iGEM 2023 — Team korea-hs

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Notebook

# Team Korea\_HS Meetings

Date: 03/13/2023
Event: 1st Whole Meeting (Virtual)
Agenda: The team was introduced to its members for the first time and to Afamin & Wnt3a protein and its importance to Professor Lee.

Date: 03/19/2023
Event: 2nd Whole Meeting (Virtual)
Agenda: The team was introduced to the team's Instagram account and further discussed the iGEM registration process.

Date: 04/02/2023
Event: 3rd Whole Meeting (Virtual)
Agenda: The team discussed the experimental process of the project with Professor Lee.

Date: 03/26/2023
Event: 4th Whole Meeting (Virtual)
Agenda: The team elected the team leaders and the subgroup leaders.

Date: 04/16/2023
Event: 5th Whole Meeting (Virtual)
Agenda: The team went through the medal criteria for the general team and the logistics of Special Award teams.

Date: 05/14/2023
Event: 6th Whole Meeting (Virtual)
Agenda: The team went through the award strategies based on the changed rubric. Professor Lee discussed the specific process and goals of the experiment.

Date: 05/28/2023
Event: 7th Whole Meeting (Virtual)
Agenda: The team went through the safety form and availability form submissions. Professor Lee discussed the various types of cell proliferation assays.

Date: 07/01/2023
Event: 8th Whole Meeting (In person)
Agenda: The team discussed the progress and roadmap for each team and completed team projects such as the offline sticker survey and citizen interview regarding artificial meat.

Date: 09/17/2023
Event: 9th Whole Meeting (Virtual)
Agenda: Professor Lee discussed the results of the experiment that has been completed.

## Education & Communication Meetings

Date: 07/22/2023
Event: Communication with the iGEM KCIS team
Agenda: Both teams shared their own project and discussed how each project connects to various SDG goals.

Date: 07/22/2023
Event: Communication with the iGEM CCU team
Agenda: Both teams shared their own project and discussed how each project connects to various SDG goals.

Date: 07/29/2023
Event: Soup Kitchen visit
Agenda: The team visited the Soup Kitchen and volunteered to distribute food for 4 hours and distributed the artificial meat recipe book, receiving feedback afterward.

Date: 08/03/2023
Event: Childcare Center visit
Agenda: The team visited the Childcare Center and educated the children about artificial meat through presentations, drawing activities, and quizzes.

## Interviews & Outreach Meetings

Date: 07/06/2023
Interviewee: Anderson Tan (Dr. Tan), Researcher of BioChem [Academia/Industry]
Agenda: The team listened to the presentation and the speech, asked questions about the presentations, shared the team’s presentations, and sought advice.

Date: 07/12/2023
Interviewee: Cherry Sung, Student Environmental Activist [Environment]
Agenda: The team listened to the presentation and the speech, asked questions about the presentations, shared the team’s presentations, and sought advice.

Date: 07/13/2023
Interviewee: Dr. Reisigner, IPCC Vice Chairman [Environment/Policy]
Agenda: The team listened to the presentation and the speech, asked questions about the presentations, shared the team’s presentations, and sought advice.

Date: 07/15/2023
Interviewee: Seb Alex, Animal Rights/Vegan Advocate & Lecturer [Ethical]
Agenda: The team listened to the presentation and the speech, asked questions about the presentations, shared the team’s presentations, and sought advice.

Date: 07/15/2023
Interviewee: Namdaemun citizens
Agenda: The team listened to the citizens’ opinions and perspectives.

Date: 07/15/2023
Interviewee: Namdaeum butcher shop owner [Meat Producers: Store]
Agenda: The team listened to the meat stores’ opinions and perspectives.

Date: 07/15/2023
Interviewee: Anseong livestock farm [Meat Producers: Farm]
Agenda: The team listened to the meat producer/farm’s opinions and perspectives.

Date: 07/24/2023
Interviewee: Pulmuone Harry Sangwoo Cho Vice President [Meat Producer 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: 07/26/2023
Interviewee: Martina Stephany, Animal rights activist [Ethical]
Agenda: The team listened to the presentation and the speech, asked questions about the presentations, shared the team’s presentations, and sought advice.

Date: 07/28/2023
Interviewee: Agri-Food Tech Start-Up Rising Expo 2023 [Academia/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: 08/05/2023
Interviewee: Erin Sharoni, Harvard Bioethics Professor [Academia]
Agenda: The team listened to the presentation and the speech, asked questions about the presentations, shared the team’s presentations, and sought advice.

Date: 08/17/2023
Interviewee: Mat McDermott, Senior Director of Communications for the Hindu American Foundation [Religion/Ethical]
Agenda: The team listened to the presentation and the speech, asked questions about the presentations, shared the team’s presentations, and sought advice.

Date: 08/18/2023
Interviewee: Margaret Patron (Ms. Maggie), Vegetarian [Ethical]
Agenda: The team listened to the presentation and the speech, asked questions about the presentations, shared the team’s presentations, and sought advice.

Date: 08/23/2023
Interviewee: Patrick Hopkins, Philosophy Professor [Academia/Ethics]
Agenda: The team listened to the presentation and the speech, asked questions about the presentations, shared the team’s presentations, and sought advice.

# Human Practices Meetings

Date: 03/12/2023
Event: 1st Team Meeting
Agenda: The team met each other for the first time, did orientation, and decided on the project topic for this year of iGEM.

Date: 03/19/2023
Event: 2nd Team Meeting
Agenda: The team gathered together to start registration and introduction to otheur topic.

Date: 03/26/2023
Event: 3rd Team Meeting
Agenda: An introduction to various roles and the leaders for each division in the team were given.

Date: 04/02/2023
Event: 4th Team Meeting
Agenda: As a team, we elected the president, co-president, and respective team leaders for each subgroup in the team.

Date: 04/12/2023
Event: 5th Team Meeting
Agenda: The team reviewed the general guidelines of the competition and started registering individually and as a team.

Date: 05/14/2023
Event: 6th Team Meeting
Agenda: The team went over the competition rubric as well as the medal criteria and started to plan out experiments for the summer.

Date: 05/28/2023
Event: 7th Team Meeting
Agenda: The team met up together to start registration and introduction to the topic.

Date: 06/11/2023
Event: 8th Team Meeting
Agenda: The team went over the topic and related contents and planned for summer's in-person meeting in Seoul.

Date: 07/01/2023
Event: Apgujeong Offline Consumer Survey
Agenda: The team visited Apgujeong Street and conducted an offline consumer sticker survey session.

Date: 07/15/2023
Event: Visit to Anseong Farm
Agenda: The team visited the Anseong Farm, interviewed the farm, and learned about the meat producer stakeholder.

Date: 07/15/2023
Event: Visit to Namdaemun Market
Agenda: The team visited the Namdaemun Market and interviewed the meat butchers (producer stakeholders) and citizens.

Date: 07/18/2023
Event: Visit to Pulmuone R&D Center
Agenda: The team visited the Pulmuone R&D center and interviewed the Executive Vice President (Producer Company stakeholder).

Date: 07/28/2023
Event: Agri & Food Rising Tech Startup Expo (AFRO)
Agenda: The team visited the AFRO and interviewed many experts and CEOs of various leading companies in the food industry.

Date: 08/10/2023
Event: Visit to SimplePlanet
Agenda: The team visited SimplePlanet and interviewed the co-president of the company learning the ethics and diverse technology related to cultured meat production.

Date: 09/24/2023
Event: 9th Team Meeting
Agenda: The team met up to finalize the responses from stakeholder surveys, interviews, and interactions.

Date: 10/02/2023
Event: 10th Team Meeting
Agenda: The team continued the Wiki Documentation Process.

# SDG Meetings

Date: 04/09/2023
Event: 1st SDG Meeting
Agenda: The team chose SDG goals to focus on and brainstormed ideas for SDG activities.

Date: 04/16/2023
Event: 2nd SDG Meeting
Agenda: The team added up the SDG activities to commit and planned for the main projects.

Date: 04/23/2023
Event: 3rd SDG Meeting
Agenda: The team went through the roadmaps and the medal criteria.

Date: 05/19/2023
Event: 4th SDG Meeting
Agenda: The team created questions for the online Google survey and planned topics for the Instagram hashtag event.

Date: 05/27/2023
Event: 5th SDG Meeting
Agenda: The team finalized the topics for the Instagram hashtag event and brainstormed interview questions.

Date: 06/03/2023
Event: 6th SDG Meeting
Agenda: The team organized the online Google survey results.

Date: 06/11/2023
Event: 7th SDG Meeting
Agenda: The team sent direct messages to other teams for their contribution to our Instagram hashtag event.

Date: 06/18/2023
Event: 8th SDG Meeting
Agenda: The team prepared materials and distributed roles for the offline survey.

Date: 07/08/2023
Event: 9th SDG Meeting
Agenda: The team brainstormed ideas for the climate reels project and created an activities outline for the visit to the childcare center.

Date: 07/16/2023
Event: 10th SDG Meeting
Agenda: The team created an artificial recipe book utilizing Canva.

Date: 07/30/2023
Event: 11th SDG Meeting
Agenda: The team finalized the notes for the interviews previously held.

Date: 08/13/2023
Event: 12th SDG Meeting
Agenda: The team finalized the ideas for the Instagram meat post and finished creating pamphlets.

Date: 08/27/2023
Event: 13th SDG Meeting
Agenda: The team brainstormed ideas for the business proposal and finalized the plan for the SDG meeting.

