iGEM 2022 — Team korea-hs

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

NOTEBOOK

# Team Korea\_HS Meeting

Date: 04/03/2022

Event: 1st Whole Meeting (Virtual)

Agenda: The team was introduced to its members for the first time, the 2022 research idea by Professor Lee, and the iGEM competition logistics by student advisor Matthew Lee.

Date: 04/17/2022

Event: 2nd Whole Meeting (Virtual)

Agenda: The team further discussed logistics in terms of application and planning.

Date: 05/01/2022

Event: 3rd Whole Meeting (Virtual)

Agenda: The team was introduced to the TFAM-DNA complex and its role in architectural packaging for the stability of DNA by Professor Lee. In addition, the team elected its leaders in the general team, the Collaboration Team, and the Human Practices Team.

Date: 05/23/2022

Event: 4th Whole Meeting (Virtual)

Agenda: The team discussed the experimentation process of the project introduced by Professor Lee and met in subteams to discuss their aspirations for the competition.

Date: 06/05/2022

Event: 5th Whole Meeting (Virtual)

Agenda: The team planned the logistics of their wet and dry lab processes and met in subteams to start projects for the competition.

Date: 06/19/2022

Event: 6th Whole Meeting (Virtual)

Agenda: Professor Lee introduced methods for DNA fragmentation and completed a Progress Checkup.

Date: 07/03/2022

Event: 7th Whole Meeting (Virtual)

Agenda: Student Advisor Matthew Lee discussed the logistics of Special Awards teams and the attendance logistics for the Grand Jamboree.

Date: 07/16/2022

Event: 8th Whole Meeting (Virtual and In-Person)

Agenda: This meeting was carried out in person for all those who could attend while also providing a virtual link for those attending from home. Student advisor Matthew Lee introduced the iGEM cycle and deadlines, and in-person members completed the necessary tasks.

Date: 07/30/2022

Event: 9th Whole Meeting (In-Person)

Agenda: Members who could make it in person met in their subteams (Collaboration, Human Practices) and completed team projects, such as a public sticker poll and the creation of accessible DNA kits.

Date: 08/06/2022

Event: 10th Whole Meeting (Virtual)

Agenda: The team completed their Progress Checkup and continued discussing necessary deadlines and tasks.

Date: 08/21/2022

Event: 11th Whole Meeting (Virtual)

Agenda: Professor Lee discussed the results of the wet and dry labs with the team. In addition, subteams were able to meet and discuss the necessary elements of their deadlines.

Date: 09/03/2022

Event: 12th Whole Meeting (Virtual)

Agenda: Professor Lee continued the discussion from the last meeting’s results with the team, and the team continued to plan and complete their projects, meeting as necessary.

Date: 09/25/2022

Event: 13th Whole Meeting (Virtual)

Agenda: Professor Lee continued the discussion from the last meeting’s results with the team, and the team continued to plan and complete their projects, meeting as necessary.

# Human Practice Meeting

Date: 06/01/2022

Event: 1st Human Practice Meeting

Agenda: Team Korea\_HS has met the advisor for specific instructions catered toward Human Practices and Education/Communication

Date: 06/12/2022

Event: 2nd Human Practice Meeting

Agenda: Team Korea\_HS look up for professional biologists or chemists to interview

Date:06/19/2022

Event: 3rd Human Practice Meeting

Agenda: Discuss possible educomm projects

Date: 06/28/2022

Event: 4th Human Practice Meeting

Agenda: Plan for the first offline team meeting, surveys, and pamphlet distributions

Date: 07/13/2022

Event: 5th Human Practice Meeting

Agenda: First in-person meeting. Create surveys and posters based on the plan we made previously.

Date: 07/18/2022

Event: 6th Human Practice Meeting

Agenda: Second in-person meeting. Go out to the streets and display the posters and surveys. In addition to that, distribute team brochures.

Date: 07/25/2022

Event: 7th Human Practice Meeting

Agenda: brainstorm about some of the possible ways to educate the general population on synthetic biology and TFAM.

Date: 08/01/2022

Event: Prepare an interview for Sungjun Yoon, a CEO of a synthetic biology company. Create a list of questions to ask related to the strengths and weaknesses of our project.

Date: 08/07/2022

Event: 9th Human Practice Meeting

Agenda: Prepare an interview for Yoseb Song, who is pursuing a postdoctoral degree at MIT. Prepare questions to ask related to our project, taking into consideration the answers we received from Sungjun Yoon.

Date: 08/14/2022

Event: 10th Human Practice Meeting

Agenda: Package tools that will be used in the Synthetic Biology Summer Camp (SBSC).

Date: 08/21/2022

Event: 11th Human Practice Meeting

Agenda: Prepare an interview for Jaehan Park; we created possible questions to ask. In addition to this, we brainstorm possible education/communication tools we can implement in our project.

Date: 09/03/2022

Event: 12th Human Practice Meeting

Agenda: Create a design for USB that will be distributed in middle and high schools and related to data security. Then create an online quiz regarding data security.

Date: 09/18/2022

Event: 13th Human Practice Meeting

Agenda: Create 5 videos for online courses, then create a quiz that corresponds with the videos.

Date: 09/25/2022

Event: 14th Human Practice Meeting

Agenda: The team finalized all the projects and worked on Wiki.

# Interviews

Date: 08/01/2022

Event: Interview with Sungjun Yoon, the CEO of Fortuaga Bio

Agenda: The team listened to the presentation and the speech, asked questions about the presentations, shared the team’s presentations, and sought advice.

Date: 08/08/2022

Event: Interview with Yoseb Song, MIT postdoc

Agenda: The team listened to the presentation and the speech, asked questions about the presentations, shared the team’s presentations, and sought advice.

Date: 08/23/2022

Event: Interview with Jaehan Park, General Manager of data management at Samsung

Agenda: The team listened to the presentation and the speech, asked questions about the presentations, shared the team’s presentations, and sought advice.

Date: 09/27/2022

Event: Sungjun Lee, Member of SAP - a software company

Agenda: The team listened to the presentation and the speech, asked questions about the presentations, shared the team’s presentations, and sought advice.

Date: 09/30/2022

Event: Stew Pienaar, Learning Support instructor at American International School of Johannesburg (AISJ)

Agenda: The team listened to the presentation and the speech, asked questions about the presentations, shared the team’s presentations, and sought advice.

Date: 10/01/2022

Event: Teagsang Yoo, Worker at SK biopharmaceuticals

Agenda: The team listened to the presentation and the speech, asked questions about the presentations, shared the team’s presentations, and sought advice.

# Education & Communication

Date: 07/30/2022

Event: Survey and Pamphlet Distribution

Agenda: The team created three physical surveys about PCR and Data Storage by DNA and distributed pamphlets to the public.

Date: 08/02/2022

Event: Education session

Agenda: The team started the meeting by giving a presentation about Data Storage by DNA and Synthetic Biology and continued the Education Session with word search, coloring books, and other interactive activities.

Date: 09/25/2022

Event: Educational Webinar

Agenda: The team showed the pre-made Education video and presented it to the webinar. The questions being asked were answered by the team after the presentation.

Date: 10/30/2022

Event: Synthetic Biology Summer Camp (SBSC)

Agenda: The team did Synthetic Biology Summer Camp virtually. The team showed the video tutorial and instructed live to help any who were in need. The team also sent out the packet to the destination address, which included all the material that was needed and the instruction sheet.

