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

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

Oct 05, 2023 174 likes •533 views

The University of Illinois iGEM 2013 Team Presents... http://i.ebayimg.com/t/Unisex-Bathroom-Sign-MEN-WOMEN-White-sticker-decal-8-x-8-/00/s/NjUwWDY1MA==/$( Cardiovascular Disease (CVD) is Responsible for 30% of Deaths Worldwide Cardiovascular

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The University of Illinois iGEM 2013 Team Presents...

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SLIDE 2 http://i.ebayimg.com/t/Unisex-Bathroom-Sign-MEN-WOMEN-White-sticker-decal-8-x-8-/00/s/NjUwWDY1MA==/$(
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Cardiovascular Disease (CVD) is Responsible for 30% of Deaths Worldwide

Cardiovascular disease 30%

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Atherosclerosis

CARDIOVASCULAR DISEASE

Red Meat and Energy Drinks L-carnitine from

http://www.chemspider.com
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Could you resist?

http://www.burgerbusiness.com/wp-content/uploads/Hardees_MemphisBBQ1.jpg
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L-carnitine Trimethylamine N-

  • xide

(TMAO) Trimethylamine (TMA)

Food Gut 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
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To create a probiotic that prevents Cardiovascular disease by outcompeting native gut bacteria for L-carnitine.

Our Goal:

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Gut Liver

http://www.chemspider.com

TMA TMAO Gut Liver

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+ L-Carnitine Trimethylamine (TMA)

Take Up and Break Down

Source:h ttp://www.gastropatienteducation.com/Gatro_Pat/Pat_Ed_web_pages/images/ small_intestine.jpeg source: http://ngm.nationalgeographic.com/2013/01/microbes/img/05-intestinal-bacteria-670.jpg

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L-Carnitine

https://wikispaces.psu.edu/download/attachments/46924783/image.jpg

CaiX

  • CaiX- L-carnitine

specific binding protein CBCwv

  • CBCwv- Active transporter

for L-carnitine

Take Up

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Break Down

http://www.chemspider.com

Carnitine Dehydrogenase

Current Pathway L-carnitine 3-dehydrocarnitine L-carnitine Trimethylamine Trimethylamine-N-oxide Current Pathway

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cdhBAC cbcWV caiX

CDH CbcWV CaiX

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  • E. coli Nissle 1917

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

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+

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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
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LCMS: Empty Backbone Comparison Initial and Final

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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.

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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
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L-carnitine Breakdown using Carnitine Dehydrogenase

L-carnitine

NAD+ NADH

3-dehydrocarnitine

CDH

http://www.chemspider.com
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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

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Delivery to the Small Intestine Goals

  • Surpass Stomach
  • Multiple methods of delivery

http://www.xtend-life.com/popup/info/Enteric_Coating.aspx

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Delivery to the Small Intestine

Enteric Coated Gel Capsule

Pill in pH 2 without Coating at time 5min Pill in pH 2 with Coating at time 1hr

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Alginate Gel

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

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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

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Human Practices

  • Institute for Genomic Biology’s

Pollen Power Camp

  • Began providing guidance to local high

schools for starting their own team in 2014

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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!

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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

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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!

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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

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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

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Thank you to our advisors!

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

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A very special thanks to our audience and sponsors!