iGEM 2021 — Team Korea_HS

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Korea HS/Notebook

KORHS

# [KORHS](https://2021.igem.org/Team%3AKorea_HS)

* TEAM
  + [Team](https://2021.igem.org/Team%3AKorea_HS/Team)
  + [Attributions](https://2021.igem.org/Team%3AKorea_HS/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AKorea_HS/Collaborations)
* PROJECT
  + [Communication](https://2021.igem.org/Team%3AKorea_HS/Communication)
  + [Contribution](https://2021.igem.org/Team%3AKorea_HS/Contribution)
  + [Description](https://2021.igem.org/Team%3AKorea_HS/Description)
  + [Engineering](https://2021.igem.org/Team%3AKorea_HS/Engineering)
  + [Experiments](https://2021.igem.org/Team%3AKorea_HS/Experiments)
  + [Implementation](https://2021.igem.org/Team%3AKorea_HS/Implementation)
  + [Notebook](https://2021.igem.org/Team%3AKorea_HS/Notebook)
  + [Partnership](https://2021.igem.org/Team%3AKorea_HS/Partnership)
  + [Results](https://2021.igem.org/Team%3AKorea_HS/Results)
* PARTS
  + [Part Collection](https://2021.igem.org/Team%3AKorea_HS/Part_Collection)
  + [Parts](https://2021.igem.org/Team%3AKorea_HS/Parts)
  + [Improve](https://2021.igem.org/Team%3AKorea_HS/Improve)
* SAFETY
  + [Safety](https://2021.igem.org/Team%3AKorea_HS/Safety)
* HUMAN PRACTICES
  + [Human Practices](https://2021.igem.org/Team%3AKorea_HS/Human_Practices)
* AWARDS
  + [Education](https://2021.igem.org/Team%3AKorea_HS/Education)
  + [Inclusivity](https://2021.igem.org/Team%3AKorea_HS/Inclusivity)
  + [Model](https://2021.igem.org/Team%3AKorea_HS/Model)

[JUDGING](https://igem.org/2021_Judging_Form?id=3824)

NOTEBOOK

# Team Korea\_HS

Date : March 9th, 2021
Event : 1st Virtual Meeting
Agenda : The team was introduced to its members (for the first time), 2021 research idea (by Professor Lee), and iGEM competition logistics (by advisors Sam Han and Jina Bae).

Date : April 17th, 2021
Event : 2nd Virtual Meeting
Agenda : The team discussed MV1/MV2-siRNA-doxorubicin construct in depth and was introduced to team divisions (Wiki, Human Practices, and Collaboration).

Date : May 1st, 2021
Event : 3rd Virtual Meeting
Agenda : The team discussed fundamental concepts of (normal and cancer) cell lines and siRNA and was engaged in the first sub-team (Wiki, Human Practices, and Collaboration) discussions.

Date : May 15th, 2021
Event : 4th Virtual Meeting
Agenda : The team discussed experimental methods (e.g. FITC/MTT assay) of validating CPP construct penetration specificity and efficacy and elected sub-team leaders and co-leaders.

Date : May 29th, 2021
Event : 5th Virtual Meeting
Agenda : The team discussed experimental methods (e.g. RT-PCR and western blotting) of validating construct transfection and gene silencing efficacy and elected main leaders.

Date : June 12th, 2021
Event : 6th Virtual Meeting
Agenda : The team discussed proof of concept methods (e.g. immunogenicity and serum degradation) with Professor Lee and scheduled wet lab meetings at Suwon University.

Date : June 26th, 2021
Event : 7th Virtual Meeting
Agenda : The team finalized proof of concept methods (e.g. modification of CPP) and wet lab meetings at Suwon University and engaged in sub-team breakout sessions.

Date : July 11th, 2021
Event : 8th Virtual Meeting
Agenda: The team discussed dry lab methods (e.g. PEP-FOLD3 and Benchling) to stimulate CPP constructs and medal criteria (particularly gold) and special/community-based awards for 2021.

Date : July 25th, 2021
Event : 9th Virtual Meeting
Agenda : The team discussed all medal and special/community-based award criteria and workload allocation until Wiki Freeze (and further task assignments) and took team photos virtually.

Date : August 14th, 2021
Event : 10th Meeting (1st in-person meeting)
Agenda : The team met for the first time in-person, took team photos, discussed workload allocation and deadlines until Wiki Freeze, and completed several team tasks.

Date : August 29th, 2021
Event : 11th Virtual Meeting
Agenda : The team discussed experimental results and analysis (e.g. FITC, MTT, and RT-PCR) with Professor Lee and updated each sub-team with events and Wiki progress.

Date : September 18th, 2021
Event : 12th Meeting (2nd in-person meeting)
Agenda : The team created lab kits for elementary & middle school lab kit webinars and board and engagement materials for live sticker poll and pamphlet distribution.

Date : October 3rd, 2021
Event : 13th Virtual Meeting
Agenda : The team discussed workload distribution and deadlines for finalized Wiki and presentation video and reviewed sub-team conclusion of events and Wiki write-ups.

Date : October 16th, 2021
Event : 14th Meeting (3rd in-person meeting)
Agenda : The team concluded deadlines and the creation of final deliverables (Wiki, presentation video, and judging form) and created promotional materials for the group’s blood donation event.

# Team Wet Lab

Date : July 4th, 2021
Event : 1st Wet Lab Meeting
Agenda : Students performed micropipette (Gilson) handling procedures, cell culture experiment (learning cell line and culture plate types), and trypsin cell detachment/count experiment.

Date : July 11th, 2021
Event : 2nd Wet Lab Meeting
Agenda : Students were introduced to modification of buforin IIb (MV1 and MV2) and performed colorimetric MTT metabolic (cytotoxicity) assay and stock solution (dilution) synthesis.

Date : July 18th, 2021
Event : 3rd Wet Lab Meeting
Agenda : Students were introduced to FITC constructs and performed a cell-penetration efficacy experiment by preparing stock-media mixtures with stock, RPMI media, and vortex mixer.

Date : August 1st, 2021
Event : 4th Wet Lab Meeting
Agenda : Students were introduced to siRNA + Religent synthetic constructs and performed an intracellular siRNA delivery experiment (total RNA extraction) via RNA extraction kit (Intron).