# Collaboration Meetings

Date: 06/01/ 2022

Event: First official collaboration meeting

Agenda: The team discussed how many teams they would like to collaborate with and what projects they would like to do, and created a slideshow for the collaboration meeting.

Date: 06/10/2022

Event: 2nd official collaboration meeting

Agenda: Team finalized the final 10 iGEM teams that they wanted to collaborate with.

Date: 06/17/2022

Event: 3rd official collaboration meeting

Agenda: the team finished contacting all the teams that they finalized during the last meeting and discussed our partnership.

Date: 06/23/2022

Event: 1st Collaboration meeting with NU Kazakhstan

Agenda: The team shared their collaboration team meeting presentations and talked about the new project that they finalized at the last meeting.

Date:06/24/2022

Event: 4th official collaboration meeting

Agenda: Team started by recapping their first collaboration meeting with NU Kazakhstan and finalized the partnership teams.

Date: 07/ 02/ 2022

Event: 1st Collaboration meeting with Gems Taiwan

Agenda: Share our project through our presentation and offer Gems Taiwan the collaboration projects that we are working on.

Date: 07/08/ 2022

Event: 5th official collaboration meeting

Agenda: Recapped the last collaboration meeting with Gems Taiwan and finalized 2 direct projects and 5 indirect projects.

Date: 07/09/2022

Event: 1st Collaboration meeting with ASIJ Tokyo

Agenda: The team discussed each other's projects and their own collaboration projects.

Date: 7/10/2022

Event: 1st Collaboration meeting with ICT Mumbai

Agenda: The team introduced their projects and offered each other’s projects

Date:7/15/2022

Event: 6th official collaboration meeting

Agenda: The team started the meeting by recapping the ASIJ Tokyo and ICT Mumbai collaboration meeting. The team also discussed who will be making the Instagram posts from the Instagram Reel Project and the iGEMer’s Badge Project.

Date: 7/15/2022

Event: 1st Meeting with Thailand RIS

Agenda: Both teams did their presentations about their projects and their collaboration projects.

Date: 7/16/2022

Event: 1st Meeting with Seoul Korea

Agenda: Both teams shared their project progress and

Date: 7/17/2022

Event: 1st Meeting with Empire Gene

Agenda: Both teams introduced the progress of their projects and shared ideas for further collaboration in the future.

Date:7/22/2022

Event: 7th collaboration meeting

Agenda: The team recapped the 1st in-person meetings and prepared for the collaboration Wiki. The team also used the meeting to prepare for the upcoming collaboration meetings.

Date: 7/30/2022

Event: 2nd GEMS Taiwan Collaboration Meeting

Agenda: Both teams updated each other on the progress of their projects. GEMS Taiwan suggested that Korea\_HS participate in their infographic translation project.

Date: 7/31/2022

Event: 2nd ICT Mumbai Collaboration Meeting

Agenda: Both ICT Mumbai and Korea\_HS updated each other on their progress and discussed other collaboration ideas and projects.

Date: 8/4/2022

Event: 2nd ASIJ Tokyo Collaboration Meeting

Agenda: During the 2nd meeting, Korea\_HS and ASIJ Tokyo further discussed plans for collaborations and projects.

Date: 8/9/2022

Event: 1st CCU iGEM Collaboration Meeting

Agenda: During Korea\_HS’s first meeting with CCU iGEM, the two teams discussed their wet lab processes and their projects to promote a healthy diet.

Date: 8/12/2022

Event: 8th official collaboration meeting

Agenda: The team was busy figuring out which team could replace our previous partnership team since there were complexions. The team also discussed making changes in our collaboration projects and updating the wet lab progress on the google slide.

Date:8/19/2022

Event: 9th official collaboration meeting

Agenda: The teams discussed collaborating with two more iGEM 2022 teams and also worked on the description for the collab wiki.

Date: 8/23/2022

Event: 2nd collaboration and partnership meeting with Empire Gene

Agenda: In their second meeting with Empire Gene, Korea\_HS discussed ideas of collaboration and began to reach out for a partnership with Empire Gene.

Date: 8/27/2022

Event: 1st collaboration meeting with Cambridge

Agenda: In the meeting with Cambridge University’s iGEM team, Cambridge invited Korea\_HS to participate in their hosted event.

Date: 9/2/2022

Event: 10th official collaboration meeting

Agenda: The team worked on Wiki and contacted ASIJ for the Northeast Asia Joint Network, International High School Network.

Date: 9/4/2022

Event: 1st collaboration meeting with KCIS iGEM

Agenda: During the first collaboration meeting, KCIS iGEM and Korea\_HS introduced their projects and their progress. Being a high school team, Korea\_HS invited KCIS iGEM to join the upcoming International High School Network Conference.

Date: 9/5/2022

Event: 3rd collaboration and partnership meeting with Empire Gene

Agenda: In their third meeting, Korea\_HS and Empire Gene continued discussing projects in order to maintain a meaningful partnership and created a resolution on which partnership projects to aid each other in.

Date:9/9/2022

Event:11th official collaboration meeting

Agenda: The team worked on the partnership projects and on the wiki.

Date:9/11/2022

Event: 3rd collaboration meeting with ICT Mumbai

Agenda: In their third meeting, ICT Mumbai assumed a mentor position on Korea\_HS’s dry lab process and discussed direct collaboration initiatives the teams were working on.

Date: 9/16/2022

Event: 12th official collaboration meeting

Agenda: The team focused mainly on the wiki portion and worked on the conferences/ networks that needed to be reached out to the participants.

Date: 9/25/2022

Event: International HS Network

Agenda: During the International High School Network, all participating iGEM teams shared their projects and gave each other feedback on how to improve their projects for the upcoming Grand Jamboree. The feedback given to each other was helpful in order for the teams understand their strengths and weaknesses with their projects.

Date: 9/26/2022

Event: 4th collaboration and partnership meeting with Empire Gene

Agenda: During their fourth meeting, Empire Gene and Korea\_HS presented their completed partnership assignments and discussed other available partnership opportunities.

# SDG

Date: 6/14/2022

Event: 1st SDG meeting

Agenda: The team gathered up and did an idea briefing about SDG and picked the main goals that they wanted to pursue.

Date: 6/28/2022

Event: 2nd SDG meeting

Agenda: The team worked on the overall plan and planned for the main projects.

Date: 7/5/2022

Event: 3rd SDG meeting

Agenda: The team went over the medal criteria, overall progress checkup, and the storyline. The team also worked on project planning.

Date: 7/12/2022

Event: 4th SDG meeting

Agenda: The team worked on a timeline for finalizing the projects.

Date: 7/19/2022

Event: 5th SDG meeting

Agenda: The team finished with the public survey and posted it on social media platforms. The team also finished finalizing the project: public survey, interviews, short videos, pamphlets/ brochures, and SDG Networking conference.

Date: 7/26/2022

Event: 6th SDG meeting

Agenda: The team worked on their project and wiki.

Date: 8/2/2022

Event: 7th SDG meeting

Agenda: The team reviewed the survey results, two interview results, and projects.

Date: 8/9/2022

Event: 8th SDG meeting

Agenda: The team worked on their project and wiki.

Date: 8/16/2022

Event: 9th SDG meeting

Agenda: The team worked on their project and wiki.

Date: 8/23/2022

Event: 10th SDG meeting

Agenda: The team worked on their project and wiki.

