COLUMBIA NYC ENGINEERING AN EDIBLE PROBIOTIC CONSORTIUM TO REGULATE - - PowerPoint PPT Presentation

ENGINEERING AN EDIBLE PROBIOTIC CONSORTIUM TO REGULATE APPETITE AND DIGESTION

COLUMBIA NYC

Jacky Cheung, Samuel Magaziner, Suppawat Kongthong, Hudson Lee, Kenya Velez

THE RISE OF METABOLIC DISEASES

CHANGES IN CALORIC INTAKE PER CAPITA PER DAY OVER TIME IN THE U.S COMPARISON OF AVERAGE CALORIC INTAKE PER CAPITA AROUND THE WORLD

OBESITY & DIABETES MEDICAL EXPENSES QUALITY OF LIFE

FOOD AS THE SOLUTION – NOT THE PROBLEM

HISTORICAL AND PROJECT SALES OF YOGURT IN THE UNITED STATES WHAT ARE PROBIOTICS? Live Bacteria & Yeasts That Improve Health DIGESTIVE FUNCTIONS IMMUNE SYSTEM FUNCTIONS SUPPORT FRIENDLY BACTERIA “Let food be thy medicine and medicine be they food” - Hippocrates

GOAL: ENGINEER A PROBIOTIC FOOD THAT COUNTERS OBESITY AND DIABETES

METHOD TO COUNTER OBESITY AND DIABETES

GLUCAGON-LIKE PEPTIDE I (GLP-I) INSULIN LEVELS SATIETY LEVELS PEPTIDE YY (PYY) APPETITE LEVELS GI MOVEMENT GHRELIN APPETITE LEVELS CARDIOVASCULAR HEALTH

ENGINEERING AN EDIBLE PROBIOTIC TO SECRETE GUT PEPTIDES TO REGULATE APPETITE AND DIGESTION

• E. Coli Nissle

Lactobacillus Reuteri phoA pelB Lp_3050 M6 GLP-1 Ghrelin PYY Signal Peptides Signal Peptides (SPs)

x

Gut Peptides (GPs)

EXPERIMENTAL SETUP: SIGNAL PEPTIDE SECRETION

Anhydro-Tetracycline (or Nisin in Lactobacillus model) pTet

Hormone SP His-Tag Lysate 1X101 1X10-1 1X10-2 1X102 1X101 1X10-1 Supernatant His-tagged Protein Concentrate supernatant + lyse cells Grow-up, spin down cell pellet, extract supernatant Blot

RESULTS: HORMONES (NO SIGNAL PEPTIDE)

Ghrelin PYY Glp-1

Note: We expect to see no secretion

Lysate (Left) 1x101 Supernatant (Right) 1x102 Lysate (Left) 1x10-1 Supernatant (Right) 1x101 Lysate (Left) 1x10-2 Supernatant (Right) 1x10-1

RESULTS: SUCCESS WITH PHOA-GLP1 (IMPORTANCE OF INDUCTION TIME)

Lysate (Left) 1x101 Supernatant (Right) 1x102 Lysate (Left) 1x10-1 Supernatant (Right) 1x101 Lysate (Left) 1x10-2 Supernatant (Right) 1x10-1 PhoA-Glp1(36hr induction) PhoA-Glp1 (12hr Induction) Lysate 1x101 Supernatant 1x101 Supernatant 1x10-1

Note: Same construct, Wildly different Results; also improvement of previous biobrick BBa_K817000

RESULTS: SUCCESS WITH PELB

Lysate 1x101 Supernatant 1x101 Supernatant 1x102 PelB-Glp1 (36hr) PelB-PYY (36hr) Here we demonstrate the generalizable nature of signal peptide secretion

IMPLEMENTING A CONSORTIUM TO PROVIDE REGULATIONS AND SAFETY MECHANISM FEATURES

COMMUNICATION REGULATION SAFETY OUR SYSTEM IS TO BE TIGHTLY REGULATED AS A SAFETY PRECAUTION

THE SYSTEM: SAFETY, REGULATION, COMMUNICATION, AND SECRETION MEET

THE SYSTEM BREAKDOWN: THE AHL FACTORY

THE SYSTEM BREAKDOWN: TARGET SECRETION

THE SYSTEM BREAKDOWN: TIME-DELAYED CELL LYSIS (BBA_K1848006)

RESULTS

• Timer-Lysis Cassette system submitted as

biobrick (BBa_K1848006)

• System successfully cloned in
• Time constraints prevented its testing
• However…

1 3 2 Heat whole milk to 185ºF Cool to 115ºF and add engineered L. reuteri + yogurt starter culture Incubate at 37ºC for 8 hr and refrigerate

IN PRACTICUM: YOGURT

Note: Yogurt was not intended for human consumption, nor did it leave the lab

SURVEYING THE PUBLIC

• Online survey
• Polled public on GMOs and probiotics, to inform design of safety measures and yogurt

products

• Informed survey takers about probiotics and GMOs
• Twitter and blog

Comfort level in consuming probiotics (On scale of 1-5) before informing the survey takers about probiotics Comfort level in consuming probiotics (On scale of 1-5) after informing the survey takers about probiotics

THE FUTURE: YOGHURT (OR LOOKING WHEY INTO THE FUTURE)

• Going Forward:
• 1. Finish screen of submitted quorum sensing system
• 2. Move system into L. reuteri
• 3. Test active system’s ability to function within yogurt

culture

• 4. Screen more hormones
• The Foreseeable future:
• Catalog of peptide secreting yogurt
• Biotech branch based around personalized consumable

medicines

IN SUMMATION…

• We managed to:

1.

Submit 6 bio-bricks (5 of which are well- characterized) (BBa_K1848001-006)

2.

Successfully secrete 3 gut hormones and 2 signal peptides in E. coli

3.

Develop a promising system quorum sensing based secretion and lysis

4.

Move an engineered L. reuteri system in yogurt

5.

Poll on public opinion of probiotics and willingness to consume GMOs

6.

Have an amazing summer and lay the foundation for future iGEM teams from our University

Student Team Members The Whole Team

ATTRIBUTIONS AND ACKNOWLEDGMENT

• Undergraduate Team Members: Suppawat Kongthong, Jacky Cheung, Kenya

Velez, Hudson Lee, Samuel Magaziner

• Mentors: Nathan Johns, Sway Chen, and Sonja Billerbeck
• PIs: Harris Wang, Virginia Cornish, Ken Shepard, Dana Pe’er

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