Team:GreenfieldCentral IN

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Official Team Profile

Chris Thompson is a senior at Greenfield-Central high school. He got involved with iGEM last year through his AP Biology instructor, Mr. Rihm. He participated in last year’s iGEM competition, and was on the winning team. He is also the Vice President of his senior class, the team captain of the varsity tennis team, the treasurer of National Honor Society, and is on student council. He is interested in synthetic biology, video games, hanging out with his friends, reading, and sleeping. But mostly sleeping. He is planning on attending Purdue University for Biological Engineering. He then plans on going to MIT to get his PhD there. His dream is to be a professor of biology at MIT and have his own research lab.

Bryce Herbert is currently a senior at Greenfield Central high school. He got involved in the iGEM team through his Biotech instructor, Mr. Rihm, and members of the previous year’s team. He is involved in extracurricular activities such as National Honor Society, Spanish Honor Society, and Voice at his high school along with being on the iGEM team. His interests include reading books listening to music, playing video games, being outside, and hanging out with friends. He plans to attend the College of Engineering at Purdue University to major in Biomedical Engineering. His plans thereafter are to begin work at a biotechnology company or firm to contribute to the new technologies and mechanisms to be used in the constantly changing and evolving field of medicine and technology.

Brent Poling is a senior at Greenfield-Central high school. He got involved with iGEM last year through his AP Biology instructor, Mr. Rihm. He participated in last year’s iGEM competition, and was on the winning team. He is also on the varsity soccer team and plays travel soccer. He is interested in reading, going to rock concerts, listening to music, video games, and hanging out with his friends. Brent is planning on attending Wabash College to study Biochemistry, and then plans on attending med school to study orthopedics. His dream is to be an orthopedic surgeon.

Emily Ditto is a senior at Greenfield-Central high school. She got into iGEM through some of the classes that she’s taking, such as Biotechnical Engineering and Engineering Design and Development. She is on the varsity swim team, National Honor Society, Women in Engineering, marching band, and Band Leadership Team. She likes swimming, hanging out with her friends, watching movies, listening to music, and sleeping. Emily is planning on attending Purdue University to study Biomedical Engineering, and is thinking about going to med school after she graduates from Purdue.

Andrew Gutierrez is a freshman at Greenfield Central high school. He joined iGEM after his Biomed teacher invited him to join; this is his first year of iGEM. He is a running back for the JV football team, a member of pathfinders (a youth leadership group), and a member of the choir. He is interested in football, baseball, videogames, TV, music, and hanging out with friends. Andrew doesn’t know where he wants to attend college yet, or what he wants to major in, but he knows it will have to do with science.

Mason Keller is a junior at Greenfield-Central High School. He got involved in iGEM because of his AP Biology teacher Mr. Rihm and his involvement in the school’s biotechnical engineering class. Mason is a state champion in the Vex Robotics program. His interests are the classics and mathematics, which he plans to double major in at Wabash College.

Tyler Girton is a sophomore at Greenfield-Central high school. He plans on going to Butler University and major in pharmaceuticals. He began iGEM after it was recommended to him by his biology teacher, Mr. Rihm. Tyler also volunteers at a soup kitchen, and other hobbies and interests to him are politics, hanging out with friends, music, and bowling.

Rachel Larsen is a sophomore at Greenfield-Central. This is her first year on iGEM, but is very excited to continue in the program. Rachel is also involved in Academic Superbowl, VEX Robotics, Drama Club, Junior Statesman of America, and 4-H. She is very interested in photography, physics, neuroscience, and psychology. She is planning on either attending Capitol University in Columbus, Ohio or Indiana University- Purdue University Indianapolis, Indiana to major in applicative neuroscience research.

Brainstorming

The first idea that we looked at was a cancer detector. We would try to create a strain of E. Coli that could detect the growth factor HER2/neu, a chemical that is present in many types of cancer. The E.Coli would work in the blood stream to detect and locate malignant cells, providing for an early detection system for cancer..

The second project that we looked into was a cure for amyloid fibril-related diseases. Amyloid fibrils are small protein fibrils that are associated with many diseases such as Parkinson’s and Alzheimer’s Disease. Our project would have prevented the mis-folding of the protein that creates amyloid fibrils.

Another idea we had involved Galactosemia. The Gal genes in yeast are used for galactose breakdown and regulation, so galactosemia is when the body cannot produce the enzyme GALT to break down galactose for use by the body. Our idea was to create a strain of yeast that would produce an enzyme that could be injected into the bloodstream to break down galactose.