Date: 8/30/2022

Event: 11th SDG meeting

Agenda: The team worked on their project and wiki.

Date: 9/6/2022

Event: 12th SDG meeting

Agenda: The team worked on their project and wiki.

Date: 9/13/2022

Event: 13th SDG meeting

Agenda: The team worked on their project and wiki.

Date: 9/17/2022

Event: SDG Networking Conference (D-1)

Agenda: The team will start the conference at 10 PM and end it by 12AM KST. The team focused more on learning and sharing presentations. The team went through the plan for this conference and allowed the participating speakers to talk and share their project presentations. The team also did menti meter after every presentation and allowed the participants to share feedback or questions.

Date: 9/18/2022

Event: SDG Networking Conference (D-2)

Agenda: The team continued the conference from SDG Networking Conference (D-1). This conference started at the same time as the previous conference. The team focused on more of the action part of the SDG. The team hosted the conference as planned, and all participants actively joined in making the solutions with the team.

Date: 9/27/2022

Event: 14th SDG meeting

Agenda: The team summarized the finished projects and worked on the SDG wiki.

# Entrepreneurship

Date: 7/10/2022

Event: 1st Entrepreneurship meeting

Agenda: The team discussed the medal criteria, the overflow/ goal of the project, and product idea brainstorming.

Date: 7/13/2022

Event: 2nd Entrepreneurship meeting

Agenda: The team discussed the projects and specific deadlines for each project.

Date: 7/20/2022

Event: 3rd Entrepreneurship meeting

Agenda: The team brainstormed for the webinar, worked on the projects, worked on the investor interview, and worked on the Market/ Customer analysis survey.

Date: 7/27/2022

Event: 4th Entrepreneurship meeting

Agenda: The team worked on the survey and the interview questions, market analysis, and searched for possible interviewees.

Date: 8/3/2022

Event: 5th Entrepreneurship meeting

Agenda: The team worked on the Korea\_ HS team’s strategy and education, interview information, additional events, and the business plan.

Date: 8/10/2022

Event: 6th Entrepreneurship meeting

Agenda: The team recap interviews with Yoseb Song and Seoung Jun Yoon. The team also worked on preparing the website.

Date: 8/17/2022

Event: 7th Entrepreneurship meeting

Agenda: The team worked on the website, online interview ( survey), and planning the conference.

Date: 8/24/2022

Event: 8th Entrepreneurship meeting

Agenda: The team worked on the new interview, general public survey, webpage, and webinar.

Date: 9/1/ 2022

Event: 9th Entrepreneurship meeting

Agenda: The team worked on the conference, mailing biologist, website content, and the general survey finalization and spreading the survey.

Date: 9/7/2022

Event: 10th Entrepreneurship meeting

Agenda: The team worked on finalization, webinar, and wiki.

Date: 9/14/ 2022

Event: 11th Entrepreneurship meeting

Agenda: The team worked on the finalization, webinar planning, and wiki.

Date: 9/25/2022

Event: Business & Technology Conference

Agenda: The team introduced the plan for the conference and who was presenting. The team presented the conference, and other speakers did so afterward. After each presentation, there were menti meters to give feedback and ask questions.

# Inclusivity

Date: 6/12/2022

Event: 1st Inclusivity meeting

Agenda: The team worked on brainstorming the main topic.

Date: 6/25/2022

Event: 2nd Inclusivity meeting

Agenda: The team worked on finalizing the topic and discussed specifically the main topic.

Date: 7/2/2022

Event: 3rd Inclusivity meeting

Agenda: Started to research the interviewers and work on the interview questions. The team also started on the Inclusivity Webinar.

Date: 7/23/2022

Event: 4th Inclusivity meeting

Agenda: The team set up the timeline, distributed lines for the webinar script, started to finalize the webinar, discussed interview/reach-out opportunities

Date: 8/28/2022

Event: Inclusivity Webinar

Agenda: The team proceeded with the webinar as planned and allowed all the presenters to share their own inclusivity project. The team also gave feedback and questions after every presentation.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

Engineering

# Design

1) What is the purpose of our design?

The purpose was to design a TFAM-DNA complex to enhance the stability of the DNA.

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

A549 (human lung adenocarcinoma cell line)

![](https://static.igem.wiki/teams/4305/wiki/eng-f1.png)
[Figure 1]: cDNA amplified from A172, MCF7, MKN45, and A549 human cell lines. The expected amplified TFAM (43 aa - 246 aa) is 636 bp.

The amplification of TFAM (43 aa - 246 aa) was performed using cDNA produced from A172, MCF7, MKN45, and A549 human cell lines. The amplified product from A549 showed a strong one band. Therefore, this amplified product was selected to be cloned into a pET28 expression vector (Figure 1).

The following primer sequences were used:
TFAM Forward primer containing BamHI site: 5' - ATAggatccatgtcatctgtcttggcaagt - 3'
TFAM Reverse primer containing XhoI site: 5' - ATActcgagttaacactcctcagcaccatattt - 3'

# Build

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

A new DNA vector with the binary image data inserted was required for the experiment. In order to get the DNA vector, we have converted the image data into binary codes, and sent them to Bioneer for manipulation.

TFAM-204, the target protein used to create a TFAM-DNA complex, usually has 246 amino acid sequences. The first 42 amino acids function as a Mitochondrial Signal Peptide (MTS), which guides TFAM from the cytosol to the mitochondria. Since our project manually mixed TFAM with the DNA, the first 42 amino acids were unnecessary. To ease later experiments such as protein expression and complex creation, the unnecessary amino acid sequences were removed.

TFAM was also collected through a series of experiments. First of all, we manipulated a DNA vector and attached a DNA encoding protein to the vector.

![](https://static.igem.wiki/teams/4305/wiki/eng-f2.png)
[Figure 2]: Two ways of adding polyhistidine in the target gene. (A) The His-tag is added by inserting the DNA encoding a protein of interest in a vector that has the tag ready to fuse at the C-terminus. (B) The His-tag is added using primers containing the tag, after a PCR reaction the tag gets fused to the N-terminus of the gene.

We used method A to insert the TFAM gene into the pET28a (+) expression vector.
The pET-28a (+) vector carry an N-terminal His•Tag®/thrombin/T7•Tag® configuration plus
an optional C-terminal His•Tag sequence.

BamHI and XhoI restriction enzymes were used to cut both insert and vector.

Next, the DNA with TFAM from ligation was inserted into the E coli.

![](https://static.igem.wiki/teams/4305/wiki/eng-f4.png)
[Figure 4]: pET28-TFAM vector was transformed into BL21(DE3) E.coli strain.

The engineering of proteins represents a modern and powerful approach to generate novel proteins for applications in different fields as biocatalysts, therapeutic agents, and biosensors. Therefore, knowledge of the basic skills of protein engineering is mandatory for future bioengineers and chemical engineers specialized in biotechnology.

The protein was finally expressed through the pET expression system.

![](https://static.igem.wiki/teams/4305/wiki/eng-f5.png)
[Figure 5]: Principle of regulating gene expression using the pET-expression system.

![](https://static.igem.wiki/teams/4305/wiki/eng-f6.png)
[Figure 6]: The TFAM protein expression test was performed using four selected colonies with or without IPTG induction.

The expected TFAM protein size was 28 kDa, and the strong bands showed in colony # 2 and # 4 only in the IPTG-added sample. IPTG induces the inserted gene expression in pET28 containing E.coli (DE3) strain.

