Gluc o Gone uOttawa iGEM 2009 Overview Background Organisms we - PowerPoint PPT Presentation

Gluc ‐ o ‐ Gone uOttawa iGEM 2009

Overview •Background •Organisms we used •Genetic Circuitry •Device Construction

Sugar, Sugar • One gram of glucose contains 3.75 kcal, or 16 kJ of energy 1 • It is the preferred energy source for the brain 2 • 1 Chapter 3: Calculation of the Energy Content of Foods ‐ Energy Conversion Factors, Food energy ‐ methods of analysis and conversion factors , FAO Food and Nutrition Paper 77, Rome: Food and Agriculture Organization, 2003, http://www.fao.org/docrep/006/Y5022E/y5022e04.htm • 2 Fairclough, Stephen H.; Houston, Kim (2004), "A metabolic measure of mental effort", Biol. Psychol. 66 (2): 177–90, doi:10.1016/j.biopsycho.2003.10.001

The Problem With Sugar • In the past 20 years, consumption in the US has increased from 26 to 135 lbs per person 1 . • Leading cause of: – Obesity Glucose – Diabetes Mellitus Ascorbic Acid 1. Sugar’s Effect on Your Health, http://www.healingdaily.com/detoxification ‐ diet/sugar.htm , accessed 14 October 2009

Obesity • Viewed as one of the most serious health concerns of the 21 st century • A leading cause of preventable death in developed countries 3 • Childhood obesity rates in Canada have increased from 11% in the 1980s to 30% in the 1990s! 4 3 Barness LA, Opitz JM, Gilbert ‐ Barness E (December 2007). "Obesity: genetic, molecular, and environmental aspects". Am. J. Med. Genet. A 143A (24): 3016–34 4 Flynn MA, McNeil DA, Maloff B, et al. (February 2006). "Reducing obesity and related chronic disease risk in children and youth: a synthesis of evidence with 'best practice' recommendations". Obes Rev 7 Suppl 1 : 7–66.

Obesity Source: Statistics Canada, 2004 Canadian Community Health Survey

Diabetes Mellitus • Between 2005 ‐ 2006, 1.9 million Canadians were diagnosed with diabetes. • By 2016, the annual cost of diabetes is expected to surpass $8 billion in Canada • 7.8% of the American population suffers from a form of diabetes 5 American Diabetes Association, All About Diabetes, http://www.diabetes.org/about ‐ diabetes.jsp, accessed 20.09.2009

Gluc ‐ o ‐ Gone! • When you just can’t say no to sugar – Gluc ‐ o ‐ Gone fights back!

Cellulose • An indigestible polymer of β‐ glucose, it locks up sugar in a form your body can’t access!

Dietary Fiber • Cellulose is an insoluble fiber , which resists acid hydrolysis and fermentation in the human GI tract • The US Academy of Sciences suggests a daily intake of 20 ‐ 35g of dietary fiber, while most Americans consume only 12 ‐ 18g per day *Spiller, Gene; Margo N. Woods, Sherwood L. Gorbach (27 June 2001). "Influence of fiber on the ecology of the intestinal flora". CRC handbook of dietary fiber in human nutrition. CRC Press. p. 257.

The Joys of Fiber 1) Fiber promotes the proliferation of the intestinal flora by providing nutrients 2) Cellulose has been specifically shown to augment metabolism of mutagens 3) By absorbing water, fiber eases the passage of food 4) Water ‐ mediated attenuation of glucose in the GI tract *Spiller, Gene; Margo N. Woods, Sherwood L. Gorbach (27 June 2001). "Influence of fiber on the ecology of the intestinal flora". CRC handbook of dietary fiber in human nutrition. CRC Press. p. 257.

