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What I learn from Genomics class

: Obese in Genomics

                                         20131571 woobeen Jo

                               Correspondence: <a href="mailto:woobin512@unist.ac.kr">woobin512@unist.ac.kr</a>

                                  

 

1. Abstract

UNIST Genomics course applied SELF (Self Evaluating Learning Framework) to evaluate student. In the system of SELF, Students have to evaluate themselves in Genomics class. Many students feel awkward when evaluating themselves. Because they don’t know how to evaluate themselves. They have been evaluated by other professors. That’s why Self-evaluation is good opportunity for developing individual or problem-solving through self-knowledge

 This paper introduces woobeen Jo’s independent experiment design using SELF, performing and conclusion based on the experimental results. Because of importance of how to think critically and independently when performing science research, set up of experimental design and evaluation were categorized into 5 parts.

 

<table border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr>
<td style="width: 236px">
Grading standard (%)
</td>
<td style="width: 365px">
Point of view
</td>
</tr>
<tr>
<td style="width: 236px">
Attendance (10)
</td>
<td style="width: 365px">
Attendance is very important in grading standard because of its indicator on student's attitude.
</td>
</tr>
<tr>
<td style="width: 236px">
Pre-study of Scientific design (30)
</td>
<td style="width: 365px">
it is integrated form of how someone critically and independently think and having questions before performing science research.
</td>
</tr>
<tr>
<td style="width: 236px">
<ul>
<li>Achieving Goal (10)</li>
</ul>
</td>
<td style="width: 365px">
Evaluate critical thinking of the motivation of research and way of acknowledgement of problem, solving problem
</td>
</tr>
<tr>
<td style="width: 236px">
<ul>
<li>Experimental Design (20)</li>
</ul>
</td>
<td style="width: 365px">
Voluntary way of designing experimental design with critical and independent way while discussing problem with other people
</td>
</tr>
<tr>
<td style="width: 236px">
Result Data (25)
</td>
<td style="width: 365px">
 
</td>
</tr>
<tr>
<td style="width: 236px">
<ul>
<li>Consistent Participation (5)</li>
</ul>
</td>
<td style="width: 365px">
How actively manage and update scientific results
</td>
</tr>
<tr>
<td style="width: 236px">
<ul>
<li>Process of acquiring scientific data (20)</li>
</ul>
</td>
<td style="width: 365px">
How critically and independently acquire data and interpret with scientific logic. Reason for think so and it’s clearance
</td>
</tr>
<tr>
<td style="height: 28px; width: 236px">
Evaluation (independent & Critical thinking) (15)
</td>
<td style="height: 28px; width: 365px">
Things woobeen Jo can learn from the genomics course

Way of writing scientific report
</td>
</tr>
<tr>
<td style="height: 9px; width: 236px">
Final presentation (20)
</td>
<td style="height: 9px; width: 365px">
presentation within time limitation with scientific problem and way of solving procedure
</td>
</tr>
<tr>
<td style="height: 9px; width: 236px">
Total (100)
</td>
<td style="height: 9px; width: 365px">
 
</td>
</tr>
</tbody>
</table>

Table 1. Grading standard

Criteria of grade: (%)

<table border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr>
<td style="width: 601px">
A+(95~100), A0(90~95), B+(85~90), B0(80~85), C+(75~80), C0(70~75), D+(65~70), D0(60~65), F (0-60)
</td>
</tr>
</tbody>
</table>

Table 2. Criteria of grade

 

After setting the criteria through SELF, total percentage of 5 criteria was 97.5% out of 100%. This is in the range of letter grade A+ through SELF.

 

2. Introduction

Obesity is a metabolic disease, which massiveness makes it a burning issue nowadays. At the end of the 20th century, the World Health Organization (WHO) declared even officially an obesity epidemic. It is estimated that there are currently more than one billion persons in the world having excess body weight, of which about 300 million persons are obese. ( Dr Ranđelović et.al. 2015) According to Wikipedia, definition of obesity is medical condition in which excess body fat has accumulated to the extent that it may have a negative effect on health.(Wikipedia_obesity) One of the problem is that obesity increases the likelihood of various diseases, particularly heart disease, type 2 diabetes, certain types of cancer, and inflammation. That’s why starting from leptin, many drugs were developed. However, because of certain side effects, new novel drug is needed. One of the novel protein that is discovered is Endotrophin. (Park, et.al. 2014) Endotrophin acts as pivotal role in shaping a metabolically unfavorable microenvironment in adipose tissue during consumption of a high fat diet. It causes fibrosis and inflammation and finally leading to insulin resistance and metabolic dysfunction. (Park, et.al. 2014) However, we don’t know how ETP works. Because of lack of information, woobeen Jo planned experimental design to find the interactors using Mass-Spectroscopy Technique. 