![](https://static.igem.wiki/teams/4305/wiki/eng-f7.png)
[Figure 7]: Protein purification using Ni-NTA magnetic silica resins.

The expression vectors contain an IPTG inducible promoter. The gene encoding a protein when cloned to an expression vector can be expressed by the induction using IPTG. The E. coli BL21 cells containing recombinant expression plasmid are grown to mid-exponential phase and then IPTG is added to the growing cells to induce the protein expression. The cell samples before and after the IPTG induction can then be analyzed for protein expression by SDS-PAGE analysis.

Colony # 1, Colony # 2, Colony # 4 samples were tested. Clear expected TFAM protein size bands were observed (Figure 7).

2) How can you screen for correct builds?

* We have performed SDS Page Gel to screen for the purification processes after experiments such as PCR and pET28 vector transfection.
* We have planned a Bradford Assay to quantify TFAM created during the experiment.
* We have planned a SDS-PAGE gel on solutions with different TFAM concentration to see if TFAM can bind with the DNA in lab conditions.

3) How does a model look like?

![](https://static.igem.wiki/teams/4305/wiki/eng-f8.png)
[Figure 8]: pET28-TFAM plasmid structure. The TFAM gene is cloned using BamHI and XhoI restriction enzymes.

Using the restriction enzyme BamHI and XhoI, the amplified TFAM gene was cloned into the pET28a expression vector. Figure 2 represents the final structure of pET28-TFAM.

# Test

1) Which types of measurements are needed?

Agarose gel electrophoresis
Agarose gel electrophoresis was conducted to check if the correct protein, with a size of 28kDa, was amplified after PCR. Agarose powder and DNA staining solution were mixed with Tris-acetate EDTA (TAE) to produce the gel. Samples were dropped on different columns and electric currents ran through to analyze the samples. The more purified the protein is, the lighter it will be, thus the more it’ll move downwards.

Bradford Assay
Bradford Assay was used to quantify the amount of protein (TFAM) in the solution. Coomassie Brilliant Blue G-250 (CBBG), a dye, was used to stain the protein. Once the reaction finished, the protein changed from maroon to blue. Using microspectroscopy, the absorbance of CBBG to protein (in this case, TFAM) of the solution was measured. The data then was plugged into Beer’s Law to quantify the protein in the solution. The more TFAM there was in the solution, the more the color changed from maroon to blue.

![](https://static.igem.wiki/teams/4305/wiki/eng-f9.png)
[Figure 9]: Bradford assay was performed to measure the stock TFAM protein concentration.

The Bradford assay is a protein determination method that involves the binding of CoomassieBrilliant Blue G-250 dye to proteins. The dye exists in three forms: cationic (red), neutral (green), and anionic (blue). Under acidic conditions, the dye is predominantly in the doubly protonated red cationic form (Amax = 470 nm). However, when the dye binds to protein, it is converted to a stable unprotonated blue form (Amax = 595 nm). It is this blue protein-dye form that is detected at 595 nm in the assay using a spectrophotometer or microplate reader [2].

In any protein assay, the ideal protein to use as a standard is a purified preparation of the protein being assayed. In the absence of such an absolute reference protein, another protein must be selected as a relative standard. We used BSA standard solution to generate the standard curve (Figure 8). The calculated TFAM protein stock concentration is 0.235 µg/µL.

* Electrophoretic Mobility-Shift Assay (ESMA): The electrophoretic mobility shift assay was performed to determine if TFAM was able to form a complex with the DNA in a lab condition.

![](https://static.igem.wiki/teams/4305/wiki/eng-f10.png)
[Figure 10]: Electrophoretic mobility shift assay (EMSA) for TFAM binding into pBHA/smile. Band shift observed in DNA plasmid containing TFAM protein.

Naked pBHA/smile (lane 1) and pBHA/smile + BSA (lane 2) shows that most of the plasmid is in free DNA form as indicated in the gel (Figure 5). BSA is used as a negative control and shows that no DNA-BSA complex is formed. pBHA/smile with 20 µg/ml of TFAM (lane 3) band shifts upward indicating TFAM-DNA complex has been successfully formed.

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

* Bradford Assay
  + The absorbance measurements were arbitrary.
  + The absorbance measurements were converted to BSA standard concentration (µg/µL)
  + The Mol concentration was finally calculated based on the TFAM recombinant protein molecular weight.
* Plasmid DNA concentration measurements using nanodrop
  + The absorbance mesruements were arbitrary.
  + The equation (1) was used to calculate DNA concentration:

    ![](https://static.igem.wiki/teams/4305/wiki/eng-math1.png)

    C = concentration of neucleic acid in the sample
    A260 = The maximum absorbance measured by the nanodrop reading
    l = pathlengh of the nanodrop reading
    DF = dilution factor
    CF = conversion factor (50 µg/mL was used for double-stranded DNA)
    - The Mol concentration was finally calculated based on the plasmid DNA molecular weight.
* Agarose gel electrophoresis
  + The EMSA data was produced with relative units compared to the negative control sample (without TFAM protein)

# Learn

1) Did your system work as anticipated?

The system worked as anticipated, and the TFAM-DNA complex was able to withstand the exposure to extensive stress conditions such as UV light and 3M of H2O2.

![](https://static.igem.wiki/teams/4305/wiki/eng-f11.png)
[Figure 11]

As shown by the data retrieved from the DNA, TFAM:pSmile vector ratios of 86.39:1 and 115.19:1 were able to protect the data in the DNA from external stress factors, whereas other samples with low TFAM concentrations weren’t. This shows the effectiveness of TFAM in forming the TFAM-DNA complex and protecting the data in the DNA from extreme stress factors, which proves our hypothesis that a Transcription Factor A Mitochondria (TFAM)-DNA complex in an aqueous solution, where TFAM encapsulates the DNA to serve as a protection from various stress factors, could enhance the stability of DNA during storage and retrieval.

# Reference

[1] Mierendorf, R C et al. “Expression and Purification of Recombinant Proteins Using the pET System.” Methods in molecular medicine vol. 13 (1998): 257-92. doi:10.1385/0-89603-485-2:257

[2] Kruger, N J. “The Bradford method for protein quantitation.” Methods in molecular biology (Clifton, N.J.) vol. 32 (1994): 9-15. doi:10.1385/0-89603-268-X:9

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

Experiments

# 1st Lab Meeting

Agenda 1: Micropipette Practice

All wet-lab participants utilized a micropipette throughout all experimental procedures. Before the experiment, every student was instructed heavily and practiced the appropriate usage of the micropipette.

General Guideline for Micropipette Usage

We have used six different micropipette volume types throughout the experimental procedure: P1000, P200, P100, P20, P10, and P2.

1. P1000 handles 1000 ~ 200 μL / Blue tip used
2. P200 handles 200 ~ 20 µL / Yellow tip used
3. P100 handles 100 ~ 10 μL / Yellow tip used
4. P20 handles 20 ~ 2 μL / Yellow tip used
5. P10 handles 10 ~ 1 μL / White tip used
6. P2 handles 0.5-2.5 µL / White tip used

Pipette Handling Procedure

![](https://static.igem.wiki/teams/4305/wiki/exp-wg1-f1.jpg)
[Figure 1]

1. Rotate rubber to set the volume to absorb.
2. Place the tip of the pipette into disposable tips.
3. Press the pressure (white) button with your thumb, for liquid absorption, before submerging.
4. Submerge the tip of the micropipette into the liquid. Press it until the first stop before uptaking the liquid.
5. Let the thumb go to absorb the liquid.
6. Stick the tip of the micropipette onto the wall of the tube.
7. Press the pressure button until the second stop to release all remaining chemicals into the container.
8. Press the second button next to the uppermost pressure button (used for absorption) to release the tip.