Date : August 8th, 2021
Event : 5th Wet Lab Meeting
Agenda : Students performed reverse transcription-polymerase chain reaction via RNA extraction kit, mRNA-cDNA synthesis via RTase enzyme, and CYP1A1 expression level design.

Date : August 15th, 2021
Event : 6th Wet Lab Meeting
Agenda : Students were introduced to agarose gel and completed the analysis of RT-PCR assay through quantification of CYP1A1 and GADPH band intensities and result expectations.

# Team Dry Lab

Date : July 27th, 2021
Event : 1st Dry Lab Meeting
Agenda : Students discussed dry lab programs applicable to the team’s project (i.e. PEP-FOLD3, Benchling, and Ensembl) with Professor Lee and simulated peptide characterization.

Date : August 8th, 2021
Event : 2nd Dry Lab Meeting
Agenda : Students discussed applicable dry lab programs (i.e. Rosetta, Pymol, CellDesigner, and MATLAB) and their methodologies, reviewed tutorials, and sought collaboration.

Date : August 21st, 2021
Event : 3rd Dry Lab Meeting
Agenda : Students finalized simulation analysis and Wiki write-up (and workload distribution) for simulated programs and considered for future use by associating analysis with wet lab results.

# Team Collaboration

Date : June 18th, 2021
Event : 1st Collaboration Meeting with IISER\_Tirupati
Agenda : Both teams introduced their projects. IISER\_Tirupati shared their modeling parts, and shared advice on medal criteria and human practices part.

Date : June 20th, 2021
Event : 1st Collaboration Meeting with UTEC-Peru
Agenda : Both teams presented their projects. UTEC-Peru shared suggestions for human practices and judging sessions.

Date : June 27th, 2021
Event : 1st Collaboration Meeting with IISER\_Berhampur
Agenda : Both teams shared their projects. IISER\_Berhampur shared their suggestions on human practices and introduced possible ideas.

Date : June 30th, 2021
Event : 1st Partnership Meeting with Greece\_United (NOUS)
Agenda : Both teams shared their presentations and had a discussion about extending the collaboration to a partnership.

Date : July 1st, 2021
Event : 1st Partnership Meeting with ASIJ\_Tokyo
Agenda : Both teams presented their projects and discussed possible areas for the partnership, including human practice and protein modeling.

Date : July 4th, 2021
Event : 1st Collaboration Meeting with Siberia
Agenda : Both teams introduced their projects and team Siberia advised on using longer double-stranded RNA for wet lab.

Date : July 5th, 2021
Event : 1st Collaboration Meeting with IISER\_Bhopal
Agenda : Both teams shared their presentations and discussed areas of collaboration: MTT assay and protein modeling.

Date : July 9th, 2021
Event : 1st Collaboration Meeting with CCU\_Taiwan
Agenda : Both teams introduced their projects and discussed areas of collaboration, wet lab, and human practices part of the project.

Date : July 10th, 2021
Event : 1st Collaboration Meeting with TU\_Darmstadt
Agenda : Both teams presented their projects. Team TU\_Darmstadt shared their understanding of gold medal criteria, especially, the proof of concept.

Date : July 12th, 2021
Event : 1st Collaboration Meeting with HU\_Berlin
Agenda : Both teams spent time presenting their projects. HU\_Berlin assisted Korea\_HS with the project modeling and gold medal criteria part of iGEM.

Date : July 13th, 2021
Event : 1st Collaboration Meeting with KCIS\_NewTaipei
Agenda : Both teams shared their projects, reviewed their wet-lab experiments, and discussed the project modeling part of the project.

Date : July 14th, 2021
Event : 2nd Partnership Meeting with ASIJ\_Tokyo
Agenda : Both teams discussed the Northeast Asia Joint Collaboration Network, set up a possible agenda, and developed possible ideas for a YouTube promotion.

Date : July 16th, 2021
Event : 2nd Partnership Meeting with Greece\_United (NOUS)
Agenda : Both teams shared ideas on ways to support one another. Greece\_United shared their progress on human practices part of the project and further discussed possible areas of collaboration.

Date : July 23rd, 2021
Event : 2nd Collaboration Meeting with USP-Brazil
Agenda : Both teams suggested possible areas of collaboration and initiated collaboration on Synthetic Biology Olympics.

Date : August 4th, 2021
Event : 3rd Partnership Meeting with ASIJ\_Tokyo
Agenda : Both teams proposed ideas on the YouTube promotion project and shared areas on ways to support one another.

Date : August 4th, 2021
Event : 3rd Partnership Meeting with Greece\_United (NOUS)
Agenda : Both teams discussed two different projects: blood donation and co-authoring a book. Teams introduced possible ways to support the project.

Date : August 12th, 2021
Event : 4th Partnership Meeting with ASIJ\_Tokyo
Agenda : Both teams shared their progress on project modeling and team Korea\_HS suggested ASIJ\_Tokyo to join the project “How to iGEM” of team NOUS.

Date : August 12th, 2021
Event : 4th Partnership Meeting with Greece\_United (NOUS)
Agenda : Team NOUS presented on how to use MatLab, a mathematical modeling software, to Korea\_HS and introduced possible modeling features for our team.

Date : August 18th, 2021
Event : 5th Partnership Meeting with ASIJ\_Tokyo
Agenda : Both teams discussed the possibility of partnering to establish a shared YouTube/TikTok channel, a joint collaboration network, and a Synthetic Biology olympiad via an extended collaboration.

Date : August 23rd, 2021
Event : 2nd Collaboration Meeting with CCU\_Taiwan
Agenda : Both teams discussed project progress updates, exchanged methods of entrepreneurship (e.g. market analysis), and suggested collaboration in inclusivity (LGBTQ+ in puberty).

Date : August 25th, 2021
Event : 6th Partnership Meeting with ASIJ\_Tokyo
Agenda : Both teams discussed updates on Northeast Asia Joint Collaboration Network and dry lab webinars, breast cancer awareness video, and intellectual exchanges on dry and wet labs.