 

3. Experimental Design & Methods

 

3.1 Pre-study of scientific design 

 

Figure 1.a. Scientific experiment design & methods.

Once we treat mETP-SpyTag protein in to differentiated adipocyte, mETP will connect with unknown target protein. Because of covalent interaction between SpyCatcher-APEX2 (Reddington SC. et al. 2015), SpyTag will make covalent bond with SpyCatcher and finally mETP-SpyTag-SpyCarcher-APEX2 appear. Then, by treating DBP(Desthiobiotin-Phenol) and H2O2, DBP will become radical and biotinalize interactors. (Lam SS. et al. 2015) By using the technique of Mass-Spectrometry with streptavidin beads, (Ruedi. et al. 2016) we can enrichment unknown protein.

 

Figure 1.b. The targeted and untargeted workflow for LC/MS-based metabolomics 

With the result acquired from Mass-Spectroscopy, identify the function of unknown gene, their location (Ruedi. et al. 2016) and their difference between obese people and lean people. Through that way, we can use this information as one of biomarker of obesity.

Since one of the most important learning object of genomics course is independent and critical thinking performing science research, (Domask. et al. 2007) Experiment was designed to connect from proteomics and Genomics using Mass-Spectroscopy.

 

4. Result

4.1. Result Data 

 

Figure 2.a. Cloning of pRA-GFP-pRL-mETP-SpyTag-Stop-Xho1 (mammalian expression vector) and its sequencing result. (biolecture_20131571_woobeen_Jo)

By certain procedures mentioned on table 3, cloning of pRA-GFP-pRL-mETP-SpyTag-stop-Xho1 success by confirming either sequencing result or double digestion using EcoR1, Xho1 restriction enzyme.

<table border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr>
<td style="width: 406px">
Details of cloning plasmid
</td>
</tr>
<tr>
<td style="width: 406px">
(1)By Using pRL-EcoR1 forward primer, mETP-SpyTag-Stop-Xho1 primer, make pRL-EcoR1-mETP-SpyTag-Stop-Xho1 by Ex-Tag PCR
</td>
</tr>
<tr>
<td style="width: 406px">
(2) Insert template gained from (1) to T-Vector to check whether it is really pRL-EcoR1-mETP-SpyTag-Stop-Xho1 or not.
</td>
</tr>
<tr>
<td style="width: 406px">
(3) Use EcoR1, Xho1 Digestion enzyme to double digest T vector
</td>
</tr>
<tr>
<td style="width: 406px">
(4) Double Digest pRA GFP vector(empty vector) and extract & purify upper part.
</td>
</tr>
<tr>
<td style="width: 406px">
(5) ligate (3), (4) product
</td>
</tr>
</tbody>
</table>

Table 3. Details of cloning pRA_mETP-SpyTag plasmid (biolecture_20131571_조우빈)

 

Figure 2.b. Cloning of pTrc-Spe1-SpyCatcher-Xho1-APEX2 (E.Coli expression vector) and its sequencing data.

By digesting mETP form Spe1-mETP-Xho1-APEX2 and ligation of SpyCatcher between two restriction enzyme site (Spe1, Xho1), we made pTrc-Spe1-SpyCatcher-Xho1-APEX2 plasmid and confirmed by either double digestion using Spe1, Xho1 restriction enzyme or sequencing data result. (biolecture_20131571_조우빈)

 

Figure 2.c. mETP-SpyTag, SpyCatcher-APEX2 protein binding test

By treating mETP-SpyTag protein that is purified through size exclusion (biolecture_20131571_조우빈) and pTrc-SpyCatcher protein that is also purified through his column size exclusion (biolecture_20131571_조우빈) later, we can check the binding near 51kDa.

 

4.2. Further study plan 

We are going to do protein interaction experiment in cell to see whether two proteins are well connected in vitro. Then, by using Mass-Spectroscopy technique, find the protein that is correlated with mETP. In that way, identify unknown protein and its gene and their location. (Gary J. Patti. et al. 2012) After finding such information, analyze how different, how overexpressed, how different of unknown ‘protein’ gene between obese people to lean people by genome sequencing of certain region of unknown ‘protein’ gene.