Pipette Handling Notice

1. If the liquid being handled is minimal, skip #6 above.
2. Make sure to hold the pipette vertically with the tip down.
3. During absorption, submerge the tip just a little bit to yield more accurate quantitative results.
4. Change the tip every time the chemical changes to prevent potential chemical contamination.
5. Try to minimize waste as much as possible.
6. Red digits in micropipettes, if they have, indicate the decimal place. Make sure to be informed of how to measure the liquid volume since they all all of them differ by micropipette brands.

Agenda 2: TFAM Gene Cloning via PCR

Type of mRNA Used for TFAM DNA

Four 4 types of mRNA can be transcribed by TFAM DNA: TFAM-201 mRNA, TFAM-202 mRNA, TFAM-203 mRNA, and TFAM-204 mRNA. Among them, we used TFAM-204 mRNA for our experiment.

TFAM-204 mRNA CDS (Protein Coding Sequence)

* 1 ~ 43 aa: Translates into Mitochondrial Signal Peptide (MTS) → Cleaved
  + MTS: Used only when TFAM protein travels from the cytosol into mitochondria in the cell.
  + We cleaved this particular coding sequence for our experiment because we will directly put the TFAM protein with the DNA.
* 43 ~ 50 aa: Linker
* 50 ~ 122 aa: Translates into HMG BOX-A
* 122 ~ 152 aa: Linker
* 152 ~ 223 aa: Translates into HBG BOX-B
* 223 ~ 246 aa: Tail

PCR Machine Principle

We conducted the PCR to multiply the TFAM-204 mRNA CDS that will be inserted into the vectors. The steps of the PCR are the following:

1. Denaturation: Heat 95℃ to separate DNA into a single strand.
2. Annealing: Cool the temperature to send DNA polymerase and several nucleotides toward DNA primer.
3. Elongation: Nucleotides form the copies of strands.

Procedural Material

1. F primer (Forward primer): 1 μL
2. R primer (Reverse primer): 1 μL
3. TFAM-204 cDNA: 1 μL
4. Water: 17 μL
5. Optimized DNA polymerase
6. dNTP
7. Buffer

Experiment Procedure

![](https://static.igem.wiki/teams/4305/wiki/exp-wg1-f2.jpg)
[Figure 2]

![](https://static.igem.wiki/teams/4305/wiki/exp-wg1-f3.jpg)
[Figure 3]

1. Mix 17.0 μL water, 1.0 μL forward primer, 1.0 μL reverse primer, and 1.0 μL cDNA inside the PCR tube.
2. Mix the liquid using the vortex.
3. Collect all the liquid on the tube wall inwards using the centrifuge.
4. Put the tubes in the PCR test machine (Thermal Cycler) and conduct the test for 1.5 hours.

![](https://static.igem.wiki/teams/4305/wiki/exp-wg1-f4.jpg)
[Figure 4]

Agenda 3: Agarose Gel Electrophoresis

Electrophoresis Principle

We conducted agarose gel electrophoresis to check if our TFAM-204 mRNA CDSs are duplicated well. The following is the principle of electrophoresis:

1. Electrical current (-) would push DNA (because DNA is generally negatively charged due to the presence of a phosphate group).
2. Small-sized DNA would move further than large-sized DNA.

Agarose Gel Explanation

Agarose is a small sugar-based molecule, and in agarose gel, agarose molecules are randomly distributed. We set the concentration of the gel as 1.5% because our DNA has about 600 bp.

Procedural Materials

1. 1.5 g Agarose powder
2. 100 ml TAE
3. Microwave heating (melting)
4. DNA staining solution (5 μL)

Experimental Procedure

![](https://static.igem.wiki/teams/4305/wiki/exp-wg1-f5.jpg)
[Figure 5]

![](https://static.igem.wiki/teams/4305/wiki/exp-wg1-f6.jpg)
[Figure 6]

1. Mix agarose powder with 100 mL TAE.
2. Mix 5 μL of DNA staining solution.
3. Heat in the microwave for 2 minutes.
4. Pour the heated liquid onto the gel caster and wait for 5 minutes.
   * Make sure that there are no bubbles when solidifying.
5. Put the agarose gel onto the gel tank.
6. Pour 400 mL of TAE buffer into the gel tank.
7. Drop 3 μL of the DNA size marker (used as the control group).
8. Drop 7 μL of the PCR testing sample.

Our Results

![](https://static.igem.wiki/teams/4305/wiki/exp-wg1-f7.jpg)
[Figure 7]

All TFAM DNA sizes were about 600 bp, as we expected.

# 2nd Lab Meeting

Agenda 1: PCR Solution Purification

Procedure Principle

This step is to purify the PCR solution by eliminating primer, DNA polymerase, and residues to only leave the pure amplified DNA.

![](https://static.igem.wiki/teams/4305/wiki/exp-wg1-f8.jpg)
[Figure 8]

Experimental Procedure

1. Add 75 μL of PB buffer and mix it with a vortex.
2. Transfer the mixture to the binding column.
3. Centrifuge for 1 min (balanced arrangement of the tubes when centrifuging).
4. Add 500 μL of NW buffer for washing.
5. Centrifuge for 1 min.
6. Discard flow-through.
7. Add 200 μL of NW buffer for washing.
8. Centrifuge for 2 min.
9. Place the column into the “new” 1.5 mL tube.
10. Add 40 μL of EB (elution buffer).
11. Incubation for 1 minute (so that more and more DNA can be dissolved).
12. Centrifuge for 1 min.

Agenda 2: Restriction Enzyme Digestion

Experimental Principle

Digestion of BamH1 & xho1 restriction enzymes onto TFAM DNA & pET28 vector DNA allows TFAM DNA insertion into the pET vector.

![](https://static.igem.wiki/teams/4305/wiki/exp-wg1-f9.jpg)
[Figure 9]

Procedural Material

DNA Solution:

* 1. DNA insert: 17 μL
* 2. 10X buffer: 2 μL
* 3. BamH1: 0.5 μL
* 4. Xho1: 0.5 μL

Vector Solution:

* 1. Vector: 17 μL
* 2. 10X buffer: 2 μL
* 3. BamH1: 0.5 μL
* 4. Xho1: 0.5 μL

Procedure

![](https://static.igem.wiki/teams/4305/wiki/exp-wg1-f10.jpg)
[Figure 10]

1. Mix 2 μL 10x buffer, 0.5 μL BamH1, 0.5 μL Xho1, and 75 μL PB buffer inside the digestion tube.
2. Vortex the mixture.
3. Incubate for 30 min

