Ross School - Senior Projects 2011

 

Student: Kisu Shin

Mentor: Carleton Schade

Domain(s): Science

 

 

 

Product                            

 

Title: Research Techniques of Protein Crystallization for Serum Response Factor

Description:

For my senior project, I worked at a Lab on Protein Crystallization and I wrote an essay on it. The lab experience gave me a good sense of idea on what research truly means. Lab work is made up of 99% failure and 1% success. I tried my best to write my essay so that anybody could read the essay and understand what my work is about. As I was writing this essay I learned more about the molecular biology, DNA, and Protein.

Details:

 

Paper

 

PowerPoint

 

Abstract

 

It was my junior year winter when I first heard about the senior project. I played duet piano piece with Angela during the Modernity Project but I wasn’t sure whether wanted to do another music piece from my senior project.

Right before the summer break in 2010, it came to my mind that I would like to do something that is related to biology. I started talking to Ms. Costello about it. She gave me some ideas on what I could go for. She told me there was a laboratory place near the school that I could make a visit and sign up for an internship. My father in Korea told me he could give me an experience similar to that in Korea.

On June 19th, I made a visit to Chosun University in Korea. There, I met professor Seung-Haeng Lee. He told me there are many laboratory experiences I could have. By then, I was reading my summer reading for English class, Never Let Me Go. That book made me want to do something that was related to cloning. I asked him if I could do something on cloning. He introduced me to the fields of protein crystallization, which requires gene cloning.

June 24th was my first laboratory day. I had a lab partner that helped me out whenever I did not understand something. I received cDNA strands in a small tube from Prof. Lee and that is how my laboratory experience started.

Because I lived about an hour and half away from the university, I usually left the house around 7 in the morning and got back home at around 9. I started learning how to use different tools and different methods that I was going to need.

Step 1 was Polymerase Chain Reaction (PCR). With the cDNA strands that I received from the professor, I initiated the PCR. For PCR, I had to mix primers, pfu, buffer, dNTP, cDNA strands and water. The mixed liquids were then placed in an automated heating machine. The machine was set to heat the liquid up to certain temperature and cool it back down to certain temperature. The buffer made liquid’s acidity stable so that the DNA would not be damaged by unexpected acidity level. While the liqid was heated up, the cDNA strands are untangled by pfu and broken up to two DNA strands. Then primers go in between, to make the other half of the DNA strands making the two broken up DNA strands to two full DNA strands. dNTP is the nucleotides that makes up the other halves of DNA strands.

Step 2 was the PCR Purification, which was initiated a day after PCR. After the PCR, the tube not only had the wanted DNA segments but it also contained nucleotides, primers, pfu, and buffer that were unwanted for the next step and therefore needed to be removed. By adding certain chemicals (specified in methodology section) into the tube, the unnecessary ingredients break down and pass through the filter. After all the liquid has gone through the filter, the DNA segments are the only things left.

Step 3 was to check whether the DNA segments remaining in the tube were the correct one (that is the DNA segment that coded for the serum response factor of Mus musculus). To check if the correct DNA segment had been sequenced, Agarose gel electrophoresis was used according to the method that has been used by Phil Sharp, Joe Sambrook and Bill Sugden at the Cold Spring Harbor laboratory in 1970s (10). Agarose gel electrophoresis is used to separate DNA segments by size. It uses an electric current to push molecules down the gel. The smaller molecules move down the gel faster than the bigger molecules so that eventually, after a certain period of time, all the different sizes of molecules are sorted down the gel.

Step 4 involved Enzyme Cutting. Enzyme cutting is used to cut the DNA strand so that the DNA strand in the future can be ligated with the vector to form a plasmid. A vector is a small piece of circular DNA strand (1). The segmented and duplicated DNA strands from PCR must be inserted into a vector to form a plasmid. A plasmid is a circular DNA strand that is very small compared to the chromosomes.  The enzymes (specified in methodology section) cut the end of the DNA strands, allowing other DNA strands to be attached to the vector.

Step 5 was Enzyme Cutting for a vector. The vectors were cut with the same enzyme that cut the DNA strand in step 4.

Step 6 was DNA purification from gel. Using a fresh block of Agarose gel, DNA purification from gel allowed me to clean everything out of the EP tube except for the wanted DNA and the vector. I used Agarose gel to purify both the DNA strand and the vector. The same methods were used as the Agarose gel electrophoresis in step 3. The DNA strands and vectors were mixed with a dye that allowed the DNA strands and vectors to be highlighted under UV light. Under UV light, highlighted parts of the gel were cut out with a knife. After the gels were cut out the GB buffer liquefied them.

Step 7 was Ligation. Ligation allows the DNA strand and vector to form a plasmid. We need to form a plasmid because during asexual reproduction, E.coli will not reproduce the DNA strand unless the DNA strand is in a plasmid form. Plasmids are easily inserted to E. coli.