Date: 09/03/2023
Event: 14th SDG Meeting
Agenda: The team started the climate activism project and finished creating the handkerchiefs.

Date: 09/10/2023
Event: 15th SDG Meeting
Agenda: The team finished the first draft of the business proposal.

Date: 09/17/2023
Event: SDG Conference
Agenda: The team started the SDG conference at 10 PM KST and ended at 12 AM KST.

Date: 09/24/2023
Event: 16th SDG Meeting
Agenda: The team finalized the business proposal and the patent proposal.

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Engineering

# Design

## Issue of Interest: Pioneering Substitute for FBS

This year, we aimed to develop conditioned media as a potential substitute for fetal bovine serum (FBS) in cultured meat production. FBS has been a standard practice; however, it involves drawbacks in terms of ethical concerns, batch-to-batch variability, and potential transmission of pathogens. Therefore, we will focus on developing a sustainable and FBS-free growth medium for cultivating bovine muscle cells that could potentially replace FBS.

## Purpose Behind the Design

The design of our machine, in part, was fueled by two proteins of interest: Afamin and Wnt3a, two crucial components that could support cell growth and proliferation without the need for FBS. We sought to engineer human embryonic kidney (HEK293) cells that stably contain and express Afamin and Wnt3a genes. Afamin is a glycoprotein that plays a significant role in binding and stabilizing various growth factors, while Wnt3a is a critical signaling molecule involved in cell proliferation and differentiation. The co-expression of these two factors was expected to create an ideal microenvironment for the growth of bovine muscle cells. HEK293 cells are commonly used for genetic engineering experiments due to their high transfection efficiency and robust growth characteristics. The transgenesis requires the following:

* Integration of Afamin and Wnt3a genes into the host genome
* Stable expression of Afamin and Wnt3a in a polycistronic mRNA-like manner
* Formation of a stable Afamin-Wnt3a protein complex without fusing two proteins

We identified the PiggyBac Vector System as a plausible medium for fulfilling these conditions. PiggyBac Vector System is a powerful implement for introducing and stably integrating genetic material into host cells, ensuring long-term and stable expression of the desired genes.

### [Background: PiggyBac Vector System]

The PiggyBac Vector System is comprised of two elements: PiggyBac Vector and Super PiggyBac Transposase Expression Vector. PiggyBac Vector contains genes of interest and is intervened by terminal inverted terminal repeat (ITR) sequences at its 5’ and 3’ ends. Super PiggyBac Transposase Expression Vector, self-explanatorily, expresses Super PiggyBac Transposase; transposases are enzymes that identify the terminal repeat sequences in a gene and bind DNA transposon between each of the terminals. Upon co-transfection of two vectors into the host cells, Super PiggyBac Transposase recognizes ITR sequences and thereby integrates the intervening sequence (containing the genes of interest) into the host genome at TTAA–a process known as ‘PiggyBac-ing.’ Overall, this system integrates the desired set of genes into the host genome.

Our first element of our PiggyBac Vector System is the BII-CMV-AfmW3A Vector (Part: BBa K4869000). Primarily, it contains Afamin (Part: BBa K4869004) and Wnt3a (Part: BBa K4869005) flanked by ITR sequences (Part: BBa K4869006). Our full genetic construct is shown in Figure 1, and our key elements (and their rationale) are as follows:

![](https://static.igem.wiki/teams/4869/wiki/eng-figure-1.png)
![](https://static.igem.wiki/teams/4869/wiki/eng-figure-1-1.png)

Figure 1 (Inverted Terminal Repeat (ITR) sequences are positioned at the 5’ and 3’ ends. CMV promoter, afamin, 2a, wnt3a, IRIS, puromycin resistance gene (PuromycinR), WPRE, and SV polyA elements are designed to transfer into HEK293 genomic DNA by Supper Piggybac transposase)

![](https://static.igem.wiki/teams/4869/wiki/eng-figure-1.png)

Figure 2 (2A self-cleaving peptides sequence used in this vector)

* ITR sequence: The Super piggyBac transposase is a powerful tool for inserting genes into a host genome using the Inverted Terminal Repeat (ITR) sequences. The piggyBac transposon system operates through a "cut-and-paste" mechanism to move genetic material from one location to another within a genome. Transposase recognizes the ITR sequence and cuts and pastes it into HEK293 genomic DNA.
* CMV promoter: The BII-CMV-AfmW3A vector's inclusion of the CMV promoter is a critical element in the design of this genetic construct. The CMV promoter, which stands for Cytomegalovirus promoter, is potent and widely utilized in molecular biology and genetic engineering. The CMV promoter is favored for its strong and constitutive (constant) expression activity in a broad range of mammalian cell types, making it an excellent choice for driving the expression of genes of interest. This promoter is known for its robustness and ability to initiate transcription efficiently.
* Afamin: The mouse afamin gene was used and positioned after the CMV promoter.
* 2A: The 2A peptide cleavage system is a powerful tool in molecular biology and biotechnology because it allows the co-expression of multiple genes from a single mRNA transcript. The 2A peptide sequence is typically placed between two genes of interest on a single mRNA transcript to produce two proteins. Therefore 2A peptide sequence is placed between Afamin and Wnt3a. The insertion of an 18-amino acid 2A self-cleaving peptide between Afamin and Wnt3a proteins enables co-expression in a single mRNA, offering a powerful and precise method for achieving stable protein expression (Figure 2).
* IRES: IRES sequence in a DNA vector is a valuable tool for enabling the expression of multiple proteins from a single mRNA transcript. It allows for cap-independent translation initiation and co-expresses genes of interest without fusing them, making it a versatile element in molecular biology and genetic engineering. The IRES sequence was positioned between our target gene and the puromycinR gene.
* PuromycinR: The puromycin resistance gene is commonly used in molecular biology and cell biology research as a selectable marker. Its purpose in the selection process is to enable the isolation and maintenance of cells or organisms that have been successfully transfected or transformed with a specific DNA construct. In our experiment, puromycin was used to isolate the stable cells that contain the Afamin and Wnt3a sequence in HEK293.
* WPRE: Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) sequence in a DNA vector is a regulatory element that enhances gene expression by increasing mRNA stability and facilitating efficient cytoplasmic accumulation. Its inclusion in vectors is a valuable strategy to boost and stabilize the expression of genes.
* SV polyA: The SV40 polyadenylation sequence in a DNA vector is crucial for proper termination of transcription, mRNA stability, and efficient translation of the gene of interest. It ensures that the mRNA is processed correctly and is equipped for stable and productive expression within the host cell.

![](https://static.igem.wiki/teams/4869/wiki/eng-figure-3.png)

Figure 3 (Super PiggyBac Transposase Expression Vector contains the Super PiggyBac Transposase gene)

The second element of our System is the Super PiggyBac Transposase Expression Vector (Part: BBa K4869001, Figure 3). Upon the expression of Super PiggyBac Transposase, it recognizes the ITR sequences and integrates the flanked genetic construct into the genome of HEK293 cells. Accordingly, HEK293 cells, genomically, contain both Afamin and Wnt3a genes. This is preceded by lipofectamine-mediated co-transfection of the BII-CMV-AfmW3A Vector and Super PiggyBac Expression Vector into HEK293 cells (Figure 4).

![](https://static.igem.wiki/teams/4869/wiki/eng-figure-4.png)

Figure 4 (Engineered HEK293 cells producing Afamin and Wnt3a proteins into conditioned media)

Upon genomic integration, engineered HEK293 cells simultaneously express Afamin and Wnt3a genes in a polycistronic mRNA-like manner, while the nature of the integrated genetic construct prevents the fusion of these proteins. The resulting proteins will be secreted into the surrounding conditioned cell culture media. This Afamin-Wnt3a enriched media can then be collected and utilized in experiments involving the growth of bovine cells.

![](https://static.igem.wiki/teams/4869/wiki/eng-figure-5.png)

Figure 5 (Predicted Afamin-Wnt3a complex structure. Afamin in gray and Wnt3a in green)

Overall, we expect a complex between Afamin and Wnt3a proteins (Figure 5). Afamin is a serum glycoprotein that can maintain the active and water-soluble form of lipidated Wnt protein in hydrophilic environments. The experiments conducted by researchers at Osaka University and Keio University School of Medicine showed that afamin binds tightly to Wnt proteins, forming a 1:1 complex that remains soluble in aqueous buffer after isolation. This complex can induce signaling in various cellular systems, including the intestinal stem cell growth assay.

## Parts for Implementation

To implement the function of our system, we will use the Parts (which we newly added to the Registry of Standard Biological Parts this year) listed below. Detailed descriptions and validation of each part can be further viewed on both Parts and Parts Collection pages.