Date : August 25th, 2021
Event : 1st Collaboration Meeting with NYC\_B1O
Agenda : Both teams discussed project ideas and discussed possible collaboration agenda on integrated human practices/education (resource exchange) and dry lab (intellectual exchange).

Date : September 1st, 2021
Event : 7th Partnership Meeting with ASIJ\_Tokyo
Agenda : Both teams discussed a rough draft and participation of the 2021 Autumn Dry Lab Webinar, a shared TikTok/YouTube channel, the TAS\_Taipei SDG Conference, and the Cancer Meetup.

Date : September 2nd, 2021
Event : 5th Partnership Meeting with Greece\_United (NOUS)
Agenda : Both teams discussed the possibility of wet lab partnership such as exchange of specific wet-lab protocols (e.g. MTT assay and RT-PCR) and measurement and result analysis.

Date : September 3rd, 2021
Event : 2nd Collaboration Meeting with NYC\_B1O
Agenda : Both teams facilitated human practices exchange (e.g. survey and stakeholders) and devised an agenda for an educational exchange program between South Korea and the U.S.

Date : September 8th, 2021
Event : 8th Partnership Meeting with ASIJ\_Tokyo
Agenda : Both teams reviewed progress on interview and animation for a shared YouTube channel and finalized participation roster for Northeast Asia Joint Collaboration Network.

Date : September 9th, 2021
Event : 6th Partnership Meeting with Greece\_United (NOUS)
Agenda : Both teams discussed mentored specific protocols of wet lab, such as MTT cytotoxicity assay (Korea\_HS) and measurement analysis (NOUS) and devised co-authored implementation.

Date : September 11th, 2021
Event : 2nd Collaboration Meeting with IISER\_Bhopal
Agenda : Both teams exchanged methods to satisfy “Partnership,” “Integrated Human Practices,” “Contribution,” “Proof of Concept,” and “Project Model” and suggested co-hosting of events.

Date : September 13th, 2021
Event : 2nd Collaboration Meeting with IISER\_Berhampur
Agenda : Both teams discussed concepts of engineering cycle (process-analyze-results-improve) and application of Python and CellDesigner to modeling deterministic cell line scenarios.

Date : September 16th, 2021
Event : 7th Partnership Meeting with Greece\_United (NOUS)
Agenda : Both teams reviewed the current partnership agenda and finalized workload for human practices (“How To iGEM” and “Give a Blood~Save a Life”) and the wet lab.

Date : September 17th, 2021
Event : 2nd Collaboration Meeting with Siberia
Agenda : Both teams discussed specific methods of RT-PCR band intensity quantification and FITC assay analysis and solutions to issues regarding Siberia’s genetic oscillator rate of differentiation.

Date : September 22nd, 2021
Event : 9th Partnership Meeting with ASIJ\_Tokyo
Agenda : Both teams discussed the finalized script and facilitation division for the 2021 Autumn Dry Lab Webinar and 1st Conference of Northeast Asia Joint Collaboration Network.

Date : September 23rd, 2021
Event : 8th Partnership Meeting with Greece\_United (NOUS)
Agenda : Both teams discussed a possible proposed implementation of fusing affinity peptide - exosome - miRNA construct with chondrocyte-specific cell-penetrating peptide KAFAK.

Date : September 29th, 2021
Event : 10th Partnership Meeting with ASIJ\_Tokyo
Agenda : Both teams debriefed the 2021 Autumn Dry Lab Webinar and 1st Conference of Northeast Asia Joint Collaboration and finalized outline for partnership write-up on the Wiki.

Date : October 4th, 2021
Event : 9th Partnership Meeting with Greece\_United (NOUS)
Agenda : Both teams discussed the “MATLAB Tutorial” provided by Greece\_United and “Give a Blood~Save a Life” event and finalized the outline for partnership write-up on the Wiki.

# Team Human Practices

Date : May 1st, 2021
Event : First HP team meeting
Agenda : Held individual introductions of sub-group members, followed by team leader elections and a short planning session.

Date : June 30th, 2021
Event : First HP interview with Dr. Sungjoon Yoon, a researcher at RWTH
Agent : Discussed theoretical clinical applicability of therapeutic and how to experimentally account for possible in vivo issues (cytotoxicity, immunogenicity in particular).

Date : July 12th, 2021
Event : Submission of experimental-tract report written by HP members to KSCY competition
Agenda: Spread awareness about our project and interacted with other students about their research via KSCY (virtual science fair/competition).

Date : July 28th, 2021
Event : Second HP interview with Dr. Munhong Kim, OB/GYN at Korea Cancer Center Hospital
Agenda : Discussed current demographics of cancer therapeutics, prospects of our peptide therapeutic, and how to account for possible in vivo deficiencies through structural modification.

Date : Aug 4th, 2021
Event : First HP interview with Dr. Youngwoong Lee, a biological science researcher at KAIST
Agenda : Discussed appropriate method of assessing product performance with special focus on cell specificity and intracellular distribution of peptides.

Date : August 14th, 2021
Event : Finalization of Korea\_HS pamphlet
Agenda : Released final version of the informational pamphlet containing information about iGEM competition, cancer, and the specific project made by Ellie.

Date : August 24th, 2021
Event : Release of public engagement survey created by Andrea
Agenda: Released 8-question google form survey assessing awareness of CPP-based treatments among the public. Members recruited around 15 respondents each.

# Team Education

Date : September 9th, 2021
Event : SAS-Puxi Synthetic Biology
Agenda : Haryeong introduced iGEM, Synthetic Biology, and simplified modules of the team's project to elementary school students in SAS-Puxi and also held the “Milk Lab.”

Date : September 25th, 2021
Event : 2021 Autumn Dry Lab Webinar
Agenda : Under a partnership with Team ASIJ\_Tokyo, Team Korea\_HS hosted the 2021 Autumn Dry Lab Webinar providing a walkthrough of mainstream dry lab programs and activities.

Date : September 30th, 2021
Event : BHA-Jeju Synthetic Biology Workshop
Agenda : Bojeong and Chaehyun held a Synthetic Biology Workshop at BHA-Jeju. They discussed an experimental analysis of our project and performed DNA extractions and gel electrophoresis.