And lastly, an idea that was thought of by the Biomedical side of Project Lead the Way was a fish tuberculosis detector. Fish tuberculosis is undetectable in aquatic environments, and is currently untreatable due to mycolic acid on the membrane of the virus. Our idea was a strain of E. coli that would detect the mycolic acid on the cell membranes, and maybe even dislodge it to make the pathogen treatable.

Project

Our first project deals with Galactosemia. Galactosemia is a disease in which an afflicted person cannot break down galactose, a simple sugar found in many food items such as milk. Classic galactosemic individuals cannot effectively produce the enzyme GALT, which is needed to catalyze the breakdown of galactose. Galactose build-up can lead to many debilitating conditions, such as ataxia, liver failure, and learning disabilities. Our goal for this project is to create a blood-galactose monitor to help galactosemic patients monitor their condition. This is needed because current blood sugar monitors only detect both sugars together, which does not help galactosemic people. We are planning on creating a strain of yeast that can detect galatose and glucose separately in the bloodstream. We are planning on using the promoters Gal1/10 to detect galactose, and HXT1 to detect glucose. Then we are going to use the mCherry fluorescent protein and cyan fluorescent protein to indicate the concentration of both sugars. Once we assemble our plasmid and transform it into E. coli to amp up DNA concentration, we will then transform it into yeast. We are also going to characterize the promoters by testing the fluorescence when introduced with different sugar concentrations. This will help standardize the test and make it easier to use. This project will hopefully be a simple test for galactosemics to monitor the status of their condition.

Our second project focuses on the disease in fish called Mycobacterium Marinum. Mycobacterium is a strain of tuberculosis. Mycobacterium Marinum kills large masses of fish, mainly in aquariums, and is currently undetectable. This disease also affects humans; if a human has an open sore and comes in contact with the contaminated water, the human will then be a carrier of mycobacterium Marinum and could possibly infect other aquariums. Our project is to find the structure of Mycobacterium Marinum and have E. coli target mycolic acid, mycobacterium's defense mechanism to current vaccines and antibiotics. When our engineered E. coli detects the mycolic acid on the cell wall of the mycobacterium, the green fluorescent protein gene we will insert into the plasmid will indicate that disease is present in the aquarium. If this step is successful, then we will take the project one step further. We will attempt to engineer E. coli to target an enzyme on the cell wall of Mycobacterium and have it release the mycolic acid so the mycobacterium will become defenseless to current treatments. In the broad view, if we are able to detect Mycobacterium through the use of fluorescent proteins, then the actual test for tuberculosis today could be simplified and made cheaper compared to the current process. A blood sample would be taken from the patient, and exposed to our E. coli. A spectrophotometer would then calculate the fluorescent levels of the sample, and determine if the patient has tuberculosis.

Procedures

PCR Extraction

Add the following to a fresh PCR tube:

1. 2.5 uL 5' Primer

2. 2.5 uL 3' Primer

3. 5 uL Thermopol Buffer

4. 1 uL dNTPs

5. 33.5 ddH2O

6. .5 uL DNA Polymerase

Gel Elctrophoresis

1. Add the following to an Eppindorf tube:

a. 5 uL of the extracted PCR part

b. 15 uL of distilled water

c. 5 uL of PV92 Xylene Cyanole Loading Dye

2. Using a micro pipette, dispense the mixture into a well of the gel.

3. Run the gel until the bands of DNA move to the middle of the gel.

4. Stop the gel and compare the band to the DNA ladder, to find if they are the right length.

5. If it is

Notebook

September 28th, 2011 - Had the first call-out meeting for iGEM.

October 5th, 2011 - Formed plans for attending the 2011 American Collegiate Regional Jamboree.

October 9th and 10th - Volunteered at the 2011 collegiate jamboree, gathered brainstorming ideas for project.

November 9th, 2011 - Met after a temporary hiatus, discussed brainstorming ideas such as cancer detector and amyloid fibrils.

November 16th - Assigned research groups to study the two projects.

November 23th - Came together to discuss the research papers, and assigned research summaries.

November 30th - We shared our research summaries, and decided that both projects were a little above what we would be able to do this year with iGEM. So it was

back to the drawing board to find projects.

December 14th - Some students shared project ideas, such as the problem of galactosemia and the detection of tuberculosis in fish. We told everyone to look

up research papers about these projects.

January 4th - After Christmas break, we shared the knowledge that we gained from the research we did. We decided that we could do both projects, and have two

groups within our team. One would detect the GALT enzyme in blood for galactosemics, and one would detect mycolic acid in aquariums for fish

tuberculosis.