* BII-CMV-AfmW3A Vector ([Part: BBa K4869000](http://parts.igem.org/Part%3ABBa_K4869000))
* Super PiggyBac Transposase Expression Vector ([Part: BBa K4869001](http://parts.igem.org/Part%3ABBa_K4869001))* Engineered HEK293 cells containing afamin and wnt3a genes inside the genome ([Part: BBa K4869003](http://parts.igem.org/Part%3ABBa_K4869003))
  * Afamin gene ([Part: BBa K4869004](http://parts.igem.org/Part%3ABBa_K4869004))
  * Wnt3a gene ([Part: BBa K4869005](http://parts.igem.org/Part%3ABBa_K4869005))
  * Terminal inverted repeat sequence ([Part: BBa K4869006](http://parts.igem.org/Part%3ABBa_K4869006))

# Build

## Overview: Building Process

### [Part 1: Cloning BII-CMV-AfmW3A Vector and Polymerase Chain Reaction (PCR)]

Since we did not have enough BII-CMV-AfmW3a Vector (purchased from AddGene) for our experiment, we had to transform this plasmid vector into E.coli DH5alpha for amplification. The amplified plasmid was purified via a MiniPrep Kit. We conducted PCR afterward to ensure the presence of Afamin and Wnt3a genes in the purified vector. The following primer sequences were used in this experiment:

* Afamin (250 bp)
  + Forward primer: 5′ - ATGAGACACTTAAAACTTACAGG - 3′
  + Reverse primer: 5′ - GAAGTGTGTTGTCAGCCCAGCAC - 3′
* Wnt3a (200 bp)
  + Forward primer: 5′ - ATGGCCCCACTCGGATACTTCTTAC - 3′
  + Reverse primer: 5′ - GCCACGCTGGGCATGATCTCCA - 3′

![](https://static.igem.wiki/teams/4869/wiki/eng-figure-6.png)

Figure 6 (Amplified Afamin and Wnt3a DNA products by PCR are visualized using the agarose gel. Lane 1: standard marker, Lanes 2 and 3: vector plasmid #1, Lanes 4 and 5: vector plasmid #2, Lanes 6, 7, and 8: vector plasmid #3. Afamin targeting primers were used in Lane 2, 4, and 6. Wnt3a targeting primers were used in Lane 3, 5, 7, and 8)

A total of 8 samples were loaded into the agarose gel, and it was run for 20 minutes at 100 V in the agarose electrophoresis tank. Each vector plasmid had both Afamin and Wnt3a gene indicators, and the bands represented the amplified products. According to the result, vector #2 and #3 bands showed multiple bands, which demonstrated the possibility of any mutations or altercations during the purification process. On the other hand, vector #1 plasmid represented clear single bands in both genes: Afamin at 250 bp and Wnt3a at 200 bp (Figure 1). Therefore, we concluded that vector #1 plasmid was the most suitable for further experiments.

### [Part 2: Validation of Engineered HEK293 Cells through Cell Morphology Analysis]

![](https://static.igem.wiki/teams/4869/wiki/eng-figure-7.png)

Figure 7 (Cell morphology of HEK293 cells of negative control, 1:2.5 transfected sample, 1:5 transfected sample)

Subsequently, we co-transfected the BII-CMV-AfmW3A Vector and Super PiggyBac Transposase Expression Vector into HEK293 cells via lipofectamine. As previously demonstrated, the Super PiggyBac Transposase recognizes our genetic construct in the BII-CMV-AfmW3A (flanked with ITR sequences) Vector–and integrates it into the HEK293 genome at TTAA sites. Such transgenesis, indeed, creates engineered HEK293 cells containing Wnt3a and Afamin genes–primary supplements of our conditioned media.

Three experimental conditions–the negative control, 1:2.5 transfected sample, and 1:5 transfected sample–show negligible differences in their cell morphology and structure (Figure 7). This, in fact, is a primary checkpoint in our research, for our procedures (e.g., puromycin treatment and co-transfection of vectors) may potentially impair the structural and cellular integrity of HEK293 cells. Observing none, we suggest that the engineering protocols did not adversely impact the cellular and structural integrity of HEK293 cells.

### [Part 3: Validation of Engineered HEK293 Cells through Bradford Assay]

![](https://static.igem.wiki/teams/4869/wiki/eng-figure-8.png)

Figure 8 (The BSA standard curve was used for quantifying the proteins presented in cell culture media)

We then performed to quantify the proteins presented in cell culture media. Bovine serum albumin (BSA) standard curve was utilized as a reference point to quantify total proteins within our cell culture media. From the trendline equation derived from the BSA standard curve, we established a relationship between the absorbance values obtained from our samples and the corresponding protein concentrations. Using this relationship, we calculated the protein concentrations for each condition tested in our experiment (Figure 8). The results of this quantification revealed the following total protein concentrations (in µg/mL) within the respective cell culture media conditions:

* Negative control HEK293: 0.81 µg/mL
* 1:2.5 transfected HEK293: 0.79 µg/mL
* 1:5 transfected HEK293: 0.75 µg/mL

This is another paramount checkpoint of our research: assessing the impact of our experimental manipulations on protein levels in the cell culture media. Observing negligible differences between the three cell culture media conditions, we suggest that our experimental protocols do not necessarily impair the molecular integrity of the engineered HEK293 cells. This finding, in conjugation with Part 3, demonstrates that our experimental exploitation of the PiggyBac Vector System (and its associated protocols) does not adversely impact HEK293 cells.

### [Measurement Protocols]

Bradford assay is a molecular technique utilized for the quantification of protein concentration. The reaction relies on the amino acid composition of target proteins and the dye’s (Coomassie Brilliant Blue G-250) color change in response to protein binding. The dye interacts with positively charged amino acid residues on the surface of proteins, causing the solution to change color. Upon treatment in an acidic solution, the dye binds with proteins to shift the color from brown to blue. The intensity of this color change is directly proportional to the protein concentration in the sample. Key procedures involve the following:

* Preparation of the BSA standard curve (Figure 8): Spectrophotometric analysis of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 µg/µL BSA solutions.
* Preparation of sample wells and spectrophotometric absorbance: Addition of Bradford reagent to protein samples.
* Calculation of protein concentration: Spectrophotometric analysis of reacted protein samples at 595 nm followed by calculations using the BSA standard curve.

It must be noted that all spectrophotometric measurements were recorded in arbitrary units. The least-square regression line of the standard BSA curve is shown below:

* Y = 0.286 + 0.834\*X, R=0.99
* Absorbance (595 nm) = 0.834 x Total Protein concentration (µg/µL) + 0.286
* Total Protein concentration (µg/µL) = [Absorbance (595 nm) - 0.286] / 0.834

### [Part 4: Validation of Engineered HEK293 Cells through Western Blotting]

Furthermore, to validate that the engineered HEK293 cells, indeed, are capable of synthesizing Wnt3a and Afamin proteins, we performed western blotting. We performed two trials: first with 1 µg of total proteins loaded per well and second with 10 µg of total proteins loaded per well. The first trial, however, was limited with low signal intensity observed for both Afamin and Wnt3a proteins, rendering accurate quantification challenging. Accordingly, we responded by loading 10 µg of total proteins per well to enhance detection sensitivity and obtain solid data.

![](https://static.igem.wiki/teams/4869/wiki/eng-figure-9.png)

Figure 9 (Quantification of Afamin and Wnt3a in conditioned media collected. For the first trial, 1 µg of total proteins were loaded per well. For the second trial, 10 µg of total proteins were loaded per well)

Per western blot analysis, we demonstrated that engineered HEK293 cells (transfected with the vector plasmid) exclusively express both Afamin (at approximately 75 kDa) and Wnt3a (at approximately 37.5 kDa) proteins (Figure 8). This validates our ‘Build’ cycle by showcasing the successful integration of our genetic construct into the HEK293 genome. However, owing to the lack of normalization references for both Afamin and Wnt3a, the molar ratio, or relative abundance, of Afamin and Wnt3a proteins is yet to be elucidated and is a ground for future investigation.

## Generating Different Builds

Throughout our experiments, we consistently utilized six new Parts we added to the iGEM Registry of Standard Biological Parts. However, for adequate validation and screening procedures, we generated different builds for the following experiments:

* Part 1: We prepared three plasmid samples (#1, #2, and #3, respectively, Figure 6). Observing proper amplification in only plasmid vector #1–with plasmid vectors #2 and #3 suggesting potential mutation or structural alteration–we utilized plasmid vector #1 for our downstream experiments, ensuring accuracy and precision in our experiments.
* Part 2: We performed morphological analysis on three experimental samples––the negative control, the 1:2.5 transfected sample, and the 1:5 transfected sample (Figure 7). From the absence of morphological variations, we ascertained that our engineering protocols do not impair the structural integrity of the engineered HEK293 cells.
* Part 3: We performed Bradford Assay and total protein quantification on three experimental samples–the negative control, 1:2.5 transfected sample, and 1:5 transfected sample (Figure 8). Accordingly, we concluded that our experimental manipulations do not impede host cells’ ability to produce proteins and engage in normal cellular processes (thereby observing the preservation of molecular integrity).
* Part 4: We performed western blot analysis on three experimental samples–the negative control, 1:2.5 transfected sample, and 1:5 transfected sample (Figure 9). This enabled us to validate bona fide engineered HEK293 cells that contain Afamin and Wnt3a genes.

## Screening for Correct Builds

Throughout our ‘Build’ cycle, we extensively screened for correct builds. Primarily, each part of our ‘Build’ cycle was implemented with a corresponding screening measure:

* Part 1: We performed PCR for three vector plasmid samples. With plasmid vectors #2 and #3 presenting multiple amplified bands, we speculated structural alteration or mutation in the vector plasmids. Accordingly, we responded by utilizing plasmid vector #1–which showed the exact size of the amplified DNA product for both Afamin (250 bp) and Wnt3a (200 bp)--for our downstream experiments (Figure 6). We screened for accurately amplified BII-CMV-AfmW3A vectors upon amplification via E. coli DH5-alpha.
* Part 2: Through brightfield microscopy, we observed negligible morphological differences between three experimental conditions: the negative control, 1:2.5 transfected sample, and 1:5 transfected sample (Figure 7). We demonstrate that our protocols that involve puromycin treatment and lipofectamine transfection do not adversely impact both the structural and cellular integrity of the engineered HEK293 cells. We screened for the viability and integrity of our engineered cells.
* Part 4: Through western blot analysis, we observed the production of both Afamin and Wnt3a proteins exclusively in the engineered HEK293 cells (co-transfected with PiggyBac Vector System, Figure 9). This validates the success of our experimental design and approach. This screening measure allowed us to conclude that the HEK293 cells successfully integrated both Wnt3a and Afamin genes–enabling us to proceed with this build for the downstream experiments (as a proof of concept).