Date : October 3rd, 2021
Event : ASIJ\_Tokyo ✕ Korea\_HS YouTube Channel
Agenda : Team ASIJ\_Tokyo and Korea\_HS launched a shared YouTube channel and uploaded two videos (interview compilation and animation) promoting breast cancer awareness.

Date : October 4th, 2021
Event : Lab Kit Webinar
Agenda : Hannah and Matthew hosted virtual lab kit webinars for elementary (“Extracting DNA from Bananas”) and middle (“Designing Your Own Petri Dish”) school students.

Date : October 6th, 2021
Event : SAS-Puxi Synthetic Biology Journalism
Agenda : Haryeong submitted a proposal to design a cancer-specific cell-penetrating peptide for the efficient delivery of siRNA into cancer cells to International Youth Scientists in SAS-Puxi.

Date : October 16th, 2021
Event : “Give a Blood~Save a Life”
Agenda : Under the partnership with Team Greece\_United, Team Korea\_HS held a group blood donation event at Gangnam, Seoul, educating the public on the necessity of safe blood donations.

Date : October 17th, 2021
Event : “How To iGEM” Booklet
Agenda : Under the partnership with Team Greece\_United, Team Korea\_HS co-authored the “How To iGEM” booklet, an engaging guideline to all aspiring iGEMers.

# Team Inclusion

Date : August 15th, 2021
Event : First Inclusion meeting
Agenda : Introduced sub-group, project decisions, role division across projects, and deadline establishment to the team members.

Date : September 9th, 2021
Event : First themed posting on Korea\_HS Instagram account by Bojeong
Agenda : Posted about Alan Hart, an American radiologist, in order to increase awareness about LGBTQ+ STEM pioneers.

Date : September 22nd, 2021
Event : Finalization of LGBTQ+ in STEM inclusion report by Hannah and Ellie
Agenda : Completed all parts of Inclusion report: Introduction, Analysis of Status-Quo, Significance, Plan of Action, and Conclusion.

Date : October 18th, 2021
Event : LGBTQ+ STEM webinar through zoom event hosted by Matthew and Hannah
Agenda : Discussed issues specific to LGBTQ+ in STEM, the significance of ameliorating such issues, a plan of action to target the issues, and a short conclusion and takeaways.

Date : October 20th, 2021
Event : Interview with LGBTQ+ affiliated STEM personnel Professor Ramirez
Agenda : Discussion of STEM LGBTQ+ in the status-quo and how to ameliorate issues faced by LGBTQ+ in STEM.

# Korea\_HS-Hosted Events

Date : August 8th, 2021
Event : Korea Meetup
Agenda : The team convened with Korean iGEMers – KUAS\_Korea and Yonsei\_KOR – sharing project ideas and discussing possibilities of local collaboration (education) initiatives.

Date : September 24th, 2021
Event : Northeast Asia Joint Collaboration Network
Agenda : The team convened with high school teams of Northeast Asia (Japan, China, Taiwan, etc) and discussed opportunities for a common communication platform for complementary support.

Date : September 25th, 2021
Event : 1st Global iGEM Diagnostics & Therapeutics Conference
Agenda : The team hosted the 1st Global iGEM Diagnostics & Therapeutics Conference to discuss the role of Synthetic Biology in future diagnostics and therapeutics with five other teams.

Date : October 7th, 2021
Event : Instagram Project
Agenda : The team created the Instagram Project. It was a collaborative outreach initiative connecting global iGEMers via an individualized posting (with contact and project information) for each team.

© 2021 Korea-HS.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Korea HS/Engineering

KORHS

# [KORHS](https://2021.igem.org/Team%3AKorea_HS)

* TEAM
  + [Team](https://2021.igem.org/Team%3AKorea_HS/Team)
  + [Attributions](https://2021.igem.org/Team%3AKorea_HS/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AKorea_HS/Collaborations)
* PROJECT
  + [Communication](https://2021.igem.org/Team%3AKorea_HS/Communication)
  + [Contribution](https://2021.igem.org/Team%3AKorea_HS/Contribution)
  + [Description](https://2021.igem.org/Team%3AKorea_HS/Description)
  + [Engineering](https://2021.igem.org/Team%3AKorea_HS/Engineering)
  + [Experiments](https://2021.igem.org/Team%3AKorea_HS/Experiments)
  + [Implementation](https://2021.igem.org/Team%3AKorea_HS/Implementation)
  + [Notebook](https://2021.igem.org/Team%3AKorea_HS/Notebook)
  + [Partnership](https://2021.igem.org/Team%3AKorea_HS/Partnership)
  + [Results](https://2021.igem.org/Team%3AKorea_HS/Results)
* PARTS
  + [Part Collection](https://2021.igem.org/Team%3AKorea_HS/Part_Collection)
  + [Parts](https://2021.igem.org/Team%3AKorea_HS/Parts)
  + [Improve](https://2021.igem.org/Team%3AKorea_HS/Improve)
* SAFETY
  + [Safety](https://2021.igem.org/Team%3AKorea_HS/Safety)
* HUMAN PRACTICES
  + [Human Practices](https://2021.igem.org/Team%3AKorea_HS/Human_Practices)
* AWARDS
  + [Education](https://2021.igem.org/Team%3AKorea_HS/Education)
  + [Inclusivity](https://2021.igem.org/Team%3AKorea_HS/Inclusivity)
  + [Model](https://2021.igem.org/Team%3AKorea_HS/Model)

[JUDGING](https://igem.org/2021_Judging_Form?id=3824)

ENGINEERING

# Design

1) What is the purpose of our design?

The purpose was to design a cancer-specific short peptide for the efficient delivery of siRNA into target (cancer) cells.

2) Which biological parts will be required to implement function?

* ◉ A549 (human lung adenocarcinoma cell line)
* ◉ MRC5 (human normal cell line)
* ◉ Gene CYP1A1-targeting gene
* ◉ Three types of peptides used in this research: buforin IIB (BIIB), MV1, and MV2

![](https://static.igem.org/mediawiki/2021/c/c6/T--Korea_HS--eng_Figure1.png)

# Build

1) Will you need to generate different variants of your build?