 

5. Discussion

 

5.1. Attendance (10%)

 

Figure 3. attendance sheet and email from Professor Bhak.

9/2: No class        10/21: mid-term

9/30: No Class       11/18: No class

10/14: late           12/2: No class

 10/28: because of seminal, I got permission from park

 

Attendance is very important in grading standard because of its indicator on student's attitude. Except absent on 10/28 due to another seminal attendance, I attend every class. (Total: 9.5%/10%)

5.2. Pre-study of scientific Design (30%)

5.2.a Achieving goal (10%)

In performing science research, independent and critical thinking is very important. Likewise, self-study with motivation and purpose is important.

The ultimate goal is that apply identified protein information to genomics to find certain gene location and find the difference between obese people and lean people. To achieve the ultimate goal, motivation had been set to take the genomics into two. One is finding the connection between Genomics and Proteomics in obesity problem. The other one is to know how to think critically when designing my own experiment and correlate with Mass Spectroscopy technique.

The relationship between Genomics and proteomics are same in that Genomics is omics study of gene and omics is analyzing the interaction of biological information objection in various omes. Because proteomics also focusing on interaction point (biolecture_definition), Studying Proteomics is necessary to research and study the Genomics.

In order to critically design my own experiment and correlate with Mass Spectroscopy technique,

Study the basic concept of Mass-Spectroscopy and its application in terms of finding unknown gene location (Ruedi. et al. 2016) was needed.

<table border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr>
<td style="width: 301px">
 
</td>
<td style="width: 301px">
Credit (%)
</td>
</tr>
<tr>
<td style="width: 301px">
Motivation
</td>
<td style="width: 301px">
3/3
</td>
</tr>
<tr>
<td style="width: 301px">
Acknowledgement of problem
</td>
<td style="width: 301px">
3/3
</td>
</tr>
<tr>
<td style="width: 301px">
Learning
</td>
<td style="width: 301px">
4/4
</td>
</tr>
<tr>
<td style="width: 301px">
Total
</td>
<td style="width: 301px">
10/10
</td>
</tr>
</tbody>
</table>

Table 4. Details of Achieving goal section.

5.2.b Experimental design (20%)

I read 4 scientific papers and 4 review papers during this semester. Those papers are closely related with my own scientific experimental design idea. In order to find the way of interaction relationship, I read the novel technique SpyCatcher-SpyTag system. (Reddington SC. et al. 2015)

In order to find the effective way of finding the protein that is well biotinalize by treating biotin phenol and H2O2, I read APEX2 system. (Lam SS, et al. 2015) Also, by consulting with professor Lee, (professor of BIO UNIST) I was able to learn the detail process of dealing APEX2. Whenever I had questions related to obesity and experimental design (for example, reason for doing IPTG test before using cloned plasmid, is there difference between metabolic condition between world, and so on), asked to professor Park and ‘bric’. Through those kind of series of process, I made scientific experimental scheme. In order to think expand which cover both of Genomics and Proteomics, I read Omics, Proteomics, Genomics definition and their way of studying from biolecture. (Biolecture_definition)

<table border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr>
<td style="width: 301px">
 
</td>
<td style="width: 301px">
Credit (%)
</td>
</tr>
<tr>
<td style="width: 301px">
Study of Scientific paper and article and discussion with other people.
</td>
<td style="width: 301px">
5/5
</td>
</tr>
<tr>
<td style="width: 301px">
Voluntary way of designing experimental design and ask for advice from other people
</td>
<td style="width: 301px">
10/10
</td>
</tr>
<tr>
<td style="width: 301px">
Making experimental scheme with critical and independent way.
</td>
<td style="width: 301px">
5/5
</td>
</tr>
<tr>
<td style="width: 301px">
Total
</td>
<td style="width: 301px">
20%
</td>
</tr>
</tbody>
</table>

Table 5. Details of experimental design section.

 

5.3. Result Data (25%)

5.3.a consistent participation (5%)

 

Figure 4. Active users list in bio-wiki

In figure 3, woobeen, Jo’s edit number is higher than average people’s edits. This can be one of evidence that I consistently manage and update my page.

