Team:Evansville Central

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Team

The Evansville Central High School iGEM team consists of students in the Project Lead the Way Biomedical Innovations course. Evansville Central is the oldest high school in continuous operation west of the Allegheny Mountains. Evansville, Indiana is located in the southwest corner of the state. The city is positioned on a horseshoe bend of the Ohio River. Central High School is one of five public high schools the Evansville Vanderburgh School Corporation. The high school has 1300 students and is the only high school in Evansville with the complete 4 year Biomedical Science program.

Project

Our project is focusing on the incomplete metabolism of casein and gluten in the human digestive tract. Individuals with dairy intolerance are unable to consume foods containing milk or any milk product that contains casein. Individuals with gluten intolerance are unable to consume foods containing gluten such as wheat/flour products. Our goal is to research the isolation of the dipeptidyl peptidase 4 gene and create a BioBrick which will eventually produce a protein that will aid in the digestion of gluten and casein invitro.

Notebook

2/3/12 - First meeting date Assigned synthetic biology websites and previous iGEM competitors wikis to generate project ideas.

2/10/12 - Brainstormed ideas for our project

2/17/12 - Narrowed our topic human casein and gluten intolerance. Identified a protein of interest to assist in the metabolism of casein and gluten

2/24/12 - Research assignments: a. Normal Gluten metabolism b. Normal Casein metabolism c. Molecular structure of gluten and casein d. Mechanism pathway for DPP-4 (Dipeptidyl peptidase-4) e. Analogs for DPP-4 f. Effects of poor metabolism of gluten and casein Assigned articles for research

3/6/12 - Introduced the Mendely citation manager

3/16/12 - Created a shared Google document for all team members to access and share information from their research assignments from 2/24/12

3/23/12 - Ice cream party

4/4/12 - Figured out that DPP-4 is naturally produced in humans and in patients with Celiac disease DPP-4 maybe deficient. We are going to target DPP-4 for isolation from human cells.

4/18/12 - Trouble finding genomic sequences for DPP-4 and could only find whole genome shotgun sequences.

4/27/12 - Donut party. Team discussed other options for proteins in bacterial organisms that would breakdown casein and gluten. Team discussed the University of Washington's project in 2011 on gluten intolerance.

5/11/12 - Using NCBI genbank database in conjunction with the ensembl database we were able to figure out that DPP-4 genomic DNA was spread out over 60,000 base pairs with many large intervening regions between the coding sequences. Our team decided what our opinions were for continuing DPP-4 as our Bio-Brick. We determined the best course of actions was to amplify a 3000-4000 base pair region for DPP-4. Used Primer3Plus web tool to design primers for our genomic region of DPP-4. We designed 3 sets of primers which were ordered from IDT (Integrated DNA Technologies).

5/25/12 - Re-hydrated primer stocks to 100 micromolar. Made working primer solutions at 10 micromolar concentration. Autoclaved materials necessary for DNA extraction and PCR (microcentrifuge tubes, water, mineral oil, and pipet tips). Discussed project poster and created a google presentation document.

5/31/12 - Collected DNA samples from two people. Extracted DNA from cells using dneasy tissue kit (QIAGEN) following manufacturing protocols.

Results/Conclusions

Through research of current literature, the dipeptidyl peptidase IV is a human gene that produces an enzyme that could assist in the digestion of gluten and casein. We successfully located the gene on chromosome 2 using the National Center for Biotechnology Information Genbank and Ensembl databases. The size of the gene is determined to be over 60,000 base pairs in length including introns and exons. Due to the excessive length of the gene, we made an attempt to isolate a 4,000 base pair portion of the gene that included exon 1 and 2. We designed primers for the isolation process. Two human samples of genomic DNA were isolated using a Qiagen DNeasy Tissue Kit. Using three different nested primer sets, we amplified our desired products from the DNA samples. The products were analyzed using an agarose electrophoresis check gel.

Safety

1. Safety Procedures included standard DNA wet lab precautions including safety glasses, nitrile gloves, and aseptic techniques, i.e. autoclaving, cleaning benchtops with acetone and ethanol, and proper disposal of used items. 2. The safety procedures were supervised by instructors at all times.

Attributions

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Human Practices

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Fun!

Our favorite team snack is Russian sweet bread that is filled with fruit. Its called Peroshki. We also like to drink Mountain Dew and eat chips.

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