Buforin IIb has been known to target cancer cells by its positive charge. In this research, we design a modified version of Buforin IIB peptide sequence (MV1 and MV2) to find the optimized peptide sequence for low cytotoxicity and enhanced siRNA delivery into targeted cancer cells.

We discovered that buforin IIb had RLLR repeats at the C-terminal end. We hypothesized that deleting the RLLR sequence may lower cell cytotoxicity and increase the siRNA delivery because the shorter peptide together with a positive charge has the advantage of entering through the cell membrane.

2) How can you screen for correct builds?

* ◉ We planned to perform a cell cytotoxicity test using MTT assay with various concentrations of different versions of peptides
* ◉ We planned to perform an siRNA transfection test and measure the target gene expression level using agarose gel-based RT-PCR.

# Test

1) Which types of measurements are needed?

* ◉ The MTT assay

It is used to measure cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity. This colorimetric assay is based on the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) to purple formazan crystals by metabolically active cells. The viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduce the MTT to formazan. The insoluble formazan crystals are dissolved using a solubilization solution and the resulting colored solution is quantified by measuring absorbance at 570 nanometers using a multi-well spectrophotometer. The darker the solution, the greater the number of viable, metabolically active cells.

* ◉ Agarose gel-based RT-PCR

Reverse transcription converts the RNA to cDNA. Then PCR amplification amplifies the template cDNA. Agarose gel electrophoresis allows detection and quantification of amplified DNA. PCR amplification can be broken down into three phases. The exponential phase occurs when the reaction components are in excess, thereby theoretically allowing for a doubling of the PCR product per cycle. In the next phase, the amplification of the PCR product occurs linearly rather than exponentially as the reagents are partially depleted. Then, the plateau phase represents the point in which the amplifications have further slowed or have completely stopped. The end product of PCR is analyzed in the agarose gel. The band intensity of the amplified gene detected on the agarose gel is correlated with the target RNA copy number at the linear phase.

2) Are your units arbitrary, relative, or absolute?

* ◉ MTT: absolute units

It is measured by the spectrophotometer – absorbance of 570 nm.

* ◉ RT-PCR: relative units

The target gene expression must be normalized by GAPDH (housekeeping gene).

# Learn

1) Did your system work as anticipated?

Cell cytotoxicity:

![](https://static.igem.org/mediawiki/2021/e/e8/T--Korea_HS--eng_Figure2.png)

![](https://static.igem.org/mediawiki/2021/c/c1/T--Korea_HS--eng_Figure3.png)

Both MV1 and MV2 have low cytotoxicity compared to buforin IIB.

2) How does your experimental data compare to your model?

![](https://static.igem.org/mediawiki/2021/e/eb/T--Korea_HS--eng_Figure4.png)

siRNA-CYP1A1 (20 µM) showed the lowest CYP1A1 expression level, meaning that MV1 successfully delivers siRNA targeting CYP1A1 only in lung cancer cells (A549). The cytotoxicity and RT-PCR results indicate that MV1 was the best peptide to achieve both low cytotoxicity and delivery of siRNA into lung cancer cells.

© 2021 Korea-HS.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Korea HS/Experiments

KORHS

# [KORHS](https://2021.igem.org/Team%3AKorea_HS)

* TEAM
  + [Team](https://2021.igem.org/Team%3AKorea_HS/Team)
  + [Attributions](https://2021.igem.org/Team%3AKorea_HS/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AKorea_HS/Collaborations)
* PROJECT
  + [Communication](https://2021.igem.org/Team%3AKorea_HS/Communication)
  + [Contribution](https://2021.igem.org/Team%3AKorea_HS/Contribution)
  + [Description](https://2021.igem.org/Team%3AKorea_HS/Description)
  + [Engineering](https://2021.igem.org/Team%3AKorea_HS/Engineering)
  + [Experiments](https://2021.igem.org/Team%3AKorea_HS/Experiments)
  + [Implementation](https://2021.igem.org/Team%3AKorea_HS/Implementation)
  + [Notebook](https://2021.igem.org/Team%3AKorea_HS/Notebook)
  + [Partnership](https://2021.igem.org/Team%3AKorea_HS/Partnership)
  + [Results](https://2021.igem.org/Team%3AKorea_HS/Results)
* PARTS
  + [Part Collection](https://2021.igem.org/Team%3AKorea_HS/Part_Collection)
  + [Parts](https://2021.igem.org/Team%3AKorea_HS/Parts)
  + [Improve](https://2021.igem.org/Team%3AKorea_HS/Improve)
* SAFETY
  + [Safety](https://2021.igem.org/Team%3AKorea_HS/Safety)
* HUMAN PRACTICES
  + [Human Practices](https://2021.igem.org/Team%3AKorea_HS/Human_Practices)
* AWARDS
  + [Education](https://2021.igem.org/Team%3AKorea_HS/Education)
  + [Inclusivity](https://2021.igem.org/Team%3AKorea_HS/Inclusivity)
  + [Model](https://2021.igem.org/Team%3AKorea_HS/Model)

[JUDGING](https://igem.org/2021_Judging_Form?id=3824)

EXPERIMENTS

# 1st Lab Meeting

Agenda 1: Micropipette Practice

Micropipette information: MicroPipette (Gilson) was utilized for all experimental expenditures. It provided a convenient, cost-effective delivery method and required fewer chemicals to deliver a certain volume of fluid (maximum volume: 1mL = 1000μL / minimum volume: 0.2mL = 200μL - 1000/200/100/20/10/2P).

Pipette Handling Procedures

![](https://static.igem.org/mediawiki/2021/8/81/T--Korea_HS--ex_Figure_1.jpg)

* 1. Place the tip of the pipette into disposable tips.
* 2. Press the pressure button (absorbs a certain volume of liquid).
* 3. Rotate rubber to set volume.
* 4. Press until first stop before uptaking liquid.
* 5. Press till 2nd stop to release all remaining chemicals (more safety and accuracy).

Pipette Handling Notice:

* 1. The solution should stay out of the pipet as contamination could occur.
* 2. Always hold the pipette vertically, tip down.
* 3. Every time the pipet enters the solution, the solution can change concentrations or be contaminated. Change the tip every time the chemical changes.
* 4. Press the release button to release the tip.
* 5. Place the tip on the left wall of the tube and let the liquid move down.
* 6. A lot of waste may be generated. Try minimizing waste for the environment.
* 7. When releasing liquid, always hold it straight, not slanted (refer to the law of gravity: all liquids flow downwards).