## Final Version of the Model

![](https://static.igem.wiki/teams/4869/wiki/eng-figure-10.png)

Figure 10 (Engineered HEK293 cells producing Afamin and Wnt3a proteins into conditioned media)

We illustrate our system’s final model as above (Figure 10). Detailed descriptions or depictions of our plasmid vectors (or other Parts added to the iGEM Registry of Standard Biological Parts) can be seen in the ‘Design’ section of this page.

# TestRationale & Objectives The previous sections focused on the development of the engineered HEK293 cells containing both Afamin and Wnt3a genes. As a proof of concept, this section investigates the effect of Afamin-Wnt3a conditioned media (CM) on bovine myoblast cell (MyoB) proliferation and differentiation. Accordingly, we performed two experiments for this phase: Prestoblue cell proliferation assay and brightfield microscopy. Test 1: Prestoblue Cell Proliferation Assay[Experimental Design] To comprehensively analyze the impact of Afamin-Wnt3a CM on MyoB cell proliferation, we utilized the Prestoblue assay, a highly versatile and pervasive technique to assess cell proliferation with marked precision and accuracy. The assay quantifies cells' metabolic activity, thereby yielding a reliable estimate of cell numbers. Foremost, we established four experimental conditions: * Standard DMEM cell culture media without FBS * Standard DMEM cell culture media with 10% FBS * Standard DMEM cell culture media without FBS but 10% Afamin-Wnt3a CM * Standard DMEM cell culture media without FBS but 20% Afamin-Wnt3a CM We utilized the PrestoBlue assay and conducted incubations at five distinct time points: 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours. The accompanying graph displays the mean and standard deviation (SD) of these measurements (n=2). Furthermore, we assessed statistical significance using the Mann-Whitney test to calculate p-values. All samples were seeded with the sample number of cells (1.25 x 103 cells). [Measurement Protocols] Prestoblue assay is a pervasively utilized molecular technique to quantify the metabolic activity of cells as a proxy for cell proliferation. Non-toxic, colorless resazurin, taken up by the living cells, is reduced by mitochondrial enzymes in metabolically active cells. The reduced resazurin, or resorufin, is a highly fluorescent and colorful compound. Accordingly, the fluorescence quantified by absorbance is an indirect measure for the number of metabolically active cells present in the cell culture–an effective reflection of the changes in cell numbers over time, or cell proliferation. The measurement protocols primarily involve the following: * Cell Seeding: Seeding of 1x103 MyoB cells to each well and the preparation of standard curve cells by varying the number of cells to 0, 1x103, 2x103, 5x103, 10x103, and 15x103. * Preparation of Test Wells and Prestoblue Treatment: Addition of Prestoblue solution at 0, 24, 48,72, and 96 hours of the treatment, followed by an incubation period. * Quantification of Cell Proliferation: Absorbance was measured at 570 nm and 600 nm after the incubation period and the calculation of cell viability. ![](https://static.igem.wiki/teams/4869/wiki/eng-figure-11.png) Figure 11 (The number of cells and absorbance measured using the Prestoblue assay shows a strong correlation (R2=0.991)) It must be noted that all spectrophotometric measurements were recorded in arbitrary units. The least-squares regression line, or the standard curve, presents a strong correlation between the number of cells and the absorbance values of the Prestoblue accuracy, validating the accuracy and consistency of our experimental results (R2=0.991, Figure 11). Our standard curve not only showcases the Prestoblue assay as a valid method of quantifying the changes in live cell numbers but also the success of our optimized experimental conditions for the Prestoblue assay. Our least-squares regression line can be illustrated as below: * Absorbance (570/600 nm) = 0.1859 x Number of cells (x103) + 0.2943, R=0.99 We quantified the absorbance of cells (or cell viability) at five incubation periods: 0 hr, 24 hr, 48 hr, 72 hr, and 96 hr (Figure 12). Accordingly, the number of live cells (x103 cells) was calculated based on the standard curve equation in Figure 11 (Figure 13). ![](https://static.igem.wiki/teams/4869/wiki/eng-figure-12.png) Figure 12 (Prestoblue assay was used to quantify cell proliferation incubation at five incubation times: 0 hr, 24 hr, 48 hr, 72 hr, and 96 hr. The measurements indicate the absorbance (570/600 nm) for duplicate experiments) ![](https://static.igem.wiki/teams/4869/wiki/eng-figure-13.png) Figure 13 (The number of live cells (x10^3 cells) was calculated based on the standard curve equation in Figure 11, and absorbance was measured in Figure 12) [Results and Implications] ![](https://static.igem.wiki/teams/4869/wiki/eng-figure-14.png) Figure 14 (Afamin-Wnt3a CM enhances cell proliferation. Prestoblue assay was used to quantify cell proliferation incubation at five incubation times: 0 hr, 24 hr, 48 hr, 72 hr, and 96 hr. The mean and standard deviation (SD) were plotted in the graph (N = 3). Mann-Whitney test was used to calculate the p-value) The results demonstrate the crucial role of Afamin-Wnt3a CM in inducing MyoB cell proliferation. Notably, we observed an elevated proliferation rate in MyoB cells cultured with 10% Afamin-Wnt3a CM, as shown by an absorbance reading of approximately 2.0 a.u. (p = 0.0295) compared to the FBS-absent sample (Figure 14). These findings articulate the prospect of Afamin-Wnt3a CM in stimulating cell proliferation, with promising implications for cell-based applications and research. We further speculated that increasing the concentration of Afamin-Wnt3a to 20% would result in a more pronounced proliferation increase. However, our observations indicated that elevating Afamin-Wnt3a to 20% did not substantially boost cell proliferation (~1.3 a.u.). Additionally, it is worth noting that the proliferation rate was lower when supplementing with Afamin-Wnt3a CM compared to 10% FBS (~4.0 a.u.). This suggests that, at present, 10% FBS cannot be replaced by Afamin-Wnt3a CM supplementation, highlighting the unique roles and potential limitations of these factors in cell culture applications. Overall, we successfully iterated the ‘Test’ phase of our engineering cycle by validating the role of Afamin-Wnt3a CM in regulating MyoB cell proliferation. We also underscore the distinct roles and limitations of Afamin-Wnt3a CM in cell culture applications. Test 2: Brightfield Microscopy[Experimental Design] To analyze the extent of MyoB differentiation via analysis of cell density and size and the formation of cell aggregates, we obtained brightfield images of MyoB cells after 96 hours of incubations. We performed the experiment with four total culture media conditions: Standard DMEM cell culture media without FBS Standard DMEM cell culture media with 10% FBS Standard DMEM cell culture media without FBS but 10% Afamin-Wnt3a CM Standard DMEM cell culture media without FBS but 20% Afamin-Wnt3a CM [Results and Implications] ![](https://static.igem.wiki/teams/4869/wiki/eng-figure-15.png) Figure 15 (Afamin-Wnt3a CM enhances cell proliferation and differentiation. The cell image was taken after 96 hours of incubation of four types of culture media (CM) conditions: without FBS, with 10% FBS, with 10% Afamin-Wnt3a CM, and with 20% Afamin-Wnt3a CM) We observed the lowest cell density and smaller cell sizes (as compared to other conditions) in the absence of FBS (Figure 15). By contrast, we noted spindle-shaped cells with robust coverage and density in 10% FBS-cultured MyoB cells. Comparably, MyoB cells cultured with 10% Afamin-Wnt3a CM presented similar cell morphology, whereas a ~50% reduction in cell density was observed as compared to the 10% FBS culture. Interestingly, 20% Afamin-Wnt3a CM-cultured MyoB cells exhibited a formation of cell aggregates. From the analysis of acquired images, we speculated that the 20% Afamin-Wnt3a CM treatment induces the differentiation of MyoB cells (from myoblasts to myocytes and myotubes). The reduction of cell proliferation as compared to the 10% Afamin-Wnt3a CM-cultured MyoB cells can be potentially attributed to the differentiation process. Accordingly, we validated another aspect of our ‘Test’ phase by demonstrating that Afamin-Wnt3a CM induces both cell proliferation and differentiation of MyoB cells; however, the extent of cell proliferation and differentiation likely varies by the concentration of Afamin-Wnt3a CM. Learn Through our ‘Build’ and ‘Learn’ phases of the Engineering Cycle, we actively responded to the core principles of engineering in iGEM and Synthetic Biology. Learn #1: Solve your project’s problems and use synthetic biology tools and/or experimental techniques to generate expected results. This year, we aimed to propose Afamin-Wnt3a conditioned CM as a potential substitute for FBS, which is limited by ethical concerns, batch-to-batch variability, and potential transmission of pathogens. We effectively addressed our project goals by accomplishing the following: * Build: PCR analysis using Afamin and Wnt3a primers to analyze the accurate, precise amplification of the BII-CMV-AfmW3A PiggyBac Vector via E. coli DH5-alpha (Figure 6). * Build: Brightfield microscopy to analyze the cell morphology (thus structural integrity) of HEK293 cells co-transfected with the PiggyBac Vector System (Figure 7). * Build: Bradford assay using standard BSA curve to analyze the molecular integrity of engineered HEK293 cells by quantifying total protein concentration (Figure 8). * Build: Western blot analysis of engineered HEK293 cells to validate the expression and production of Afamin and Wnt3a proteins exclusively in transfected cells (Figure 9). * Test: Cell proliferation assay to analyze the effect of Afamin-Wnt3a conditioned CM on proliferation of MyoB cells as compared to FBS (Figures 11~14). * Test: Brightfield microscopy to analyze the effect of Afamin-Wnt3a conditioned CM on differentiation of MyoB cells as compared to FBS and previous experiments (Figure 15). Overall, we performed six rounds of experimentation (involving unique synthetic biology tools) to generate the expected results. We demonstrated that the engineered HEK293 cells co-transfected with the PiggyBac Vector System inserted Afamin and Wnt3a genes and robustly expressed them. Furthermore, we confirmed that Afamin-Wnt3a conditioned CM induced MyoB proliferation and differentiation--while its extent varies by the concentration. This validates the potential of Afamin-Wnt3a-conditioned CM as a substitute for FBS. Learn #2: Think about and document what changes in design you would make for the next iteration(s) of the cycle. While our first iteration of the Engineering Cycle was fruitful, we were limited by the following: * Inability to elucidate the relative abundance of Afamin and Wnt3a proteins * Lacking a clear-cut explanation of the relationship between MyoB proliferation and differentiation as evinced by Prestoblue assay and brightfield microscopy To quantify the relative abundance of Afamin and Wnt3a proteins, we must derive a normalization standard for both Afamin and Wnt3a proteins, which is currently lacking in the literature. We highlight the potential of developing novel measurement standards (or new Parts to characterize/quantify Afamin and Wnt3a proteins) to accomplish this gap. This, indeed, will enable us to further understand the distinct roles and limitations of Afamin-Wnt3a conditioned CM in cell culture applications. Second, to offer a clear-cut explanation of the relationship between MyoB proliferation and differentiation, another round of experimentation/investigation must be implemented. This may involve molecular analysis (such as PCR or western blot analysis) of differentiation-related factors, such as MYOD and MYF5, under varying concentrations of Afamin-Wnt3a conditioned CM. This, when conjugated in relation to cell proliferation assay, can potentially provide a categorical effect that the manipulation of Afamin-Wnt3a conditioned CM’s concentration wields. Learn #3: Design and build a new Part, measure its performance, document whether it worked or not, and propose how the results would inform the next design or steps. We added six new Parts to the iGEM Registry of Standard of Biological Parts. We accurately designed six rounds of experiments--spanning one full iteration of the Engineering Cycle--to validate the performance and functionality of the optimized PiggyBac Vector System. The limitations of our experiments attest to the next steps and designs we may take. * BII-CMV-AfmW3A Vector ([Part: BBa K4869000](http://parts.igem.org/Part%3ABBa_K4869000)) * Super PiggyBac Transposase Expression Vector ([Part: BBa K4869001](http://parts.igem.org/Part%3ABBa_K4869001)) * Engineered HEK293 cells containing afamin and wnt3a genes inside the genome ([Part: BBa K4869003](http://parts.igem.org/Part%3ABBa_K4869003)) * Afamin gene ([Part: BBa K4869004](http://parts.igem.org/Part%3ABBa_K4869004)) * Wnt3a gene ([Part: BBa K4869005](http://parts.igem.org/Part%3ABBa_K4869005)) * Terminal inverted repeat sequence ([Part: BBa K4869006](http://parts.igem.org/Part%3ABBa_K4869006))

