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

COLUMBIA NYC ENGINEERING AN EDIBLE PROBIOTIC CONSORTIUM TO REGULATE APPETITE AND DIGESTION Jacky Cheung, Samuel Magaziner, Suppawat Kongthong, Hudson Lee, Kenya Velez

THE RISE OF METABOLIC DISEASES CHANGES IN CALORIC INTAKE PER CAPITA PER COMPARISON OF AVERAGE CALORIC INTAKE PER DAY OVER TIME IN THE U.S 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 phoA Ghrelin PYY GLP-1 pelB Lactobacillus Reuteri Gut Peptides (GPs) Signal Peptides Lp_3050 (SPs) x M6 Signal Peptides

EXPERIMENTAL SETUP: SIGNAL PEPTIDE SECRETION Concentrate supernatant + His-tagged lyse cells Protein Anhydro-Tetracycline (or Nisin in Lactobacillus model) Blot SP Hormone His-Tag pTet Supernatant Lysate 1X10 2 1X10 1 Grow-up, spin 1X10 1 1X10 -1 down cell pellet, extract 1X10 -1 1X10 -2 supernatant

RESULTS: HORMONES (NO SIGNAL PEPTIDE) Ghrelin Glp-1 PYY Lysate (Left) 1x10 1 Supernatant (Right) 1x10 2 Lysate (Left) 1x10 -1 Supernatant (Right) 1x10 1 Lysate (Left) 1x10 -2 Supernatant (Right) 1x10 -1 Note: We expect to see no secretion

RESULTS: SUCCESS WITH PHOA-GLP1 (IMPORTANCE OF INDUCTION TIME) PhoA-Glp1 (12hr Induction) PhoA-Glp1(36hr induction) Lysate (Left) 1x10 1 Supernatant (Right) 1x10 2 Lysate 1x10 1 Lysate (Left) 1x10 -1 Supernatant (Right) 1x10 1 Supernatant 1x10 1 Supernatant 1x10 -1 Lysate (Left) 1x10 -2 Supernatant (Right) 1x10 -1 Note: Same construct, Wildly different Results; also improvement of previous biobrick BBa_K817000

RESULTS: SUCCESS WITH PELB PelB-Glp1 (36hr) PelB-PYY (36hr) Lysate 1x10 1 Here we demonstrate the generalizable Supernatant 1x10 1 nature of signal peptide secretion Supernatant 1x10 2

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…

IN PRACTICUM: YOGURT 1 2 3 Cool to 115ºF and Heat whole milk to Incubate at 37ºC for add engineered L. 185ºF 8 hr and refrigerate reuteri + yogurt starter culture 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 Comfort level in consuming probiotics (On scale of 1-5) after informing the survey takers about probiotics 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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