PCR purification

1. First, add 3 volumes of FB Buffer to the PCR product and then 1 volume of absolute isopropanol.
2. If the PCR product is 20 µl, add 60 µl of FB Buffer then 20 µl of absolute isopropanol. Mix them completely by vortexing. It is not necessary to remove mineral oil.
3. Transfer the mixture to the binding column and centrifuge the column at 14,000 rpm for 1 min. We recommend performing all centrifugation steps at room temperature.
4. Pour off the flow-through and re-assemble the binding column with the 2 ml collection tube.
5. Add 500 µl of W2 Buffer to the binding column and centrifuge at 14,000 rpm for 1 min.
6. Pour off the flow-through and re-assemble the binding column with the collection tube.
7. Repeat steps 4 and 5.
8. Centrifuge once more at 14,000 rpm for 1 min to completely remove residual washing buffer, and check that there is no droplet at the bottom of the binding column. Then, transfer the binding column to the new 1.5 ml microcentrifuge tube (not provided).
9. Add 30 µl of EA Buffer to the center of the binding column and wait for at least 1 min at room temperature for elution.
10. If DNA fragments are larger than 3 kb, increase incubation time to 10 min at 60°C. In the case of pure water, eluted fragment DNA may be denatured and unstable. EA Buffer, as well as TE buffer (pH 8.0) are suitable for ordinary downstream applications such as such as sequencing, restriction enzyme digestion, and ligation. On the contrary, the elution of fragment DNA with pure water may give the DNA denaturation and/or instability. TE buffer (pH 8.0) can be used as EA buffer, except that EDTA may interrupt the subsequent enzymatic reactions.
11. Elute the fragmented DNA by centrifugation at 14,000 rpm for 1 min.
12. If more DNA yield is required, elute the sample twice and use after concentration.

\*Incubate the mixture solutions in the incubator for 10 minutes so that we provide enough time for enzymes to cut the gene.
\*Do the second PCR purification to eliminate BamH1 & Xho1 restriction enzymes for ligation.

Agenda 3: Ligation

Experimental Principle

We inserted the TFAM DNA into the pET28 vector of E. coli, which can produce the TFAM proteins in the next experiment.

Procedural Material

1. 10x ligation buffer: 2 μL (shifting the solution into optimal condition for reaction with DNA ligase)
2. Digested vector: 7.5 μL
3. Digested DNA Insert: 10 μL
4. DNA ligase: 0.5 μL

Experimental Procedure

1. Mix 10x ligation buffer, digested vector, digested DNA Insert, and DNA ligase into a 1.5 mL tube.
   Vortex the solution.
2. Centrifuge for 1 minute.
3. Incubate at 15°C for 4 hours.

# 3rd Lab Meeting

Agenda 1: BL21 (DE3) Cell Harvest

Experimental Principle

The BL21 (DE3) cell harvest will allow the cells to be collected and duplicated through PCR. Cells were collected by the harvested E-coli sample solution.

Procedural Principle

Once the samples are collected from the harvested E. coli solution, samples go through centrifugation. Centrifugation allows the comparatively dense cells and bacteria to be separated from the solution and gain a cell palette. After centrifugation, PBS and SDS loading dye was added to allow the nucleus and the membrane to break. Sonification is done to support the breaking of the cell nucleus. After sonification, the tubes go through centrifugation to remove the possible leftover solution in the tube. This allows the removal of any possibility of leftover solution mixed with the cell/bacteria.

Experimental Procedure

![](https://static.igem.wiki/teams/4305/wiki/exp-wg2-f1.jpg)
[Figure 1]

![](https://static.igem.wiki/teams/4305/wiki/exp-wg2-f2.jpg)
[Figure 2]

1. Label two tubes, one with IPTG and one without IPTG. Samples labeled ‘Sample number + IPTG’ and ‘Sample number - IPTG’ are samples with and without IPTG, respectively.
2. Swirl the flask to mix the bacteria.
3. Take the sample and use the P1000 micropipette (set to 100) and the largest tip to collect 1 mL in the tube, minimizing the opening of the lid and the formation of any bubbles.
4. Collect 500 mL of the control sample without IPTG (Sample number - IPTG) by using a P1000 micropipette (set to 050) and the largest tip.
5. Centrifuge the samples, balanced within the centrifuge.
6. Add 160 µL PBS using the P200 micropipette with a maximum volume of 160 µL.
7. Absorb and release the solution 10 times to loosen the cells all clumped together due to centrifugation through pipetting.
8. Add 40 µL 5xSDS loading dye using the P200 micropipette with a maximum volume of 160 µL.
9. Absorb and release the sample a few times to make the solution even.
10. Sonicate samples with a total running time of 1 min per sample.
    1. Pulse on for 1 second and off for 10 seconds, repeated to prevent protein denaturation due to the sample heating.
    2. Power set to 1% because there was only a small amount of substance, and the power of the sonicator is too strong in comparison.
    3. Wipe the tip of the sonicator with alcohol after every repetition to prevent samples from being mixed.
11. Centrifuge the samples at 12000 RPM for 1 min.

Agenda 2: SDS-PAGE Gel

Experimental Principle

The SDS-PAGE gel was performed to identify the E.coli sample with the most TFAM protein.

Procedure Principle

The buffer solution was set to 95’C to denature the protein (TFAM) and compare the proteine sizes to choose a colony of E coli.

By providing electric current, smaller proteins will move faster (positioned lower) and larger proteins will move slower (positioned higher / above). Thick bands in the SDS-PAGE gel indicate larger amounts of proteins, while thinner bands indicate smaller amounts of proteins. TFAM protein with His tag has a size of approximately 20 kDa. Therefore, the band thickness was specifically focused on.

Experimental Procedure

![](https://static.igem.wiki/teams/4305/wiki/exp-wg2-f3.jpg)
[Figure 3]

![](https://static.igem.wiki/teams/4305/wiki/exp-wg2-f4.jpg)
[Figure 4]

1. Add buffer set to 95’C (50 mL buffer solution to 450 mL water).
2. Add standard protein marker to both ends in the gel.
3. Load all 8 samples into the gel.
4. Give electric current.
   1. 100V for one hour

# 4th Lab Meeting

Agenda 1: TFAM Protein Purification (Ni-NTA Magnetic Nanobeads)

Experimental Principle

The protein purification process will separate the desired protein (TFAM) from all other proteins present within E. coli.

Experimental Procedure

![](https://static.igem.wiki/teams/4305/wiki/exp-wg2-f5.jpg)
[Figure 5]

![](https://static.igem.wiki/teams/4305/wiki/exp-wg2-f6.jpg)
[Figure 6]