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Experiments

# Experiments Meetings

## First Experiments Meeting

### [Agenda 1: Pipette Practice]

Experimental Principle

* Micropipette practice
  + Before carrying out the PCR test, micropipette practice is required for better accuracy and precision.

Materials Needed

* Micropipette: precise volume control range from 1000µL ~ 0.1µL
* Used a small capacity pipette for our experiment (2, 10, 20 µL)

Procedure

1. Connect the pipette to the tip.
   + The button on the side should be close to the hand.
2. Press the button on the top and release all the air inside.
   + Push the button until the first stop.
3. Place the pipette inside the liquid.
   + Make sure that the pipette is perpendicular to the liquid.
4. Slowly release the button and absorb the liquid.
5. Pour the liquid inside the pipette into a small container.
   + Pour the liquid close to the container in contact.
   + Pour the liquid onto the bottom middle of the container.

![](https://static.igem.wiki/teams/4869/wiki/exp-figure-1.jpg)

Figure 1 (Korea-HS team member pipetting with a small container, practicing for better accuracy and precision for the PCR test)

### [Agenda 2: Polymerase Chain Reaction Test]

Experimental Principle

+ Polymerase Chain Reaction (PCR) Test
  - The objective of the experiment is to check if the two proteins Afamin and Wnt3a are in the plasmid accurately or intact.
  - In addition, the PCR test is done to rapidly produce tons of copies of a DNA segment between Primer F and Primer R.

Materials Needed

+ Primer F 1 µL
+ Primer R 1 µL
+ Plasmid DNA 1 µL
+ Water 17 µL
+ DNA Polymerase
+ Nucleotide (dNTP) - [ATGC]
+ Buffer: 5,6,7 are already placed in the tube

Procedure

1. Denaturation
   + 95°C Single Strand for 1 minute.
2. Annealing Step
   + Attach 60°C Primer for 1 minute.
3. Extension
   + 72°C DNA synthesis for 1minute.
4. N\*( 2) as many times as cycle number.
5. 10\*(2) as many as 35 ~ 40 times during the polymerase chain reaction.
   + If there is a reaction after amplification, it indicates that the result is positive.
   + If there is no response after amplification, it indicates that the result is negative.

![](https://static.igem.wiki/teams/4869/wiki/exp-figure-2.jpg)

Figure 2 (Korea-HS team member running a PCR test by setting up the conditions of the RT-PCR machine)

## Second Experiments Meeting

### [Agenda 3: Agarose Gel Electrophoresis]

Experimental Principle

+ Agarose Gel Electrophoresis
  - Our team aimed to check the quantity and quality of the DNA throughout the Gel DNA Marker Test. We used an Agarose gel standard DNA marker to identify DNA size.
  - It is possible to see the band size getting thinner from top to bottom, which means that the smaller the DNA, the thinner the mark goes down. To measure the DNA quantity, we examined the band thickness, which directly correlates to how much DNA is contained.

Materials Needed

+ Agarose Gel electrophoresis
+ 1x TAE buffer: (Buffer with Tris Acetate EDTA)
  - Tris-acetate is used to match the pH
+ Agarose Powder
  - 1.5% concentrated Agarose gel
+ Gel tank

Procedure

![](https://static.igem.wiki/teams/4869/wiki/exp-figure-3.jpg)

Figure 3 (Procedure 4: Korea-HS team members inserting the afamin and wnt3a protein-DNA samples into the holes of the gel tank)

![](https://static.igem.wiki/teams/4869/wiki/exp-figure-4.jpg)

Figure 4 (Procedure 7: Korea-HS team members examining the standard DNA marker, checking for DNA quality and quantity)

1. Add the TAE Buffer and the agarose powder used at 70~80° Celsius to make them soluble enough and add DNA staining solution.
   + DNA staining is essential because it lets us physically identify DNA when we reflect the UV light or phe.
2. Pour the gel into the plastic mold and shape them.
3. Put a plastic mold on top of the main gel caster and solidify the gel into cube shapes at 40° Celcius.
4. Put our samples carefully (using the pipette) into the holes of the gel tank.
   + If the PCR test has been operated correctly - the results.
5. Place the gel mold into the gel tank, and fill the tank with the buffer.
   + The gel tank sends electricity onto the mold.
   + The pipette needs to go under the liquid (buffer) because the procedure is quite difficult to inject the protein inside the well plate.
6. The gel tank sends electricity from the top to the bottom of the gel tank, gathering all of the DNA at the bottom of the well.
7. Wait 20 minutes to check how far it moves in the standard DNA marker.
   + The DNA bend can move farther down when we turn on electric chem.
   + Main objective: To find DNA purity.

### [Information to Help Data Analysis]

![](https://static.igem.wiki/teams/4869/wiki/exp-figure-5.jpg)

Figure 5 (Strap bands: a picture of a standard DNA marker)

1. Analysis method
   + If the band is shown as one clear band the DNA is pure enough
   + IPrimary non-specific binding may result in an unpredicted band size
2. PCR Troubleshooting method
   + Multiple bands convey that we need to increase the temperature
   + No bands convey that we need to decrease the temperature
3. Reason of error
   + Even though the chemicals and apparatus used are the same, the result resolution may differ due to skill level disparities.

## Third Experiments Meeting

### [Agenda 4: Determine the optimal ratio BII-CMV-AfmW3A Vector (AW) to Super PiggyBac Transposase Expression Vector (T) for efficient transfection of HEK293 cells]

Experimental Principle

+ Determine the optimal ratio.
  - In this experiment, our objective was to determine the optimum ratio between the AW vector plasmid and T plasmid that would enable the AW vector to integrate into the nuclear genome of the host cell.
  - To achieve this efficiently and test for different ratios with a sufficient number of trials, we divided into four separate groups.