During the Ligation, the nutrition broth that contained E. coli started forming molds so I had to restart the process.

Step 8 was Transformation. On a jellified nutrition plate, small amount of E. colis were spread evenly. After 8 hours, certain parts of the gel became bumpy. Those bumpy places were where E. coli started reproducing itself. With a tip of a pipette, two lumps were scratched off and they were placed in a liquefied nutrition plate.

Step 9 was Protein Expression. During the protein expression, the proteins that had been made according to my inserted DNA segments were extracted and ready to be crystallized.

            Once these nine steps were done, the proteins were crystallized. During the Ligation (step 7), I lost one of the samples and when the DNA segments were sent to be sequenced, only one showed up with right genetic information. By the time I was crystallizing protein I only had one sample. Proteins were crystallized twenty-four times however, only one of them succeeded to form a crystal.

            On August 3rd, the crystal was sent to a crystallographer to be mapped out and I left Korea to come back to Korea on September 9th.

            When I got back to the school, I had to be assigned to a new mentor because Ms. Costello had left the school during the summer break. My new mentor was Mr. Schade. He gave me two choices of what I could do with my Laboratory experience. One was to write an essay and the other one was to make a poster. I knew my English is still poor and I was going to go to college in United States, so I decided to write an essay and improve my English skill. Mr. Schade agreed with my decision and he said he could help me with English. He revised my essay 7 times in total. About half way through, when I had 4 revisions done, the paragraphs still did not seem like they were in order. He told me to have subheadings to be more organized. I also cut all the paragraphs out to move them around and put them in order. I also learned APA form of citation from Ms. Parkes because my paper was a scientific paper.

            On February 16, I ended my paper with 37 pages. Mr. Schade finally gave me the final approval to present.

 

 

Works Cited

 

(1) Cloning Vectors [internet]. [Updated in 1998]. Sofer WH. DW Brooks Site;

[cited 08 Dec. 2010]. Available from

http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer/cloningvectors.html

 

(2) Kraus D. 1984. Concepts in Modern Biology: fifth edition. New York. Globe Book Co.

 

(3) E. coli Genome Project [internet]. University of Wisconsin-Madison

[cited 24 Dec 2010]. Available from

http://www.genome.wisc.edu/

 

(4) Campbell RM. 1999. Biology: fifth edition. Illinois. Benjamin Cummings

 

(5) Introduction to Protein Crystallization [internet].

[cited 6 Jan 2011]. Available from

http://www.google.com/url?sa=t&source=web&cd=9&ved=0CHkQFjAI&url=http://www.mitegen.com/products/jen_screens/Background_Information.pdf&rct=j&q=protein%20crystallization&ei=lsVMTc1nwbzyBsy6uO4O&usg=AFQjCNEHY-DrA4V82I8s44QZSSKZ40dXiw&sig2=FbRQTO9pkVhM6FGkUN

 

(6) Molecule of the Month - DNA Ligase [internet]. DNA Ligase. RCSB Protein Data Bank

[cited 08 Dec. 2010]. Available from

http://nist.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb55_1.html

 

(7) Molecule of the Month - Restriction Enzymes [interent]. Restriction Enzyme. RCSB Protein Data Bank

[cited 08 Dec. 2010]. Available from

http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb8_1.html

 

(8) Protein Data Bank [interent]. Understanding PDB Data: Looking at Structures

[cited 12 Feb. 2011] Available from

http://www.pdb.org/pdb/static.do?p=education_discussion/Looking-at-Structures/intro.html

 

(9) Milestone 2 : Nature Milestones in DNA [interent]. Nature Publishing Group : Science Journals, Jobs, and Information.

[cited 08 Dec. 2010]. Available from

http://www.nature.com/milestones/miledna/full/miledna02.html

 

(10) Gel Electrophoresis [internet]. Biology Animation Library. Dolan DNA Learning Center.

[cited 12 Dec. 2010]. Available from

http://www.dnalc.org/resources/animations/gelelectrophoresis.html

 

(11) DNA Purification - DEAE Nitrocellulose Membranes [internet]. Lawrence J. Roth Lab;

[cited 13 Dec. 2010]. Available from

http://rothlab.ucdavis.edu/protocols/P01.html

 

(12) Gene Sequencing [internet]. Gene Sequencing.

[cited13 Dec. 2010]. Available from

http://www.genelearning.com

 

(13)History of Genetics [internet]. THE MEDICAL NEWS | from News-Medical.Net - Latest Medical News and Research from Around the World. 

[cited 13 Dec. 2010]. Available from

http://www.news-medical.net/health/History-of-Genetics.aspx

 

(14) Brown TA. 1996. Gene Cloning: an Introduction. London. Chapman and Hall

 

Outside Consultant

 

Professor Seung-Haeng Lee. He is a professor at Chosun University. He crystallizes protein at Chosun University.