1. Preparation of E. coli lysate
   1. Prepare 3 mL of pre-induced bacterial culture with IPTG (Sample #2).
   2. Harvest the cells by centrifugation for 1 min at 12,000 rpm at 4℃ and discard the supernatant.
   3. Resuspend the cells in 500 µL of binding/washing buffer using a P1000 micropipette.
   4. Sonicate the cells using a sonicator (equipped with a micro-tip on ice).
      1. Pulse on: 3 sec / off: 10 sec
      2. Total running time: 3 min
      3. Power: 25%
      4. Repeat twice to make sure membranes are properly broken
   5. Centrifuge the E. coli lysate for 5 min at 12,000 rpm at 4℃ to pellet the cell debris & save the supernatant.
   6. Take 10 µL of the supernatant (lysate sample) for SDS-PAGE analysis using a P20 micropipette.
2. Equilibrating Ni-NTA magnetic nanobeads
   1. Transfer 1 mL of Ni-NTA magnetic nanobeads to a 1.5 mL tube & place the tube on a Neodymium (Nd) magnet for 1 min using a P1000 micropipette (set to 1 mL).
   2. Remove supernatant using P1000 micropipette.
   3. Equilibrate by adding 1 mL of binding/washing buffer to the bead slurry & mix briefly using a P1000 micropipette.
   4. Place the tube on Nd magnet for 1 min & remove supernatant.
   5. Repeat steps c & d once more.
3. Protein binding
   1. Load up to 500 µL of cleared lysate onto pre-equilibrated magnetic nanobeads using a P1000 micropipette.
   2. Mix by inverting 5-10 times.
   3. Place the tube on the Nd magnet for 1 min & remove supernatant (unbound sample).
4. Washing magnetic nanobeads (to remove E. coli proteins that are still present through nonspecific binding)
   1. Add 1 mL of binding/washing buffer & wash magnetic nanobeads by gently pipetting,
   2. Place the tube on Nd magnet for 1 min & remove supernatant (washing sample),
   3. Repeat a & b three times.
5. Eluting target proteins
   1. Add 200 µL of elution buffer & gently mix (to elute target proteins from magnetic nanobeads).
   2. Incubate for 1 min at room temperature.
   3. Place the tube on the Nd magnet for 1 min & collect supernatant (elution sample).
   4. Repeat elution step (a) once more.
   5. Identify purification steps by analyzing each sample with SDS-PAGE.

Agenda 2: SDS-PAGE Gel

Experimental Principle

The SDS-PAGE gel was performed to ensure that the TFAM protein was properly purified and separated from all other proteins.

Experimental Procedure

1. Load all samples from the TFAM protein purification process.
2. Add a standard protein marker to one end.
3. Give electric current.
   1. 100V for 30 minutes
   2. 120V for 3 hours

Experimental Results

TFAM protein was successfully purified.

# 5th Lab Meeting

Agenda 1: Bradford Assay (Protein Quantification)

Materials Needed

1. Bradford reagent (Bio-rad)
2. Bovine serum albumin (BSA) (2 mg/mL)
3. 96 well plate
4. UV/vis spectrometer
5. Distilled water
6. 1.5 mL, 2 mL tube

Experimental Principle

Bradford assays are used to perform a quantitative analysis of the unknown protein. Comassie Brillian Blue G-250 (CBBG), an absorbing medium, reacts with the protein, TFAM. Once the reaction finishes, the protein changes from maroon to blue: as more protein reacts with CBBG, the blue becomes darker. Once the reaction is over, using microspectroscopy, the absorbance of Comassie Brillian Blue G-250 (CBBG) to protein (in this case, TFAM) can be measured. The data then can be plugged into the equation from Beer’s Law to quantify the protein in the solution.

![](https://static.igem.wiki/teams/4305/wiki/exp-wg3-f6.jpg)
[Figure 6]

Experimental Procedure

![](https://static.igem.wiki/teams/4305/wiki/exp-wg3-f1.jpg)
[Figure 1]

![](https://static.igem.wiki/teams/4305/wiki/exp-wg3-f2.jpg)
[Figure 2]

1. In the 1.5 mL tube, mix the BSA and distilled water to make a BSA standard curve sample and calculate the density of the solution.

   ![](https://static.igem.wiki/teams/4305/wiki/exp-tb1.png)
2. Use the 2 mL tube to dilute the Bradford reagent by mixing water (by making the ratio 1:5) for the unknown sample.
3. Add 195 µL of the diluted Bradford reagent to the 96 well for each sample, a total of 7.
4. Add 5 µL of BSA standard sample curve and the unknown sample to match a total volume of 200 µL.
5. Mix the sample with the reagent using a pipette, minimizing the air bubbles being created in the sample.

   ![](https://static.igem.wiki/teams/4305/wiki/exp-tb2.png)
6. Wait for five minutes.
7. Use a microplate reader to record the absorbance at a wavelength of 595 nm.

Agenda 2: TFAM-DNA Binding Test

Experimental Principles

Agarose is a small sugar-based molecule, and in the gel, agarose molecules are randomly distributed. Agarose gel was used to test the binding of TFAM to the pSmile vector (DNA). The electrical current (-) would push the DNA because DNA is generally negatively charged due to the presence of a phosphate group, and small-sized DNA would move further than the larger ones due to its mass. As more proteins are present, the bends get thicker when the gel is loaded. Through this experiment, we’re able to compare the size and amount of protein in different samples. As the pSmile vector binds with the TFAM, the sample will get heavier and larger. Thus, as more TFAM is added, the sample would move at a slower rate.

Experiment Procedure

![](https://static.igem.wiki/teams/4305/wiki/exp-wg3-f3.jpg)
[Figure 3]

1. Prepare nine tubes.
2. Put 3 µL of DNA to each test tube.
3. Starting from 0 µL, increase 1 µL of TFAM per test tube so that the 9th test tube contains 8 µL of TFAM.
4. Starting from 8 µL, decrease µL of elution buffer being added to the test tube so that the total volume of solution is set to 11µL.
5. Add 2 µL of DNA loading dye at the end to load the gel.

# 6th Lab Meeting

Agenda 1: TFAM-DNA Stress Test

Procedural Principle

To test the stability of the TFAM-bound DNA, UV stress and H2O2 were added to the DNA solution.

Experimental Procedure

![](https://static.igem.wiki/teams/4305/wiki/exp-wg3-f4.jpg)
[Figure 4]

3M H2O2 Test

1. Prepare 2 different tubes, labeled ‘Elution, 3M H2O2’ and ‘TFAM, 3M H2O2.’
2. Add 3 µL of DNA, 8 µL of the elution buffer, 1 µL of 3M H2O2, and 2 µL of DNA loading dye to the tube labeled as ‘Elution, 3M H2O2.’
3. Add 3 µL of DNA, 8 µL of TFAM, 1 µL of 3M H2O2, and 2 µL of DNA loading dye to the tube labeeled as TFAM, 3M H2O2.’
4. Centrifuged the samples for 10 sec at 13000 rpm
5. Wait for 60 minutes.
6. Run the agarose gel electrophoresis

UV Test

![](https://static.igem.wiki/teams/4305/wiki/exp-wg3-f5.jpg)
[Figure 5]

1. Prepare 2 tubes, labeled ‘Elution, UV’ and ‘TFAM, UV.’
2. Add 3 µL of DNA, 8 µL of the elution buffer, and 2 µL of DNA loading dye.
3. Add 3 µL of DNA, 8 µL of TFAM, and 2 µL of DNA loading dye.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

RESULTS

# PCR of TFAM mRNA

There are four types of mRNA that can be transcribed by TFAM DNA: TFAM-201 mRNA, TFAM-202 mRNA, TFAM-203 mRNA, and TFAM-204 mRNA. Among them, TFAM-204 mRNA was used as it is the major mRNA that produces the longest amino acid-sequence (246 aa). Among 246 amino acid-sequences, the sequence that produces the first to 43rd amino acids is translated into the Mitochondrial Signal Peptide (MTS) that helps TFAM protein to travel from the cytosol the into mitochondria in the cell. However, in our experiment, TFAM will be attached directly to the data-stored DNA, so the amino acids are cleaved. Regarding the amino acid sequence, the 43rd to 50th amino acids act as a linker, the 50th to 122nd amino acids are translated into the HMG BOX-A, the 122nd to 152nd amino acids also act as a linker, the 152nd to 223rd amino acids are translated into the HBG BOX-B, and the 223rd to 246th amino acids act as a tail.