Materials Needed

+ BII-CMV-AfmW3A Vector (AW)
+ PiggyBac Transposase Expression Vector (T)
+ Lipofectamine 2000 Transfection Reagent (Thermofisher, CAT#11668027)
+ DMEM cell culture media (Gibco)

Procedure for Groups 1 and 2

Groups 1 and 2 conduct the experiment with a 1:2 ratio of AW Vector Plasmid and T Plasmid.

1. AW Vector Plasmid: 500 ng/µL (ratio: 1 = 1 µL) + T Plasmid: 500 ng/µL (ratio: 2 = 2.5 µL) + Liposome (8 µL)
   + Total volume: 11.5 µL
2. Place 1 µL of AW vector plasmid in tube 1
3. Place 2.5 µL of T plasmid in the same tube → Place it at the bottom of the tube so it can be mixed with the previous fluid as well
4. Place 8 µL of Liposome in tube 1
5. Repeat procedures 2-4 for tube 2
6. Add 200 µL of cell culture media (DMEM) to both solutions
7. Incubate for 10 minutes to make the complex

Procedure for Groups 3 and 4

Groups 3 and 4 conducted the experiment with a 1:5 ratio of AW Vector Plasmid and T Plasmid.

1. AW Vector Plasmid: 500 ng/µL (ratio: 1 = 1 µL) + T Plasmid: 500 ng/µL (ratio: 2 = 5 µL) + Liposome (8 µL)
   + Total volume: 14 µL
2. Place 1 µL of AW vector plasmid in tube 3
3. Place 5 µL of T plasmid in tube 3
4. Place 8 µL of Liposome in tube 3
5. Repeat procedures 2-4 for tube 4
6. Add 200 µL of cell culture media (DMEM) to both solutions
7. Incubate for 10 minutes to make the complex

### [Agenda 5: Preparing HEK293 Cells for Transfection]

Experimental Principle

+ DNA plasmid transfection
  - In this experiment, we completed DNA plasmid transfection. In our project, transfection is the process of adding Afamin and Wnt3a into a human embryonic kidney cell called HEK293.
  - In order to facilitate this effectively, we added liposomes to allow the AW Vector Plasmid to enter the human cell. The liposome is crucial in this procedure, as the DNA cannot be inserted into the HEK293 cell without it.
  - We used stable transfection, which involves the transposase in the T plasmid. When T plasmid is present, the DNA (AW Vector Plasmid) can enter the host cell’s nuclear genome. It is efficient as it can sustain long-term expression of a transgene by integrating foreign DNA into the host nuclear genome.

Materials

+ HEK293 cell line
+ DMEM culture media (Gibco)
+ Trypsin-EDTA cell dissociation buffer (Gibco)
+ Puromycin (10 mg/mL) stock solution (Sigma)
+ 6 well-culture plate

Procedure

1. Use the aspirating machine to draw out base old media
2. Incubate the HEK293 cell with Trypsin-EDTA buffer to dissociate the HEK293 cells from the cell culture plate
3. Incubate the cell for 5 minutes in order for protease (Trypsin) to cut the membrane proteins from the cells
4. Condition 37 degrees Celsius and 5% CO2 level for mammalian cell culture
5. Incubate the cells with the prepared transfection complex for 6 hours
6. After aspirating the old media, add the fresh DMEM media to each sample
7. Add the puromycin in a final concentration of 10 µg/mL to select the HEK293 cells inserted with the Afamin and Wnt3a genes
8. Add the fresh DMEM media with puromycin (10 µg/mL) every three days
9. Culture the cells until the puromycin-resistant HEK293 cells are maintained

## Fourth Experiments Meeting

### [Agenda 6: Utilizing the Western Blot and Bradford Assay]

Experimental Principle

+ Western Blot
  - The western blot can be used to determine the amount of Afamin and Wnt3a proteins produced in the cultured media.
    * This method will be performed by group 3.
+ Bradford Assay
  - Before conducting the western blot, it is necessary to perform a Bradford assay to measure the total protein concentration. This will ensure that the same amount of protein is used during the western blot.

Materials Needed

+ Conditioned media samples from the previous experiment
  - Contain HEK293 cells that include the vectors in the conditioned media
  - Standard sample: sample #6
  - Our sample: sample #7
+ Coomassie Brilliant Blue
  - Dye used to determine the protein concentration for the Bradford assay
+ Graphing tool
  - Used Google Sheets to graph
  - Bovine serum albumin (BSA)

Procedure

![](https://static.igem.wiki/teams/4869/wiki/exp-figure-6.jpg)

Figure 6 (Table 1: Preparation of standard curve sample)

1. Bradford Assay
   + The following condition was used to prepare the six samples of standard curve sample (Figure 6).
   + Add 5 µL of standard samples to the well in the microplate
   + Add 95 µL Bradford reagent to each well
   + Mix the solution in the microplate wells gently by pipetting up and down (Figure 7)

     ![](https://static.igem.wiki/teams/4869/wiki/exp-figure-7.jpg)

     Figure 7 (Bradford assay standard sample using 94-well plates)
2. Prepare Sample Wells
   + Take the protein samples (5 µL) and add them to the microplate.
   + Add 95 µL Bradford reagent to each well
   + Mix the solution in the microplate wells gently by pipetting up and down
   + Allow the standard and sample wells in the microplate to incubate at room temperature for approximately 10 minutes. During this time, the Bradford reagent will bind to the proteins.
3. Measure Absorbance
   + Set your microplate reader to measure absorbance at 595 nm.
   + Zero the microplate reader using the blank well containing distilled water.
   + Measure the absorbance of each standard solution and your protein samples in the microplate.
4. Calculate Protein Concentration
   + Plot a standard curve using the known concentrations of the BSA standard and their corresponding absorbance values using a spreadsheet.
   + Determine the absorbance of our protein samples.
   + Using the standard curve, find the corresponding protein concentration for each sample based on their absorbance values.

![](https://static.igem.wiki/teams/4869/wiki/exp-figure-8.jpg)

Figure 8 (Korea-HS team member inserting each sample into the wells)

![](https://static.igem.wiki/teams/4869/wiki/exp-figure-9.jpg)

Figure 9 (Korea-HS team member inserting bovine serum (BSA) into the wells containing samples)

![](https://static.igem.wiki/teams/4869/wiki/exp-figure-10.jpg)

Figure 10 (Image of completely inserted samples and a mixture of BSA and samples. The wells written S0~5 row A and row E are where the standard samples were inserted into. The wells written 1~7 row C and row G are where our samples were inserted into.)

## Fifth Experiments Meeting

### [Agenda 7: Analyzing Our Western Blot Results]

Experimental Principle

+ Western Blot Results Analysis
  - The goal of this experiment is to specifically look at the amount of Afamin and Wnt3a protein using the Western Blot.
  - After going through this process, only the proteins with Afamin and Wnt3a will appear (chemiluminescence or HRP).
  - Hence, after the utilization of Western Blot, it is possible to determine the optimal condition in which Afamin and Wnt3a are most produced as the greater number of Afamin of Wnt3a protein will indicate a brighter color.

Materials Needed

+ Protein Samples: Conditioned media samples were collected in previous experiments
+ Electrophoresis Equipment
  - Gel electrophoresis apparatus (tank, power supply, gel trays, and combs)
  - Polyacrylamide gel (usually SDS-PAGE gels) or precast gels
  - Electrophoresis Buffers
  - Running Buffer (Tris-glycine buffer)
  * Sample loading buffer (containing reducing agents and tracking dyes)
+ Protein Transfer
  - PVDF (polyvinylidene difluoride) membrane
  - Transfer apparatus (wet transfer systems)
+ Blocking and Antibody Dilution
  - Blocking buffer (5% BSA in TBS)
  - Primary antibody diluent (usually the same as blocking buffer).
+ Primary Antibodies: Afamin (Cat# AA 260-599) from antibodies-online and Wnt3a (Cat# 2391) from cell signaling targeting primary antibodies
+ Secondary Antibodies: Horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit)
+ Chemiluminescent Substrates
  - Chemiluminescent substrate reagents (e.g., ECL or SuperSignal)
  - Developer and fixer solutions
+ Imaging Equipment
  - A chemiluminescence detection system (chemiluminescence imager)

Procedure

1. Gel Electrophoresis for Western Blot
   + Before the experiment, put the assigned loading buffer into each well
   + Well 1: DMEM-FBS (20mL)
   + Well 2: DMEM+FBS (20mL)
   + Well 3: No Treatment (20mL)
   + Well 4: Afamin & Wnt3a in 1:2.5 ratio (30mL)
   + Well 5: Afamin & Wnt3a in 1:5 ratio (30mL)
   + Well 6: Afamin & Wnt3a in 1:2.5 ratio (30mL)
   + Well 7: Afamin & Wnt3a in 1:5 ratio (30mL)
2. Heat the samples at 90 degrees for 5 minutes in an incubator as this process will denature the proteins inside.* Put in Tris-Glycine SDS buffer (running buffer) in the gel tank up to 2 gel labels to help electron glow in during electrophoresis.* Load the samples into an SDS-PAGE gel loading well using a micropipette (1 µg of total protein for the first trial and 10 µg of total protein for the second trial).* Put on the lid and set the gel electrophoresis tank to 120V
         + Transfer the proteins into the PVDF membrane using 100 V overnight.
         + Incubate Skim milk solution for blocking for 1 hour.
         + The primary antibody was incubated overnight
         + Using the PBST solution wash the PVDF membrane three times (For each washing - 5-minute incubation)
         + The secondary antibody was incubated for 1 hour
         + Using the PBST solution wash the PVDF membrane three times (For each washing - 5-minute incubation)
         + Chemidoc was used to detect the Afamin and Wnt3a protein band
       * Cell Proliferation Assay from Bovine-Cultured Cells
         + Detach the bovine cells attached to the bottom of the media container and put them in the well
         + Divide the wells into 7 groups for examination for a week (1 column for each day)
         + Use suction to remove the media from the now detached bovine cells
         + Use cell dissociation reagent and insert 1000 µL of it into the bovine sample
         + Incubate for 5 minutes
         + Put DMEM solution into the detached bovine cells 6 times with a 1000µL pipette and mix well
         + Use a 200 µL pipette and put 100 µL of the bovine cell and DMEM solution 7 times, one for each of the 7 well groups.