5.3.b Process of acquiring scientific data (20%)

I wrote an experimental paper per every week. Before doing experiment, I asked to myself about my plan of experiment scheme and way of doing and why I need this experiment according to scientific essay structure. During every experiment I discuss about the progress of projects with graduate student and professor. For every unexpected data, I always independently think the reason and search many website to support my logic. Then I raised many scientific questions and got feedback from other scientists. By having chance to learn from professor Lee’s lab for a while, I was directly and actively able to learn process of acquiring scientific data especially in biotinalizing process. Through this process, I can actively ask the science field and what scientific and creative question is. That is critical and independent thinking when performing science research.

Figure 5. Questions and Respond from professor Lee.

 

<table border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr>
<td style="width: 340px">
 
</td>
<td style="width: 261px">
Credit(%)
</td>
</tr>
<tr>
<td style="width: 340px">
Critical and independent way of solving unexpected problem
</td>
<td style="width: 261px">
5/5
</td>
</tr>
<tr>
<td style="width: 340px">
Way of interpreting data with scientific logic
</td>
<td style="width: 261px">
5/5
</td>
</tr>
<tr>
<td style="width: 340px">
How actively participate and joy experiment
</td>
<td style="width: 261px">
10/10
</td>
</tr>
<tr>
<td style="width: 340px">
Total
</td>
<td style="width: 261px">
20
</td>
</tr>
</tbody>
</table>

Table 6. Details of Process of acquiring scientific data section

 

5.4. Evaluation (15%)

Before starting my own experiment, I had question. what can I learn from genomics course?

In this question, my answer is Independent and critical thinking in science research. Through learning critical and independent thinking during experimental design of my experiment, I can study and think more about topic logically. Also, by getting more information especially in science from facebook, bric, it improved me way of critical thinking. Unlike previous, I become active on searching Bio-start up and actively questioned why they start Bio startup, what is their main target, and so on. Through consistent questioning to other scientific paper and science field, way of thinking and its process become independent and more importantly critical. Also, I was able to learn writing science paper. On my first meeting with professor Bhak, he pointed out the wrong way of writing scientific paper. After that, I once again search <a href="http://biolecture.org/">http://biolecture.org/</a> to find how to write scientific report. Finally, I was able to write this report using scientific report format and self-evaluation format.

 

<table border="1" cellpadding="0" cellspacing="0" style="width:614px">
<tbody>
<tr>
<td style="width: 200px">
 
</td>
<td style="width: 414px">
Credit(%)
</td>
</tr>
<tr>
<td style="width: 200px">
Close connection on scientific papers and articles.
</td>
<td style="width: 414px">
5/5
</td>
</tr>
<tr>
<td style="width: 200px">
Ask questions actively
</td>
<td style="width: 414px">
5/5
</td>
</tr>
<tr>
<td style="width: 200px">
Knowing of writing scientific paper
</td>
<td style="width: 414px">
5/5
</td>
</tr>
<tr>
<td style="width: 200px">
Total
</td>
<td style="width: 414px">
15
</td>
</tr>
</tbody>
</table>

Table 7. Details of evaluation section

 

5.5. Final presentation (20%)

I presented my topic obese in Genomics using SpyCatcher-SpyTag and APEX2 system. In the maximum 10 minutes limitation rule, I presented 8 minutes. During presentation, in order to present the scientific presentation logically, I focus on presenting scientific problem and its limitation on ETP. Then, I suggest the way of solving procedure with smooth presentation speed. However, I didn’t finish my data because of time limitation on this semester. Therefore, there are deduction on that category.

<table border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr>
<td style="width: 301px">
 
</td>
<td style="width: 301px">
Credit (%)
</td>
</tr>
<tr>
<td style="width: 301px">
Presentation within time limitation
</td>
<td style="width: 301px">
5/5
</td>
</tr>
<tr>
<td style="width: 301px">
Moderate presentation speed and well organized categories.
</td>
<td style="width: 301px">
3/3
</td>
</tr>
<tr>
<td style="width: 301px">
Finding scientific problem and way of solving procedure
</td>
<td style="width: 301px">
10/10
</td>
</tr>
<tr>
<td style="width: 301px">
Clearance of suggesting result
</td>
<td style="width: 301px">
0/2
</td>
</tr>
<tr>
<td style="width: 301px">
Total
</td>
<td style="width: 301px">
18/20
</td>
</tr>
</tbody>
</table>

Table 8. Details of final presentation section.