Agenda 2: Cell Culture Experiment

Procedural Materials

![](https://static.igem.org/mediawiki/2021/3/39/T--Korea_HS--ex_Figure_2.jpg)

* 1. MRC5 (normal lung cell/fibroblast presenting slow cell division, thus displaying higher sensitivity and requiring careful handling)
* 2. A549 (lung cancer/carcinoma cell that proliferates much more rapidly)
* 3. Plastic cell culture plate (transparent for microscopy)
* 4. 6-well (with bigger individual wells) and/or 96-well plate (with smaller individual wells)

Procedural Principle

During cell culture experiments, two types of cell lines might ensue: adherent cell (growing on the bottom of medium) and suspension cell (not growing on the bottom of medium).

Agenda 3: Cell Detachment Experiment

![](https://static.igem.org/mediawiki/2021/0/03/T--Korea_HS--ex_Figure_3.jpg)

One method to cell detachment is by physical force, or scraping of cells. However, this method is not recommended as cells often burst and die off when scraped. Enzyme method, correcting limitations described hitherto, was utilized. Trypsin protease degrades intracellular proteins that bind cells together, thus inducing cleavage. It is a recommended protocol to keep a maximum of a 10-minute interval as cells will die when isolated - minimized interval with trypsin to avoid cell damage (i.e. suspended cells = apoptosis). 5-minute enzyme incubation time for trypsin to detach all cells was set. Cells in subculture were counted (cells/mL) to check if cells detach.

# 2nd Lab Meeting

Pre-Experimental Reference

In this study, three CPPs (derivative buforin IIb, analog MV1 and MV2) were tested. MV1 and MV2 were modified because smaller peptides enter cancer cells more easily.

Buforin IIb: RAGLQFPVGRLLRRLLRRLLR 21aa + charge (~3000Mw, 1000Mw = 1mg)
MV1: RAGLQFPVGRLLRRLLR 17aa + charge (2524 Mw)
MV2: RAGLQFPVGRLLR 13aa + charge (1985 Mw)

Agenda 1: Colorimetric MTT Metabolic (Cytotoxicity) Assay

Procedural Principle

Cell counter holds a lack of sensitivity. Thus, an MTT assay was performed in this study. MTT-yellow tetrazolium (substrate) is inserted in each well and mitochondrial reductase enzyme inside viable cells reduces substrate to purple-dyed formazan (reaction: yellow → purple). Purple-dyed formazan doesn’t absorb purple. Hence, the spectrometer measures absorbance.

The mathematically presented graph presents the x-axis (live cell number) and y-axis (absorbance at 570nm) with the following assumption: positive linear relationship = higher concentration = higher absorbance. That is, in reality, the graph measures the correlation between live cell number and mitochondria reductase activity. Overall, we aim to use a peptide with low cytotoxicity which leads to higher cell viability and minimizes side effects on normal cells.

Procedural Overview

* 1. 5 μL MTT solution insertion into 9 wells
* 2. 1-hour incubation for reaction completion
* 3. Supernatant (remaining media) removal
* 4. 100X or 100 μL solubilization solution application (cytolysis and formazan dye release)
* 5. 10 minutes incubation for reaction completion
* 6. 550-600nm spectrometer analysis

96-Well Plate Design

![](https://static.igem.org/mediawiki/2021/d/d6/T--Korea_HS--ex_Figure_4.jpg)

* ▪ A549 lung cancer cell in each well
* ▪ 1st nine: Cell media [control (healthy cancer cells)]
* ▪ 2nd nine: Dead cells [SDS → leads to degradation of proteins 20%]
* ▪ 3rd nine: Concentration-dependent (5, 10, 20, 40, 80, 100 micromoles)
  + ▫ Assumption: The higher the concentration, the higher the cytotoxicity.
  + ▫ Model: 5 μM first column MV2, second column MV1, third column: BIIB
* ▪ 4th nine: 10μM first column MV2, second column MV1, third column: BIIB
  + ▫ Repeat the remaining nine(s) in a concentration-dependent manner.
* ▪ Each well = 100 μL total volume

Agenda 2: Stock Solution/Peptide Synthesis

Procedural Principle

On one hand, chemotherapeutic antineoplastic intercalates DNA of both normal and cancer cells. On the other hand, cancer leads to a loss of normal function. Below devises a common approach and/or paradigm to synthetic-biological studies.

* 1. DNA modeling, cloning, synthesis
* 2. Host cell insertion (decide if normal cell or bacteria will be used)
* 3. Protein translation
* 4. Protein extraction
* 5. Protein purification

However, CPP is a very small macromolecule of <30 amino acids. Thus, it renders purification difficult and cost-inefficient. Hence, without transfecting the bacterial host, the peptide synthesizer will bio-synthesize desired amino acid sequence via peptide bond reaction, yielding powdered peptides.

Procedural Overview

![](https://static.igem.org/mediawiki/2021/b/ba/T--Korea_HS--ex_Figure_5.jpg)

Stock Solution Creation: 1mL of 1mM (1000μM)

* ▪ Buforin IIb: (~3000Mw, 1000Mw = 1mg)
* ▪ MV1: (2524 Mw) : 2.524mg/mL → .002524g
* ▪ MV2: (1985 Mw) : 1.985mg/mL → .001985g

* 1. Place the weighing boat on the scale and rescale to 0.
* 2. Add powder and measure weight.
* 3. Place powder inside the tube in the machine (units only in grams).
* 4. Place label on tube CPP type and concentration (1mM).