![](https://static.igem.wiki/teams/4869/wiki/exp-figure-11.jpg)

Figure 11 (Image of cultured bovine cells in an incubator observed by an electron microscope)

![](https://static.igem.wiki/teams/4869/wiki/exp-figure-12.jpg)

Figure 12 (Image of gel tank filled with Tris-Glycine SDS buffer (running buffer) and bovine cell culture sample of various concentrations (Well 1:12.1 µL, Well 2:10.1 µL, Well 3:9.7 µL Well 4:8.9 µL, Well 5:11.1 µL, Well 6:10.3 µL, Well 7:9.7 µL)

![](https://static.igem.wiki/teams/4869/wiki/exp-figure-13.jpg)

Figure 13 (Korea-HS team member inserts Tris-Glycine SDS buffer (running buffer) into the wells containing samples grown with Afamin and Wnt3a)

## Sixth Experiments Meeting

### [Agenda 8: Biochemical assay]

Experimental Principle

+ Through this experiment, we wanted to see the effect of Afamin and Wnt3a protein on cell proliferation and compare that effect to the positive control which had FBS.
  - We measured this effect by adding PrestoBlue, a chemical reagent that allows us to perform a biochemical assay to examine the growth of the bovine cells.

Materials Needed

- 96-well plate
- MyoB (bovine myoblast cell line)
- PrestoBlue reagent
- Four different media samples (350 μL of each):
  * Without FBS
  * With 10% FBS
  * With 10% Afamin-Wnt3a CM
  * With 20% Afamin-Wnt3a CM

Procedure

1. Cell proliferation analysis procedure
   - To prepare the cell samples, 96-well plates were used, and 1x103 MyoB cells were added to each well for the experiment. Additionally, prepare standard curve cells by varying the number of cells to 0, 1x103, 2x103, 5x103, 10x103, and 15x103. All samples were prepared for the duplicate measurements.
   - After the cells were attached, aspirate the culture medium from the wells containing MyoB cells.
   - Rinse the cells once with phosphate-buffered saline (PBS) to remove any residual medium.
   - Add the 90 µL of treatment (Without FBS, With 10% FBS, With 10% Afamin-Wnt3a CM, With 20% Afamin-Wnt3a CM) on each appropriate position of the 96 well plate.
   - Incubate the cells for 0, 24, 48,72, and 96 hours of the treatment.
   - For each time point, PrestoBlue solution was added to the sample (10 µL) to each
   well-containing cell (final concentration should not exceed 10%).- Incubate the cells with the PrestoBlue reagent at 37°C for 30 minutes.
2. Absorbance procedure used in cell proliferation assay analysis
   - First, we measured the absorbance at 570 nm and 600 nm, which was our reference.
   - The absorbance at 570 nm increases as PrestoBlue becomes redder, indicating that there were more bovine cells in that specific sample, while the absorbance at 600 nm measures the light transmission
   - The ratio of the 570 nm absorbance and 600 nm absorbance that represents proliferation is about 1.0

![](https://static.igem.wiki/teams/4869/wiki/exp-figure-14.jpg)

Figure 14 (Korea-HS team member adding the bovine cell media to the media samples tested to check the cell proliferation level of the various media samples being tested to create cultured meat using Afamin and Wnt3a proteins)

![](https://static.igem.wiki/teams/4869/wiki/exp-figure-15.jpg)

Figure 15 (Korea-HS team member images bovine cells to check the overall growth when using various samples such as ones that contain FBS or Afamin and Wnt3a proteins)

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Results

# Research Project Goal

Our iGEM project has a primary goal of creating an environmentally friendly and sustainable growth medium for raising bovine muscle cells that do not require fetal bovine serum (FBS). FBS is commonly used in cell culture but has many drawbacks, including ethical concerns, inconsistencies between batches, and the potential transmission of pathogens. Previous research indicates that Wnt3a and Afamin can play essential roles in organoid culture, particularly in the establishment and maintenance of organoids. Therefore, to address these challenges, we focused on developing a genetically engineered HEK293 cell (Afamin and Wnt3a gene-inserted cells) to produce Afamin and Wnt3a-enriched conditioned media, two crucial components that could support cell growth and proliferation without the need for FBS.

# Results

## Result 1: Cloning BII-CMV-AfmW3A vector and PCR

![](https://static.igem.wiki/teams/4869/wiki/res-figure-1.png)

Figure 1 (Amplified Afamin and Wnt3a DNA products by PCR are visualized using the agarose gel. Lane 1: standard marker, Lane 2 and 3: vector plasmid #1, Lane 4 and 5: vector plasmid #2, Lane 6, 7, and 8: vector plasmid #3. Afamin targeting primers were used in Lane 2, 4, and 6. Wnt3a targeting primers were used in Lane 3, 5, 7, and 8)

After purchasing the BII-CMV-AfmW3A plasmid vector, which contains afamin and Wnt3a genes, from AddGene, we transformed this plasmid vector into E. coli DH5alpha to increase the copy number of the plasmid vector. After the plasmid was purified from the E. coli using miniprep, we performed the polymerase chain reaction (PCR) to check whether Afamin and Wnt3a gene sequences were presented in the purified vector. The primer sets targeting Afamin and Wnt3a were used to amplify the genes using the purified BII-CMV-AfmW3A vector as a template.

Three BII-CMV-AfmW3A plasmid vectors were purified from the E.coli. Then, Afamin and Wnt3a genes were amplified using specific primers. The Afamin gene amplified DNA product was designed to be 250 bp. Wnt3a gene amplified DNA product was designed to be 200 bp.

The following primer sequences were used in this experiment:

* Afamin (250 bp)
  + Forward primer: 5′ - ATGAGACACTTAAAACTTACAGG - 3′
  + Reverse primer: 5′ - GAAGTGTGTTGTCAGCCCAGCAC - 3′
* Wnt3a (200 bp)
  + Forward primer: 5′ - ATGGCCCCACTCGGATACTTCTTAC - 3′
  + Reverse primer: 5′ - GCCACGCTGGGCATGATCTCCA - 3′

Eight samples were loaded into agarose gel, and it was run for 20 min at 100 V in the agarose electrophoresis tank (Figure 1). The band indicated amplified products. Vector plasmid #2 and #3 bands showed multiple bands of amplified products, which indicates that this vector may be structurally altered or have mutations. However, only vector plasmid #1 products showed clear single-band amplified products in both genes. The band size indicated in Figure 1 showed the exact size of the DNA amplified product from both Afamin (250 bp) and Wnt3a (200 bp). Therefore, we concluded that vector plasmid #1 would be used for the downstream experiments.

## Result 2: Engineering HEK293 cells containing Afamin and Wnt3a genes in genomic sequence using DNA plasmid transfection and puromycin selection

![](https://static.igem.wiki/teams/4869/wiki/res-figure-2.png)

Figure 2 (Two vectors must be transfected (BII-CMV-AfmW3A vector and Super PiggyBac Transposase Expression vector) to insert the Afamin and Wnt3a genes into the HEK293 cell's genomic DNA)

Figure 2 illustrates the essential steps involved in the process of creating HEK293 cells capable of producing Afamin and Wnt3a proteins stably. To achieve this, the insertion of the Afamin and Wnt3a genes into the genomic DNA of HEK293 cells requires the transfection of two specific DNA plasmid vectors: the BII-CMV-AfmW3A vector and the Super PiggyBac Transposase Expression vector.

In detail, the procedure begins with the transfection of these two plasmid vectors into HEK293 cells, as shown in Figure 2. The first plasmid vector, BII-CMV-AfmW3A, carries the genetic information for Afamin and Wnt3a, while the second vector, Super PiggyBac Transposase Expression Vector, facilitates the integration of these genes into the cellular genome.

To determine the optimal conditions for the transfection process, we conducted experiments involving two different plasmid vector molar concentration ratios (1:2.5 and 1:5 for BII-CMV-AfmW3A: Super PiggyBac Transposase Expression Vector). These variations were carried out in conjunction with using the lipofectamine transfection reagent. Subsequently, we employed puromycin selection to identify and isolate the HEK293 cells that successfully incorporated and expressed the Afamin and Wnt3a genes. Remarkably, our efforts yielded a successful selection of puromycin-resistant HEK293 cells under both transfection conditions, indicating the establishment of stable Afamin and Wnt3a protein-producing cells.

![](https://static.igem.wiki/teams/4869/wiki/res-figure-3.png)

Figure 3 (Cell morphology of HEK293 cells of negative control, 1:2.5 transfected sample, 1:5 transfected sample)

Figure 3 shows the cellular morphology of HEK293 cells subjected to different experimental conditions, namely the negative control, 1:2.5 transfected sample, and 1:5 transfected sample. These visual observations hold significant implications for our study. After a rigorous two-week treatment with puromycin, we successfully isolated HEK293 cells that had undergone transfection with the BII-CMV-AfmW3A and Super PiggyBac Transposase Expression Vector, employing both the 1:2.5 and 1:5 ratios.