January 11th - Our galactosemia group found a promoter called Gal1, which we could use to detect GALT in blood. This will help galactosemics monitor the

seriousness of their galactosemia. Our tuberculosis group found a promoter called mmaA2. This promoter detects mycolic acid, which is a defense

mechanism on the cell membrane of tuberculosis.

January 18th - Today we decided the vectors that we would use for our systems. Our galactosemic group is going to use yeast because the Gal genes are in yeast.

The tuberculosis group is going to use E.Coli, because it will survive better in an aquatic environment.

February 1st - We looked at reporters, and how we want to indicate with our promoters. As much as we want to do something complicated and impressive, we decided

on simple fluorescent proteins due to their ease and availability.

February 8th - Our galactosemic group contacted the author of one of the papers that they read, Dr. Fridovich-Keil of Emory University. She informed us that the Gal

promoters would not let us detect GALT, so that did not bode well for our project. But she also informed us that one thing clinicians need is a way to

detect galatose in the blood, since current blood sugar tests detect all types of sugar, not just galactose. We decided to change our project to that, and

still use the same Gal genes. We asked her if she could send us the promoter Gal1/10, so we would not have to extract it ourselves.

February 15th - In this meeting we practiced our presentation for the Chamber of Commerce meeting. We finished the presentation and ran through it a few times.

February 22nd - We looked at the procedures for part extraction and restriction enzymes, and went over them to know what to do when we started lab work.

February 29th - We created a list of materials we needed to buy, such as labcoats, rubber gloves, Restriction Enzymes, Phusion Polymerase, CaCl2, NDNTP, Ligase,

Exonuclease, Agarose, YPD, Yeast, and E. Coli. Hopefully we can get donation money from the Kiwanis Club presentation in a few days.

March 7th - Today we talked some of our newer members through the extraction procedures, and how primers work to extract parts from a plasmid.

March 12th - We went over the presentation to the Greenfield Area Kiwanis Club. We found out that one of the members of the club donated 2,000 dollars to fund our

project this year, and another individual donated 200 dollars!

March 21th - We recieved our promoters from Dr. Fridovich-Keil and researched the exact sequence of the Gal1/10 promoter so that we can make primers. We made

the 40 base pair primers and ordered them so we can extract the promoter from the plasmid.

April 3rd - Today we went over the activity that we are going to do with the 5th graders. They will put three strips of paper together and tape them together. The three

strips of paper represent a promoter,a translational unit, and a selective marker. The tape represents a sticky end created by restriction enzyme sites.

Through this activity, the kids will learn all about synthetic biology and will have fun doing it.

April 4th - Today we re-suspended the plasmids that Dr. Fridovich-Keil sent us. We spun the tubes in the centrifuge to make a pellet, then we dried out the excess

liquid. After that, we added 100uL of TE buffer to re-suspend it.

April 11th - We were supposed to get our primers today, but they did not arrive yet. We poured gel electrophoresis plates instead, to prep for tomorrow's lab. We put 2

grams of the agarose into 200 mL of distilled water, boiled it, poured the gels, and then let them set. Hopefully we will get our primers and we can extract

our gal1/10 promoter out of the plasmid that Dr. Fridovich-Keil sent us.

April 16th - Today we finally received our primers, which we then re-suspended in distilled water.

April 17th - Today we started extracting the promoter from the plasmid. We used a PCR machine to run the extraction. After the extraction was completed, we ran the

resulting DNA on a gel. Sadly, the DNA streaked across the gel and we could not get an accurate reading.

April 18th - We attempted to redo the gel electrophoresis that failed yesterday with the DNA that was still left over from the extraction. Once we finished it, we could not

see any visible bands in the gel. We used the last of the DNA, so we will have to redo the extraction procedure.

April 25th - We re-did the PCR extraction and then used the DNA in a gel. We unfortunately made the gel too dense and thick, so the DNA did not travel through it. We

used all of our DNA in this gel, so we will have to redo the extraction procedure once again. We do not have enough primer DNA or plasmid DNA to do

another extraction, so we will have to ask Dr. Fridovich-Keil for more plasmid DNA and order more primers.

April 30th - We talked to Dr. Fridovich-Keil, and she told us that we could use what little plasmid DNA we had left and insert it into an E.coli sample, which would then

be purified using a selection marker. Then we would have an infinite supply of the plasmid. We knew that you could do this, we just didn't think about

attempting it. We don't have amoxicillin for the selection marker though, so we will have to order that.