# 3rd Lab Meeting

Agenda 1: Cell-Penetration Efficacy Experiment (FITC Assay)

Procedural Materials

* ▪ Cell line MRC5 (human lung normal)
* ▪ Cell line A549 (human lung cancer)
* ▪ Stock FITC-MV1 stock CCP
* ▪ Stock FITC-MV2 stock CCP

\* Stock concentration - constant at 1mM

Procedural Overview

![](https://static.igem.org/mediawiki/2021/6/60/T--Korea_HS--ex_Figure_6.jpg)

Preparing Stock-Media Mixtures

* 1. Pre-experimental cell sample incubation (30 minutes)
* 2. Create a mixture using 960µl RPMI media and 40µl stock (40µM)
* 3. Put sample tube into Vortex-Genie 2 vortex mixer
* 4. Create a mixture using 500µl RPMI media and 500µl previous mixture (20µM)
* 5. Put sample tube into Vortex-Genie 2 vortex mixer
* 6. Create a mixture using 500µl RPMI media and 500µl previous mixture (10µM)
* 7. Put sample tube into Vortex-Genie 2 vortex mixer
* 8. Create a mixture using 500µl RPMI media and 500µl previous mixture (5µM)

\*Repeat this 4 times each for MV1 and MV2 of both cell lines MRC5 and A549

Checking for Highest Efficiency

![](https://static.igem.org/mediawiki/2021/3/3c/T--Korea_HS--ex_Figure_7.png)

* 1. Take cell samples out of incubation.
* 2. Discard media solution covering cell samples using a micropipette.
* 3. Gently and slowly place each stock-media mixture onto cell samples using a micropipette (20µl for each concentration).

# 4th Lab Meeting

Agenda 1: Intracellular siRNA Delivery Experiment (Total RNA Extraction)

Procedural Design

![](https://static.igem.org/mediawiki/2021/8/8f/T--Korea_HS--ex_Figure_8.png)

Construct: siRNA (-) + CPP (MV1) (+)

* ▪ 1st sample: siCON + R (20mM)
* ▪ 2nd sample: siCYP1A1 + R (20mM)
* ▪ 3rd sample: (siCYP1A1) 20mM
* ▪ 4th sample: (siCYP1A1) 40mM
* ▪ 5th sample: (siCYP1A1) 80mM

\*In case the efficiency is below Religent, change the concentrations)

Procedural Overview

RNA Extraction with an RNA extraction kit (Intron)

* 1. Mix R-buffer and β-M
* 2. Cell lysis (5 minutes)
  + a. Insert Step 1 mixture into cell tube+ b. Incubate (5 minutes)
* 3. 70% ethanol
  + a. Precipitation
* 4. Move solution to a new column
  + a. New column
    - i. Silica membrane that is able to bind RNA
    - ii. Extract only the RNA
* 5. Insert washing buffer A and shake
  + a. Washing buffer takes away DNA, protein, and impurities
* 6. Insert washing buffer B and shake
* 7. Insert elution buffer
  + a. RNA is dissolved inside the solution

# 5th Lab Meeting

Procedural Principle

In this study, reverse transcription-polymerase chain reaction (RT-PCR) was performed for the analysis of PI-siCYP1A1 transfection and gene silencing efficacy. Amongst heterogeneous characterizations of RNA (e.g. mRNA, rRNA, tRNA, miRNA), mRNA was the material of interest. mRNA via RTase enzyme can be converted to synthesize complementary DNA (cDNA).

Procedural Materials

* ▪ RNA extraction kit
  + ⟶ R-buffer+ + ⟶ Washing Buffer A+ + ⟶ Washing Buffer B+ + ⟶ Elution Buffer+ + ⟶ 70% Ethanol
* ▪ Chemical
  + ⟶ β-mercaptoethanol (β-M)

Procedural Design

Herein, construct siRNA (- due to phosphate backbone) + CPP (MV1) (+) was tested. siCon was set as a negative control and siCYP1A1 and liposome reagent (R) were set as positive controls. Collectively, 10 samples from MRC5 and A549 were prepared (MV1 40mM).

* ▪ 1st sample: siCON + R (20mM)
* ▪ 2nd sample: siCYP1A1 + R (20mM)
* ▪ 3rd sample (siCYP1A1) 20mM
* ▪ 4th sample (siCYP1A1) 40mM
* ▪ 5th sample (siCYP1A1) 80mM

Expected CYP1A1 Expression Levels
![](https://static.igem.org/mediawiki/2021/5/56/T--Korea_HS--ex_tb1.png)

Procedural Overview

![](https://static.igem.org/mediawiki/2021/e/e3/T--Korea_HS--ex_Figure_9.png)

* 1. RNA extraction
  + a. Cell lines containing DNA, RNA, protein, and lipid.
    - i. Total RNA purification and extraction
    - ii. Lipid, protein, and DNA removal
* 2. RNA → DNA conversion (i.e. reverse transcription PCR)

# 6th Lab Meeting

Procedural Principle

RNA extraction allows for analysis of expression intensity but with low sensitivity. Following cDNA (complementary to mRNA) synthesis, however, sensitivity is induced under the assumption of mRNA quantity = cDNA quantity. In this assay, the band intensity of two genes - CYP1A1 and GAPDH (housekeeping gene) - will be quantified for comparison.

Procedural Design

![](https://static.igem.org/mediawiki/2021/f/f0/T--Korea_HS--ex_Figure_10.png)

Agarose Gel

* ▪ 1x TBE buffer
* ▪ 2.6g of Agarose (1.3%)
  + ▫ High Melting Point
* ▪ DNA staining solution
  + ▫ 20,000:1 ratio

Expected CYP1A1 and GAPDH Band Intensities
![](https://static.igem.org/mediawiki/2021/7/7a/T--Korea_HS--ex_tb2.png)

© 2021 Korea-HS.

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# Team:Korea HS/Results

KORHS

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RESULTS

# Colorimetric MTT Metabolic Assay

Colorimetric MTT metabolic (Prestoblue) assay was performed to analyze in vitro cytotoxicity to cell lines A549 and MRC5. After confirming complete solubilization of the purple formazan crystals, the absorbance of the samples using a microplate reader was measured under 570 nm. Samples included no treatment (negative control), 10% sodium dodecyl sulfate (SDS, positive control), buforin IIb (5mM~100mM), MV1 (5mM~100mM), and MV2 (5mM~100mM).

![](https://static.igem.org/mediawiki/2021/d/d4/T--Korea_HS--rs_Figure_1-1.png)
(Figure 1.1 - PI in vitro cytotoxicity evaluation via MTT metabolic assay, A549.)