![](https://static.igem.wiki/teams/4305/wiki/rts-f1.jpg)
[Figure 1] : Different parts of the amino acid sequence in TFAM

The TFAM cDNAs from different cell origins, A172 (brain), MCF7 (breast), MKN45 (stomach), and A549 (lung), were tested via PCR and put into an agarose gel to test which cell has the gene to produce TFAM protein the most. The TFAM-204 mRNA coding sequence (CDS) was multiplied via PCR. The solution included distilled H2O, forward primer, reverse primer, TFAM-204 cDNA, the mixture of optimized DNA polymerase, dNTP, and the buffer. For an hour and a half, denaturation, annealing, and elongation were repeated to amplify TFAM-204 cDNA. After the PCR, the solutions were put into an agarose gel, where the protein was expressed.

The results of the agarose gel showed that human lung cDNA expressed the most TFAM-204 protein, indicated by the width of the bend with 636 base pairs; thus, human lung cells were used throughout the experiment. The results also implied that all the TFAM cDNA was amplified without errors.

# pET vector digestion, ligation and transfection

pET28 vector and TFAM cDNA were then digested by BamH1 and Xhol. TFAM cDNA was inserted into the pET28 vector, and the pET28-TFAM vector was transformed into BL21 (DE3) E. coli strain. After harvesting the E. coli, the IPTG induction test was conducted to check if IPTG serves its purpose in removing the lac repressor, which interferes TFAM protein from being translated. Using SDS-PAGE gel, the differences were found between the E. coli genome with and without the IPTG. The E. coli genome with IPTG was able to produce the TFAM, and, thus, moved less than the lighter solution without IPTG and TFAM in the gel. This led to the conclusion that IPTG is able to serve its goal in supporting T7 polymerase from expressing the target gene.

# E. coli Lysate Purification

The E. coli lysate that contains the TFAM protein was collected to be purified. It was purified using the property that a nickel column, HIS-TAG on the end of the TFAM protein, can be attracted to the Ni-NTA magnetic nanobeads. The protein was purified by binding the protein to the magnet, washing magnetic nanobeads, and eluting target proteins. After the purification, using the SDS-PAGE Gel, protein was analyzed whether the purification process was effective after the purification. If the TFAM is not fully purified, in other words if it is still binded with other materials, the bend will move slower than the expected TFAM size, 28kDa, and thus, will be higher than the expected TFAM. The results showed that all the TFAM proteins were 28 kDa, proving the effectiveness of the purification process.

# Bradford Assay

After checking the degree of purification, the amount of TFAM protein produced (concentration) was tested via Bradford Assay. Using microspectroscopy, the absorbance of Coomassie Brillian Blue G-250 (CBBG) to TFAM was measured to check how much TFM was produced. The TFAM absorbance was then plugged into Beer’s Law to quantify the protein in the solution. The calculation showed that the TFAM protein concentration was 0.235 µg/µL and 8.38 µM as a mol concentration.

# TFAM-DNA Binding Test

To test the optimal mol ratio between the TFAM and the DNA, the TFAM-DNA Binding Test was conducted. As more TFAM binds with the DNA, the DNA will be heavier, and thus, will be slower than less TFAM binding DNA. Solutions with the same amount of DNA but with different amounts of TFAM were placed into the gel to be compared. The total volume of each solution was kept the same by buffers.

As the placement of the bend differed by the concentration of TFAM, it was concluded that the TFAM is able to form TFAM-DNA complex even in in-vitro conditions. Moreover, as the bend placements didn’t differ much between () and (), it was also concluded that the most effective TFAM:pSmile (2087 bp) binding mol ratio is 115.19:1, similar to the known TFAM:DNA binding mol ratio, 113.47:1.

# Stress Test

To test whether TFAM protein can effectively protect data-stored DNA from various damages like UV light and H2O2, each TFAM-DNA complex was exposed to UV irradiation and H2O2 for 5 hours. After applying UV and H2O2 stress, Sanger sequencing was performed to determine the nucleotide sequence in the DNA. Then, the sequence was converted into binary code and was decoded back to the original binary image form.

Naked pBHA/smile (lane 1) is completely disintegrated by an aggressive UV irradiation (lane 2).

![](https://static.igem.wiki/teams/4305/wiki/rts-f3.jpg)
[Figure 3]: Evidence of DNA protection of TFAM against UV irradiation. Agarose gel electrophoresis of pBHA/smile (lane 1-2), and pBHA/smile + TFAM (lane 3-5) treated as described in the protocol. Lane 1 to 5 from left to right.

After forming the TFAM-DNA complex with the molar ratios 86.39 and 115.19 (lane 4-5) the majority of the DNA still remained even after the UV stress for five hours. However, a TFAM-DNA complex with a low molar ratio of TFAM (14.40) was insufficient to protect DNA from UV irradiation (lane 3).

Although TFAM was able to protect the DNA to a certain degree, the band intensity of both 10 or 20 g/ml of TFAM (lane 4-5) was relatively low compared to the intensity of naked pBHA/smile (lane 1). This result suggests that the TFAM-DNA complex protects the DNA from UV irradiation, but it is insufficient to protect 100% of the DNA molecules in the sample.

Naked pBHA/smile (lane 1) is completely disintegrated by aggressive H2O2 stress (lane 2).

![](https://static.igem.wiki/teams/4305/wiki/rts-f4.jpg)
[Figure 4]: Evidence of DNA protection by TFAM against H2O2 as a stress factor. Agarose gel electrophoresis of pBHA/smile (lane 1-2), and pBHA/smile + TFAM (lane 3-5) treated as described in the protocol. Lane 1 to 5 from left to right.

In the solution with the TFAM-DNA complex with the molar ratio of 86.39 and 115.19 (lane 4-5), the plasmid DNA remained even after the 3 mM H2O2 stress for five hours. However, a TFAM-DNA complex with a low molar ratio of TFAM (14.40) was insufficient to protect DNA from H2O2 stress (lane 3). Similar to UV stress data presented, the band intensity of either the molar ratio of 86.39 and 115.19 (lane 4-5) was relatively low compared to the band intensity of naked pBHA/smile (lane 1). Therefore, similar to the UV irradiation test, the result indicated that TFAM protects DNA from H2O2 stress but does not protect 100% of DNA molecules in the sample.

In both UV irradiation and H2O2 stress, pBHA/smile without TFAM showed low recovery rates: 9% and 11%, respectively (lane 2).

![](https://static.igem.wiki/teams/4305/wiki/rts-f5.jpg)
[Figure 5] : After DNA stress was induced, the DNA was sequenced and decoded to retrieve the original image. (A) UV irradiated DNA (B) H2O2 stressed DNA. The intactness of the DNA was measured by how much of the original image has been preserved after each stress has been induced. The percentage indicates the average recovery rate of the original image (n= 3).

pBHA/smile with 1 µg/mL TFAM also showed a low recovery rate in both UV irradiated and H2O2 stressed samples: 21 % and 13%, respectively (lane 3). However, even with the UV irradiation and H2O2 stress, pBHA/smile with 10 or 20 µg/ml TFAM recovered 100% of the original image (lane 4-5). This result indicated that the TFAM-DNA complex protected the DNA even in aggressive aqueous storage conditions. Also, non-biological information, the smiley face image, stored in plasmid DNA could be retrieved without any errors.

{% endblock %}