The University of Illinois iGEM 2013 Team Presents... - PowerPoint PPT Presentation

The University of Illinois iGEM 2013 Team Presents...

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Cardiovascular Disease (CVD) is Responsible for 30% of Deaths Worldwide Cardiovascular disease 30%

L-carnitine from Red Meat and Energy Drinks Atherosclerosis CARDIOVASCULAR DISEASE http://www.chemspider.com

Could you resist? http://www.burgerbusiness.com/wp-content/uploads/Hardees_MemphisBBQ1.jpg

L-carnitine Food Trimethylamine (TMA) Gut Trimethylamine N- oxide (TMAO) Liver http://hcgdietdrop.net/wp-content/uploads/2012/06/zzzzzz.png http://www.chemspider.com http://www.westonaprice.org/blogs/cmasterjohn/files/2011/04/Hyp

Our Goal: To create a probiotic that prevents Cardiovascular disease by outcompeting native gut bacteria for L-carnitine.

Gut Gut Liver Liver TMAO TMA http://www.chemspider.com

source: Source:h http://ngm.nationalgeographic.com/2013/01/microbes/img/05-intestinal-bacteria-670.jpg ttp://www.gastropatienteducation.com/Gatro_Pat/Pat_Ed_web_pages/images/ small_intestine.jpeg + L-Carnitine Trimethylamine (TMA) Take Up and Break Down

Take Up ● CaiX- L-carnitine specific binding CaiX protein L-Carnitine ● CBCwv- Active transporter for L-carnitine CBCwv https://wikispaces.psu.edu/download/attachments/46924783/image.jpg

Break Down Current Pathway Current Pathway L-carnitine L-carnitine Carnitine Dehydrogenase Trimethylamine 3-dehydrocarnitine Trimethylamine-N-oxide http://www.chemspider.com

CbcWV CaiX CDH cdhBAC cbcWV caiX

E. coli Nissle 1917 Source: http://www.mutaflor.com.au/wp-content/uploads/ 2012/04/pack-shot_opt.jpg

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Characterization of Uptake System with Liquid Chromatography Mass Spectroscopy (LCMS) Ready for LCMS testing! http://ian.umces.edu/imagelibrary/displayimage-topn--92-5469.html http://biowake.com/sample-storage/storage-tubes/microcentrifuge-tube/ http://www.nextdayscience.com/06ml-hercules-tube-p-1089.html?osCsid=03n1pc2e6ugl6ema7icmp84av4

LCMS: Empty Backbone Comparison Initial and Final

LCMS: Concentration Comparison at initial and final time point. This drastically lower concentration of L-carnitine at the final time point shows that our protiens are functional.

Characterization of Breakdown System http://ian.umces.edu/imagelibrary/displayimage-topn--92-5469.html http://www.nextdayscience.com/06ml-hercules-tube-p-1089.html?osCsid=03n1pc2e6ugl6ema7icmp84av4

L-carnitine Breakdown using Carnitine Dehydrogenase NAD + NADH CDH L-carnitine 3-dehydrocarnitine http://www.chemspider.com

Spectrophotometric Results Greater rates of NADH accumulation in cytoplasmic extracts from cells containing our biobricks shows that our enzymes are functional. CDH CDH Empty Backbone Empty Backbone L-carnitine Control L-Carnitine Control

Delivery to the Small Intestine Goals ● Surpass Stomach ● Multiple methods of delivery http://www.xtend-life.com/popup/info/Enteric_Coating.aspx

Delivery to the Small Intestine Enteric Coated Gel Capsule Pill in pH 2 with Coating Pill in pH 2 without Coating at time 1hr at time 5min

Alginate Gel http://chaos-its-not-just- a-theory.com/ 2012/02/02/from-slime- to-gel/

Human Practices ● Developed a class for the local G.A.M.E.S. bioengineering summer camp ○ Girls’ Adventures in Math, Engineering, & Science ○ Offers high school girls a chance to experience math, engineering, and science at a high level

Human Practices ● Institute for Genomic Biology’s Pollen Power Camp ● Began providing guidance to local high schools for starting their own team in 2014

Achievements ● Successfully showed activity in take-up and degradation of L-carnitine ● Modified probiotic actually will grow in the gut flora and process L-carnitine We have designed a working viable preventative solution to cardiovascular disease!

Future Implications ● Public awareness and relations ○ Genetically modified products ○ Nissle ● Integration of parts into genome ● Extensive testing required ○ Competition experiment ○ Testing on mice ○ Testing on human subjects ○ Seek FDA approval

Bronze Requirements ● Completed all registration and paperwork ● Developed a team wiki page ● Designed three new biobricks ○ BBa_K1205000 - Carnitine Dehydrogenase ○ BBa_K1205001 - CaiX Carnitine Uptake Carrier ○ BBa_K1205002 - CbcWV Carnitine Uptake Protein ● Competed and presented a poster here at the jamboree!

Silver Requirements ● Experimentally validated and documented multiple biobricks ● Submitted part BBa_K1205000 to the registry ● Safety & Health: ○ Nissle ○ Biobricks do not present valid threat in bacterial cells ○ Of course, before this product heads to market it must be extensively tested first

Gold Requirements A Novel Approach to Safety: Ethidium Bromide (EtBr) with Food Dye ● Most low-budget biology labs use EtBr ● EtBr is a strong and dangerous carcinogen ● EtBr is often dilute ○ clear color ○ difficult to detect upon splashing onto surfaces SOLUTION Color EtBr with Food Dye

Thank you to our advisors! Soo Rin Kim Ahmet Badur Energy Biosciences Institute Chemical & Biomolecular Engineering Uros Kuzmanovic Professor Kaustubh Bhalerao Molecular & Cellular Biology Agricultural & Biological Engineering Professor Ting Lu Bob Chen Bioengineering Molecular & Cellular Biology Melissa McKillip Institute for Genomic Biology Kori Dunn Chemical & Biomolecular Engineering Professor Chris Rao Chemical & Biomolecular Engineering Todd Freestone Chemical & Biomolecular Engineering Cara Schornak Molecular & Cellular Biology Michelle Goettge Divya Tankasala Microbiology Bioengineering Professor Yong-Su Jin Food Science & Human Nutrition

A very special thanks to our audience and sponsors!