uconn presentation - igem 2012

The Transformation of S. cervisiae for the Production of Vitamin D3

VITAYEAST

OVERVIEW OF PRESENTATION

• What is Vitamin D and why is it a problem.

• Our approach to solving the problem of vitamin D deficiency

• Method we used to solve this problem

• Ongoing work

WHAT DOES VITAMIN D3 DO?

• Used in for a wide variety of functions in the human body

• Assists in absorption of calcium

• Helps prevent diabetes

• May reduce hypertension

• Linked to cancer prevention

• Decreases the negative effects of alcohol

ABSORPTION OF CALCIUM

• Essential for both phosphorus and calcium metabolization.

• Absorbed with the aid of bile after bonding to calcium

• Regulates the cellular absorption of calcium in bone and skeletal muscle tissue

• Stimulates reabsorption of phosphate in the nephrons

WHY IS VITAMIN D DEFICIENCY A PROBLEM

• Most people do not get enough sun.

• Supplements

WHAT CAUSES VITAMIN D DEFICENCY

• “Anything that diminishes the transmission of solar UVB radiation to the earth’s surface or anything that interferes with the penetration of UVB radiation into the skin will affect the cutaneous synthesis of vitamin D3”

LESS KNOWN CAUSES OF UVB DEFLECTION

• Sunscreen with an spf of 15 is enough to block 99% of vitamin D3 production.

Image courtesy of the Skin Caner Foundation

LESS KNOWN CAUSES OF UVB DEFLECTION • Geography

OUR SOLUTION

• Modify the vitamin D2 path in Saccharomyces cerevisiae to produce Vitamin D3

• S. cerevisiae already has most of the genes necessary to make D3

• Multiple applications for distributing the D3 after production

• Bread and other leavened products

• Supplements

• Fermented liquids

GENES FOR PRE-VITAMIN D3 SYNTHESIS

• 7-Dehydrocholeserol is the precursor to D3

• Only three Genes are needed to alter the D2 pathway to produce 7-Dehydrocholeserol. DHCR24, EBP, andSC5DL

• It turns out that Sc5DL is already present in S. cerevisiae under the name ERG3 in the D2 pathway.

METHODS

• We used Gateway® Recombination Cloning Technology from Invitrogen to insert the necessary genes into the S. cerevisiae cells.

• pENTR221 entry vector from Genecopia

• transformed into One Shot®

OmniMAX™ 2TI® E. Coli competent

cells

METHODS CONT.

• LR Clonease ™ II Enzyme Mix To transfer The genes into the Gateway ® vector, pYES2-DEST52

ONGOING WORK

• Transform the yeast

• Confirm expression of genes and protein production

• Confirm synthesis of 7-Dehydrocholesterol

QUESTIONS FOR THE FUTURE

• Will the yeast uptake the genes and will the genes be expressed?

• Will the yeast survive if some of the resources for D2 production are aliquot to D3 production?

• Will the final product be stable?

• Will it be able to be added into a food product?

PROBLEMS ENCOUNTERED

• Deciding on an appropriate project

• Funding

• Establishing the club

• Organizing the club

• Funding

• Finding a good meeting time

• Filling officer positions

• And Funding yet again

• Finding time to work on the project

ACKNOWLEDGMENTS

We would like to thank the following for their support

• Our advisors: Dr. Rachel O’Neill, Laura Hall, Sarah Mitchell, Brianna Flynn

• Dr. Linda Stausbaugh, center for Applied Genetics and Technology, University of Connecticut

• Dr. Ben Carone, University of Massachusetts

• Dr. Judy Brown, University of Connecticut

• Our Sponsors:

REFERENCES • Holick, M.F. and C.C. Tai. 2008. Vitamin D dificency: a world wide problem with health concequences. American Journal of

Clinical Nutrition. 87:10805-65.

• United States of America National Institutes of Health- Office of Dietary Suppliments. 2011. Dietary Supplements Facts Sheet: Vitamin D. http://ods.od.nih.gov/fsctsheets/VitaminD-HealthProfessional/

• Webb, A.R., L. Knine, and M.F. Holick. 1988. Influence of season and latitude on the cutanious synthesis of vitamin D3: exposure to winter sunlight in Boston and Edmonton will not promote Vitamin D3 synthesis in human skin. Journal of Clinical Endocrinology and Metabolism. 67:373-378

• Arms, L.G., B.W. Hollis, and R.P.Heaney. 2004. Vitamin D2 is much less effective than vitamin D3 in humans. Journal of Clinical Endocrinology and Metabolism. 89:5387-5391. Web.

• Steroid biosynthesis – Refference Pathway. Kyoto encyclopedia of genes and genomics. www.genome.jp/kegg-bin/show_pathway?ko00100

• Kasiappan, r., Z. Shen, A. K. W. Tse, U, Jinwal, J. Tang, P. Lungchuklet, Y. Sun, P. Kruk, S. V. Nicosia, X. Zhang, and W. Bal. 2012. 1,25-Dihydroxyvitamin D3 Suppresses Telomerase Expression and Human Cancer Growth through MicroRNA -498. Journal of Biological Chemistry. Online, 1-26

• Mercer, K. E., R.A. Wynne, OP. Lazarenko, C. K. Lumpkin, W. R. Hogue, L. J. Suva, J. Chen, A. Z. Mason, T. M. Badger, M. J.J. Ronis. 2012. Vitamin D supplementation prevents EtOH-mediated bone loss. Journal of Pharmacology and Experimental Theraputics. Online, 1-43

• Invitrogen.com

• http://en.wikipedia.org/wiki/File:Marmite.jpg

• http://www.marksdailyapple.com/definitive-guide-grains/

• http://www.durtynellies.com/beer.asp

• http://wineworldspirits.com/

• http://www.vitamincottage.com/nutritional_yeast_flakes_by_kal_22_oz_powder_s_104104_gm_ -p-6250.html

• http://vitamins.lovetoknow.com/Red_Yeast_Rice_Supplement

THANK YOU