![](https://static.igem.org/mediawiki/2021/c/c8/T--Korea_HS--rs_Figure_1-2_.png)
(Figure 1.2 - PI in vitro cytotoxicity evaluation via MTT metabolic assay, MRC5.)

Cell line A549 with no treatment indicated 0.16 procedure-defined unit (pdu), a baseline value indicating no cell deaths herein; 10% SDS treatment indicated 0.4 pdu. Buforin IIb from 5~40 mM indicated approximately 0.16 pdu, excluding the margin of error. However, the cytotoxic activity of buforin IIb was observed at high concentrations (80-100mM) with approximately 0.11 pdu.

Dose-dependent manner of cytotoxicity was also observed under MV1 and MV2 treatments with a comparable trend. No significant cell deaths were observed until 40mM – A549 cell viability was higher under MV1 and MV2 treatments (0.17 pdu). Significant cytotoxicity (<0.15 pdu) was observed at 80-100 mM. A comparable trend was reported under MRC5 treatments.

# FITC Specificity Assay (Confocal Microscopy)

PI displaying the highest cell viability – MV1 – was covalently conjugated with FITC for the assessment of MV1 cell specificity and post-internalization intracellular distribution. Fluorophores were excited with an argon laser at 488nm and merged with the DAPI staining image via confocal microscopy.

![](https://static.igem.org/mediawiki/2021/f/fb/T--Korea_HS--rs_Figure_2-1.png)
(Figure 2.1 - PI cancer specificity evaluation via FITC confocal microscopy, A549.)

![](https://static.igem.org/mediawiki/2021/2/28/T--Korea_HS--rs_Figure_2-2.png)
(Figure 2.2 - PI cancer specificity evaluation of PI via FITC confocal microscopy, MRC5.)

DAPI staining of A549 presented more frequent, smaller nuclei from its counterpart. FITC fluorescence of MV1-FITC synthetic construct was significantly observable under 40mM MV1 (than 20mM MV1) for A549 and MRC5. At MV1 40 mM, MV1-FITC fluorescence accumulated and was detected in A549 cytoplasm and nuclei, presenting high intracellular distribution and transfection efficacy.

At MV1 40mM, MV1-FITC was poorly internalized to the nucleus and cytoplasm of MRC5, comparable to internalization and distribution presented at MV1 20 mM. It was statistically deduced further that internalized MV1-FITC did not display transfection efficacy to yield observable effects.

# RT-PCR (Gene Silencing/Transfection Efficacy Analysis)

Following cDNA synthesis via Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV-RT) of CYP1A1 mRNA, polymerase chain reaction (PCR) assay was performed for the analysis of transfection efficacy and target gene silencing of CPP/siRNA complexes. Subsequently, band intensity (agarose gel electrophoresis) quantification was performed via the Image J program.

![](https://static.igem.org/mediawiki/2021/9/94/T--Korea_HS--rs_Figure_3-1.png)
(Figure 3.1 - Gene silencing/transfection efficacy analysis via RT-PCR, A549.)

![](https://static.igem.org/mediawiki/2021/c/c4/T--Korea_HS--rs_Figure_3-2.png)
(Figure 3.2 - Gene silencing/transfection efficacy analysis via RT-PCR, MRC5.)

![](https://static.igem.org/mediawiki/2021/5/5f/T--Korea_HS--rs_Figure_3-3.png)
(Figure 3.3 - Normalized CYP1A1 expression via RT-PCR, A549 and MRC5.)

Under A549 and MRC5, expressions of gene CYP1A1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, control) were investigated. Samples included a random arrangement of PI (RNAimax Lipofectamine) and siRNA negative control (scrambled siRNA) - control siRNA (20µM) + RNAimax Lipofectamine, CYP1A1 siRNA (20µM) + RNAimax Lipofectamine, CYP1A1 siRNA (20µM) + 40 mM MV1, CYP1A1 siRNA (40µM) + 40 mM MV1, and CYP1A1 siRNA (80µM) + 40 mM MV1.

Under A549, band intensity quantification presented the highest silencing efficacy under CYP1A1 siRNA (20µM) + 40 mM MV1 (0.2 pdu, normalized). Silencing efficacy of CYP1A1 siRNA (40µM) + 40 mM MV1 and CYP1A1 siRNA (80µM) + 40 mM MV1 and control siRNA (20µM) + RNAimax Lipofectamine (0.3 pdu, normalized) and CYP1A1 siRNA (20µM) + RNAimax Lipofectamine (0.4 pdu, normalized) remained comparable. GAPDH expressions were comparable, validating MV1-CYP1A1 siRNA complex viability to other intracellular mechanisms.

Confirmed through literature, band intensity and normalized expression level of CYP1A1 under MRC5 were inherently lower (range: 0.09 pdu - 0.14 pdu, normalized) and CYP1A1 siRNA (20µM) + 40 mM MV1 presented no effect on decreasing the normalized CYP1A1 expression level, further confirming neoplasia specific cytolytic activity of the synthetic construct herein developed.

# Colorimetric MTT Metabolic Assay (Cell Death Analysis)

Colorimetric MTT metabolic (Prestoblue) assay was performed to analyze cell death to cell lines A549 and MRC5. After confirming complete solubilization of the purple formazan crystals, the absorbance of the samples using an ELISA reader was measured under 570 nm. Samples included positive and negative controls of siRNA (MV1-DOX and MV1-DOX + siRNA Control) and final synthetic construct (FSC, MV1-DOX + siRNA-CYP1A1).

![](https://static.igem.org/mediawiki/2021/2/2b/T--Korea_HS--rs_Figure_4.png)
(Figure 4 - Cell death analysis of CPP-DOX/siRNA complex via MTT metabolic assay, A549 and MRC5.)

Under A549, MV1-DOX and MV1-DOX + siRNA Control displayed higher cell viability (0.125 pdu), and FSC showed the highest cell death efficacy (0.06 pdu). Under MRC5, all samples presented higher viability than their MRC5 counterpart with statistical assimilation at 0.14 pdu, further confirming FSC cytolytic activity specific to the adenocarcinoma cell line.

© 2021 Korea-HS.