Goal: A Probiotic Bacterium That Converts Sugar Into Cellulose Specifications: 1.Target organism must be present in the gut flora 2.Cellulose precursor must be part of target organism metabolism

Lactobacillus plantarum •Gram ‐ positive strain used in milk production •Commonly used as a probiotic due to its ability to attach to the gut wall

Gut Presence

Acetobacter xylinum Figure 1: Glucose to cellulose pathway in A. xylinum 1 1. Ross, P., Mayer, R. and Benziman, M. (1991). Microbiological Reviews. 55 (1). Pp 35 ‐ 58

Cellulose Synthase •Codon comprised of 4 genes •Well ‐ characterized by Brown, Saxena et Figure 2: Cellulose synthesis in vitro al. •10 kb total Figure 3: acs operon in A. xylinum 1 1. Saxena, I. M., Kudlicka, K., Okuda, K. and Brown, M. B. (1994) Characterization of genes in the cellulose ‐ synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization. 176 (18) pp. 5735 ‐ 5752

What’s Important Glucose ‐ 1 ‐ Phosphate Glucose ‐ 6 ‐ Phosphate Glucose UDP ‐ Glucose Cellulose

+ Figure 4: Proposed model of Gluc ‐ o ‐ Gone function 1 1. Modified from: 1. Ross, P., Mayer, R. and Benziman, M. (1991). Microbiological Reviews. 55 (1). Pp 35 ‐ 58

Constructs Basic Expression Plasmid •P45 is a strong, constitutive promoter • NisI , RepA as described by Takala et al •RFP for expression testing

Plasmid acs Expression •“ Cut ‐ and ‐ paste ” of acs operon under P45 expression

•Prophage Genomic acs Expression integrase used to integrate acs operon into genome •More stable expression over generations

Biobricks • Submitted parts: •CMV promoter •CYC1 terminator •Gal 1 / 10 divergent promoter • Characterization: Gal 10 promoter by dose response experiment

Modelling •Jones and Smith predict spatial separation of strains in their two ‐ strain plug ‐ flow model •Desirable for our strain; takeover by Gluc ‐ o ‐ Gone would present many issues •Simulation of the genetic network is difficult without preliminary expression data

Challenges •Two new organisms; microbiological techniques and non ‐ standard plasmids •Optimization of PCR for cellulose synthase size •Difficult to BioBrick cellulose synthase •Cross ‐ species ribosome binding site issues

Results •Development of A. xylinum media recipe and growth protocol, an optimized miniprep protocol for A. xylinum and protocols for transformation into Lactobacillus plantarum. •Successful amplification of the cellulose synthase codon (~10 kb). •RFP construct was also completed and confirmed, although transformation was unsuccessful.

Figure 4 . PCR of the Cellulose Synthase operon from Acetobacter xylinum . Top: lane 1, lambda Hind III digest marker; lane 2 ‐ 8, PCR unsuccessful. B ottom: lane 1, Hind lambda III digest marker; lane 2, First sample of cellulose synthase operon at ∼ 10 Kb; lane 3 , Second sample of cellulose synthase operon at ∼ 10 Kb; lane 4 ‐ 7, PCR unsuccessful; lane 8 , Positive control. Each PCR product was amplified from a miniprep sample of wildtype Acetobacter xylinum genomic DNA

Future Directions •Construct optimization •Expression testing •Gut flora competition testing •Testing in mice

Bi ‐ Regulatory System

Fimbriae and Injectisomes

Safety and Ethics • Lactobacillus is generally recognized as a safe probiotic organism – Genomic integration to eliminate lateral transfer – Cellulose synthase decreases fitness of bacteria • Proposed biofilms and mice testing • Please consult our wiki for our assessment of ethics and human practices

Thank You! Advisors: Sponsors: Team: Dr. Mads Kaern The University of Ottawa Chris Euler Dr. Nadine Wiper ‐ Bergeron Faculty of Medicine Michael Lynn Dr. Doug Johnson Faculty of Engineering Radi Zinoviev VP Research Buildman Biyong Epocal Special Thanks: Candyce Sun CREM Julienne Lumingu Sharyl Chow GE Healthcare Victor Malkov The ImFresh Team Sam Graitson IPM ‐ int Olga Vorobyova Sophie Pilon Jonathan Mayo Daniel Jedrysiak Matt Orton

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