The significance of these findings lies in their role as a critical checkpoint in our research. Cellular morphology serves as an indicator of the overall health and stability of genetically engineered cells. The absence of discernible differences in cell appearance across these experimental conditions is a reassuring outcome, suggesting that the transfection process did not induce any apparent adverse effects or alterations in cellular structure. This preservation of normal cell morphology is a pivotal step towards the establishment of stable HEK293 cell lines capable of producing Afamin and Wnt3a proteins, reinforcing the reliability and potential success of our experimental approach.

## Result 3: Quantifying the produced Afamin and Wnt3a proteins in the conditioned media using Bradford Assay and Western Blot

![](https://static.igem.wiki/teams/4869/wiki/res-figure-4.png)

Figure 4 (The BSA standard curve was used for quantifying the proteins presented in cell culture media)

In Figure 4, we employed a BSA (Bovine Serum Albumin) standard curve as a crucial tool for quantifying proteins within our cell culture media. This standard curve served as a reference point against which we measured the protein concentrations present in the media.

The process of quantification involved the utilization of the trendline equation derived from the BSA standard curve. This equation indicates a relationship between the absorbance values obtained from our samples and the corresponding protein concentrations. Using this relationship, we calculated the protein concentrations for each condition tested in our experiment. The results of this quantification revealed the following total protein concentrations within the respective cell culture media conditions:

* Negative control HEK293: 0.81 µg/mL
* 1:2.5 transfected HEK293: 0.79 µg/mL
* 1:5 transfected HEK293: 0.75 µg/mL

These values provide us with critical insights into the protein concentrations associated with different experimental conditions, allowing us to assess the impact of our experimental manipulations on protein levels in the cell culture media.

![](https://static.igem.wiki/teams/4869/wiki/res-figure-5.png)

Figure 5 (Quantification of Afamin and Wnt3a in conditioned media collected. For the first trial, 1 µg of total proteins were loaded per well. For the second trial, 10 µg of total proteins were loaded per well)

In Figure 5, we present the quantification results for Afamin and Wnt3a proteins in conditioned media collected during our experiments. To detect these proteins, we employed the western blot technique. In the initial trial, we loaded 1 µg of total proteins per well. Subsequently, in a second trial, we increased the total protein loading to 10 µg per well.

The western blot analysis conclusively demonstrated that the expression of both Afamin (at approximately 75 kDa) and Wnt3a (at approximately 37.5 kDa) proteins was exclusively observed in cells transfected with the vector plasmid. This outcome confirms the successful integration of the target genes into the cellular genome, validating our experimental approach.

Notably, the first trial, which involved the loading of 1 µg of total proteins, yielded very low signal intensities for Afamin and Wnt3a proteins, making accurate quantification challenging. In response to this limitation, we conducted a second trial with a higher protein loading (10 µg per well), aiming to enhance the detection sensitivity and obtain more robust data (Figure 5).

However, it is essential to acknowledge that our study encountered limitations in determining the molar ratio of Afamin and Wnt3a proteins. This limitation arises from the absence of a reference gene for normalization purposes. While our results confirm the presence or absence of these specific proteins, quantifying their exact abundance relative to each other remains a subject for future investigations.

## Result 4: Analyzing the effect of Afamin-Wnt3a conditioned media (CM) on Bovine myoblast cell line (MyoB)

![](https://static.igem.wiki/teams/4869/wiki/res-figure-6.png)

Figure 6 (Afamin-Wnt3a CM enhances cell proliferation of the bovine muscle cells (MyoB) cell line. Prestoblue assay was used to quantify cell proliferation incubation at five incubation times: 0 hr, 24 hr, 48 hr, 72 hr, and 96 hr. The mean and standard deviation (SD) were plotted in the graph (N = 2). Mann-Whitney test was used to calculate the p-value)

Figure 6 presents a comprehensive analysis of the impact of Afamin-Wnt3a conditioned media (CM) on MyoB cell proliferation. To quantify this effect, we utilized the PrestoBlue assay and conducted incubations at five distinct time points: 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours. The accompanying graph displays the mean and standard deviation (SD) of these measurements (N = 2), and we assessed statistical significance using the Mann-Whitney test to calculate p-values. All samples were seeded with the sample number of cells (1.25 x 103 cells).

We harvested the cell culture media derived from our engineered HEK293 cells enriched with the Afamin-Wnt3a complex. Our goal was to assess its impact on the proliferation of bovine myoblast cells (MyoB) via a dedicated cell proliferation assay. In this study, we examined four specific conditions: standard DMEM cell culture media without FBS, standard DMEM cell culture media with 10% FBS, standard DMEM cell culture media with 10% Afamin-Wnt3a CM, and standard DMEM cell culture media without FBS but supplemented with 20% Afamin-Wnt3a CM.

The results, as shown in Figure 6, affirm that Afamin-Wnt3a CM plays a crucial role in regulating MyoB cell proliferation. Remarkably, 10% Afamin-Wnt3a CM led to a significantly enhanced proliferation rate, as evidenced by an absorbance reading of approximately 2.0 A.U (p = 0.0295) compared to the FBS-absent sample. These findings underscore the substantial potential of Afamin-Wnt3a CM in stimulating cell proliferation, with promising implications for cell-based applications and research.

We further hypothesized that increasing the concentration of Afamin-Wnt3a to 20% would result in a more pronounced proliferation increase. However, our observations indicated that elevating Afamin-Wnt3a to 20% did not lead to a substantial boost in cell proliferation (~1.3 A.U.). Additionally, it is worth noting that the proliferation rate was lower when supplementing with Afamin-Wnt3a CM compared to 10% FBS (~4.0 A.U.). This suggests that, at present, 10% FBS cannot be replaced by Afamin-Wnt3a CM supplementation, highlighting the unique roles and potential limitations of these factors in cell culture applications.

![](https://static.igem.wiki/teams/4869/wiki/res-figure-7.png)

Figure 7 (Afamin-Wnt3a CM enhances cell proliferation and differentiation. The cell image was taken after 96 hours of incubation of four types of culture media (CM) conditions: without FBS, with 10% FBS, with 10% Afamin-Wnt3a CM, and with 20% Afamin-Wnt3a CM)

Next, we acquired bright field images of MyoB cells after 96 hours of incubation with these four culture media conditions. In the absence of FBS, we noted the lowest cell density, with smaller cell sizes than the other conditions. Conversely, 10% FBS resulted in spindle-shaped cells with robust coverage and density. Interestingly, 10% Afamin-Wnt3a CM treatment led to a morphology similar to that seen with 10% FBS, with a ~50% reduction in cell density compared to the 10% FBS-incubated samples. Notably, in the presence of 20% Afamin-Wnt3a CM, we observed the formation of cell aggregates.

This result suggests that the 20% Afamin-Wnt3a CM treatment may induce the differentiation of MyoB cells, transitioning them from myoblasts to myocytes and myotubes. Consequently, this differentiation process appears to result in a lower cell proliferation rate when compared to the 10% Afamin-Wnt3a CM treatment.

![](https://static.igem.wiki/teams/4869/wiki/res-figure-8.png)

Figure 8 (Muscle cell differentiation and factors involved in different stages of myoblasts (referenced from PubMed Central))

Cultured meat, produced through in vitro cultivation of animal cells, represents a sustainable and environmentally friendly alternative to traditional livestock farming. In this context, bovine cell culture is pivotal in generating lab-grown meat products. The ability to enhance cell proliferation and differentiation, as demonstrated by the effects of Afamin-Wnt3a CM, is critical for advancing the efficiency and scalability of cultured meat production.

Our project showed an increase in cell density and differentiation under the influence of Afamin-Wnt3a CM, which suggests a potential strategy for optimizing the culture conditions of bovine cells used in lab-grown meat production. By carefully modulating the concentration of Afamin-Wnt3a CM in the culture medium, we may achieve the desired characteristics in the final cultured meat product.

Moreover, the findings regarding cell differentiation hint at the possibility of directing bovine cells toward specific lineages relevant to meat production, such as myocytes and myotubes. This could lead to the development of meat products with improved texture, taste, and nutritional profiles, ultimately enhancing the consumer appeal of lab-grown meat.

In summary, these outcomes presented in our project provide valuable insights that can inform and advance the field of bovine cell culture for cultured meat production. The ability to modulate cell behavior through Afamin-Wnt3a CM opens up exciting avenues for optimizing the production process and fine-tuning the characteristics of lab-grown meat, bringing us closer to a sustainable and ethical future in food production.

# References

Tüysüz, N., van Bloois, L., van den Brink, S., Begthel, H., Verstegen, M. M., Cruz, L. J., Hui, L., van der Laan, L. J., de Jonge, J., Vries, R., Braakman, E., Mastrobattista, E., Cornelissen, J. J., Clevers, H., & Ten Berge, D. (2017). Lipid-mediated Wnt protein stabilization enables serum-free culture of human organ stem cells. Nature communications, 8, 14578. <https://doi.org/10.1038/ncomms14578>.

Jang, Y. N., & Baik, E. J. (2013). JAK-STAT pathway and myogenic differentiation. JAK-STAT, 2(2), e23282. <https://doi.org/10.4161/jkst.23282>.

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