compensatory survival pathways (e.g., RAF activation, PI3K/AKT crosstalk), though mechanistic confirmation would require pathway readouts.

Overall, NRAS^G12D shows a pro-survival phenotype under MEK inhibition, contrasting with WT and Control, and aligning with the mutation’s oncogenic character.

### Conclusion

In this study, we established an integrated in vitro workflow to investigate how the oncogenic mutation NRAS^G12D shapes cellular phenotypes relevant to multiple myeloma progression. By engineering NIH3T3 cells to express NRAS^WT or NRAS^G12D alongside an mCherry reporter, we validated successful transfection and transcription (Results 1–3), providing a reliable system for functional assays.

Our analyses revealed that:

* NRAS^G12D promotes uncontrolled proliferation compared to WT and control cells (Result 4).
* The mutation enhances migratory behavior in both 2D scratch wound assays and 3D chip-based infiltration models (Result 5, ongoing LOC experiments).
* Under pharmacological challenge with the MEK inhibitor U0126, NRAS^WT and control cells showed growth suppression, whereas NRAS^G12D cells paradoxically maintained or increased viability, highlighting mutation-specific tolerance to MEK inhibition (Result 7).

Together, these findings demonstrate that NRAS^G12D confers a pro-survival, pro-migratory phenotype, consistent with its oncogenic role in hematologic malignancies. Importantly, by combining conventional assays with a PDMS-based lab-on-a-chip that mimics the bone marrow extracellular matrix, we provide a reproducible experimental pipeline that links genetic perturbations to proliferation, motility, and drug response in a tissue-like context.

This platform has the potential to:

* Serve as an animal-reducing alternative for mutation-driven cancer research.
* Enable rapid testing of AI-nominated drug candidates.
* Support future co-culture models incorporating stromal or endothelial cells to better recapitulate the tumor microenvironment.

Overall, our results not only highlight the oncogenic features of NRAS^G12D but also establish a scalable framework for studying gene-driven cancer behaviors in vitro.

### Reference

1. [1] OncoKB™ - MSK’s Precision Oncology Knowledge Base. (2025). OncoKB™. <https://www.oncokb.org/gene/NRAS/G12D>
2. [2] Prior, I.A., Hood, F.E., & Hartley, J.L. Cancer Res (2020). The Frequency of Ras Mutations in Cancer. 80(14): 2969–2974, <https://doi.org/10.1158/0008-5472.CAN-19-3682>
3. [3] Li, N., Lin, P., Zuo, Z., You, M. J., Shuai, W., Orlowski, R., Manasanch, E. E., Li, S., Xu, J.,Garces, S., Jelloul, F. Z., Tang, Z., Wang, W., Medeiros, L. J., & Yin, C. C. Cancer Medicine (2023). Plasma cell myeloma with RAS/BRAF mutations is frequently associated with a complex karyotype, advanced stage disease, and poorer prognosis. 12(13),14293–14304, <https://doi.org/10.1002/cam4.6103>
4. [4] Meiying Chi, et al., Biomed Microdevices (2015), A microfluidic cell culture device (μFCCD) to culture epithelial cells with physiological and morphological properties that mimic those of the human intestine, 17:58, <https://doi.org/10.1007/s10544-015-9966-5>
5. [5] S. Y. Lee and J. H. Sung, Biotechnology and Bioengineering (2018), Gut–liver on a chip toward an in vitro model of hepatic steatosis. 115:2817-2827, <https://doi.org/10.1002/bit.26793>

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