May 21st - We hosted a last school meeting to discuss our summer schedule. Our instructors are going to be at Purdue for the first week of June, so that's when we

will work on our poster and presentation.

May 29th - We met at Mason's house and started brainstorming about our presentation. We looked at the college presentations and posters from last year, and got

some good ideas. We set up the outline for our presentation and started making the slide graphics in Photoshop.

May 30th - We got together at Mason's house again and worked on the poster. We got a good design for it and started working on the text and graphics.

May 31st - We met at the school today and continued to work on the poster and presentation. The presentation is completely outlined and the presenters have started

their own specific parts.

June 4th - We continued to work on the poster and presentation.

June 6th - We once again worked on the presentation and poster, and updated the wiki.

Results/Conclusions

Attributions

Galactosemia Group: Chris Thompson, Brent Poling, Mason Keller, Bryce Herbert, Emily Ditto, Andrew Gutierrez, Tyler Girton, Rachel Larson.

Tuberculosis Group: Meg Summers, Taylor Titus, Rhi Teeter, Taylor Kennedy, Marin Young.

Human Practices Group: Chris Thompson, Brent Poling, Meg Summers, Taylor Titus, Mason Keller, Bryce Herbert, Marin Young.

Software Group: Chris Edge, Chad Williamson.

Safety

Safety Rules:

1. Safety gear must be worn at all times in the lab. Safety equipment includes: goggles, a lab coat, and safety gloves before participating in the lab.

2. There is a red line separating the computer area from the lab area. If you cross this line, you must wash your hands and change your gloves.

3. No food or drink in the lab.

4. Dispose of any contaminated lab equipment in the proper biohazard container.

5. Make sure your lab area is clean before starting a lab.

5. Sterilize all equipment before starting a lab.

6. No horseplay in the lab.

7. Make sure a teacher or adult is supervising while in the lab.

8. Clean all lab equipment and surfaces when finished in the lab.

9. Record everything that takes place in the lab in your lab journal.

Any other restrictions or rules can be found in Bio Safety Level 1 Guidelines.

Human Practices

So far, we have presented to the Greenfield Area Chamber of Commerce, and educated them on the positive impacts of iGEM and synthetic biology on students, the economy, and the world. There were over 100 Greenfield businesses there that heard our presentation, and many of them did not know what synthetic biology was before we presented.

We have also presented to the Greenfield Kiwannis Club and gave a similar presentation about the effects of synthetic biology, and informed them on the positives of synthetic biology. In this presentation, we asked for funds to support our lab work and safety materials. One of the members donated 2,000 dollars to our iGEM team, and we can not wait to buy our new lab coats and team shirts.

We then did an activity with the 5th graders at the Greenfield Intermediate School that taught them how we created plasmids and put them into a cell. The students learned about DNA, restriction enzymes, promoters, translational units, selective markers, and how we transform the DNA into a cell. Many of them were very excited about the project, and said that they wanted to do this when they grow up.

Lastly, we presented to the Greenfield-Central School Board about iGEM and what we do. We actually had them do the activity like the 5th graders, but went more in-depth. There were very surprised at how much we knew, and were very supportive of the work that we are doing.

Fun!

References

1. Cozzo, B. (2005). Galactosemia 101: An Introductory Overview. [Powerpoint slides]. Retrieved from http://www.galactosemia.com/resources/galactosemia_101.pdf

2. Elsas, L.J. (2000). Classic Galactosemia, GALT Deficiency, Galactose-1-Phosphate Uridyltransferase Deficiency. Includes: Variant Galactosemias. GeneReviews

[Internet].

3. Fridovich-Keil, J.L., & Jinks-Robertson, S. (1993). A yeast expression system for human galactose-l-phosphate uridylyltransferase. Proceedins of the National

Academy of Science of the United States of America, 90, (398-402).

4. Ko, D.H., Chang, H.E., Song, S.H., Park, K.U., Kim, J.Q., Kim, M.C., Song, Y.H., Hong, Y.H., Lee, D.H., and Song, J. (2010). Molecular and biochemical

characterization of the GALT gene in Korean patients with galactose-1-phosphate uridyltransferase deficiency. International Journal of Clinical Chemistry, 20,

(1506-10).

5. Lai, K., Langley, S.D., Khwaja, F.W., Schmitt, E.W., and Elsas, L.J. (2003). GALT deficiency causes UDP-hexose deficit in human galactosemic cells.

Glycobiology, 13, (285-94).

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 <p>3. Run the gel until the bands of DNA move to the middle of the gel.</p>
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