 

6. Conclusion

 

<Total Credit summary>

<table border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr>
<td style="width: 301px">
Grading standard (%)
</td>
<td style="width: 301px">
Credit (%)
</td>
</tr>
<tr>
<td style="width: 301px">
Attendance
</td>
<td style="width: 301px">
9.5/10
</td>
</tr>
<tr>
<td style="width: 301px">
Pre-Study of Scientific design
</td>
<td style="width: 301px">
30/30
</td>
</tr>
<tr>
<td style="width: 301px">
Result Data 
</td>
<td style="width: 301px">
25/25
</td>
</tr>
<tr>
<td style="width: 301px">
Evaluation (independent & Critical thinking)
</td>
<td style="width: 301px">
15/15
</td>
</tr>
<tr>
<td style="width: 301px">
Final presentation
</td>
<td style="width: 301px">
18/20
</td>
</tr>
<tr>
<td style="width: 301px">
Total
</td>
<td style="width: 301px">
97.5/100
</td>
</tr>
</tbody>
</table>

Table 9. Final credit through SELF.

My final points of total credit based on SELF is 97.5% out of 100%. This belongs to the A+ standard in Table 1

 

7. Reference

Achieving goals in higher education: An experiential approach to sustainability studies

<a href="http://www.emeraldinsight.com/doi/abs/10.1108/14676370710717599">http://www.emeraldinsight.com/doi/abs/10.1108/14676370710717599</a>

OBESITY - a Modern Lifestyle Disease

<a href="http://www.igalospa.com/blog/obesity-a-modern-lifestyle-disease.html">http://www.igalospa.com/blog/obesity-a-modern-lifestyle-disease.html</a>

Bhak, J. (2016, 6 13). Openfree biolecture. Retrieved from Openfree biolecture: <a href="http://biolecture.org/index.php/SELF:_Self_evaluating_learning_framework">http://biolecture.org/index.php/SELF:_Self_evaluating_learning_framework</a>

Endotrophin triggers adipose tissue fibrosis and metabolic dysfunction. <a href="https://www.ncbi.nlm.nih.gov/pubmed/24647224">https://www.ncbi.nlm.nih.gov/pubmed/24647224</a>

Adipocyte-derived endotrophin promotes malignant tumor progression

<a href="https://www.jci.org/articles/view/63930">https://www.jci.org/articles/view/63930</a>

Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher.

<a href="https://www.ncbi.nlm.nih.gov/pubmed/26517567">https://www.ncbi.nlm.nih.gov/pubmed/26517567</a>

Directed evolution of APEX2 for electron microscopy and proximity labeling.

<a href="https://www.ncbi.nlm.nih.gov/pubmed/25419960">https://www.ncbi.nlm.nih.gov/pubmed/25419960</a>

From genomics to proteomics (Nature Review)

<a href="http://www.nature.com/nature/journal/v422/n6928/full/nature01510.html">http://www.nature.com/nature/journal/v422/n6928/full/nature01510.html</a>

Innovation: Metabolomics: the apogee of the omics trilogy

<a href="http://www.nature.com/nrm/journal/v13/n4/abs/nrm3314.html">http://www.nature.com/nrm/journal/v13/n4/abs/nrm3314.html</a>

Mass-spectrometric exploration of proteome structure and function

<a href="http://www.nature.com/nature/journal/v537/n7620/abs/nature19949.html">http://www.nature.com/nature/journal/v537/n7620/abs/nature19949.html</a>

Definition of Proteomics

<a href="http://biolecture.org/index.php/Proteomics">http://</a><a href="http://biolecture.org/index.php/Proteomics">biolecture.org/index.php/Proteomics</a>

Definition of Omics

<a href="http://biolecture.org/index.php/Omics">http</a><a href="http://biolecture.org/index.php/Omics">://</a><a href="http://biolecture.org/index.php/Omics">biolecture.org/index.php/Omics</a>

Biolecture_20131571 조우빈

<a href="http://biolecture.org/index.php/20131571_%EC%A1%B0%EC%9A%B0%EB%B9%88">http://biolecture.org/index.php/20131571_%EC%A1%B0%EC%9A%B0%EB%B9%88</a>

Bhak, J. (2016, 9 4). Openfree biolecture: http://biolecture.org/index.php/How_to_write_a_scientific_essay%3F

 

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