BME494 Project Group3

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ABSTRACT Generalized Bacterial Cell Structure of Bacillus Type There are various bacteria that can be found in pasteurized milk, that over time, cause the milk to spoil. These bacteria produce enzymes that break down milk components, such as lactose, lipids, and proteins, and cause off-putting smells and tastes. In this study, we utilized a plasmid containing a pH induced promoter in E. coli that senses when the environment is at the acidity level required for spoilage. Once transcription is activated, the bacteria will visually signal consumers with firefly luciferase. To incorporate this biosensor into milk products, we proposed to modify Lactobacilli, a heat-stable bacteria native to pasteurized milk, with our plasmid. BACKGROUND Spoiled milk smells bad! Sense of smell (or olfaction) is highly dependent on several factors. Mental health as well as physical health and age can affect a person’s ability to distinguish between various scents. Several studies show that men, older people, smokers, and schizophrenics tend to have an impaired sense of smell. It’s not uncommon for an unsuspecting person to find themselves sipping on a glass of spoiled milk because the expiration date was not correct or else he/she could not sense its spoilage. Besides this, some people do not enjoy the “after-smell” effects of sniffing spoiled milk. Ultimately, this would allow for a way to utilize the most utilized human sense (sight) in order to evaluate if the product (in this case milk) is unspoiled. PROOF OF CONCEPT DESIGN Key Pre-existing Part: The key pre-existing part we will be using is a part created by the 2006 iGEM team from Berkeley and also used by the 2010 Madras team. This key component is a constitutive promoter that is 35bp long and is induced by a pH range of 4.5-5. This promoter, part BBa_J23119, has been shown to overexpress luciferase when placed in the plasmid pGL3Basic and is the same model we will be using. Increased activity was not seen at a range of 5-6.5 and we will be modifying this assembly to act as an on-off switch when the pH value of milk reaches 5.0, instead of gradually expressing downstream genes as the pH decreases. The part is well characterized with a known sequence. We believe this part and the assembly that is constructed using the pGL3Basic promoter can be trusted based on data collected by both the 2006 Berkeley and 2010 Madras iGEM teams. Assembly Scheme Promoter: Promoter: BBa_J23119, RBS: BBa_B0034, Luciferase: BBa_I712019, Terminator: Part:BBa_B1002 Plasmid: pGL3-Basic TESTING Measurement A luminometer allows for the quantification of luciferous expression. An arbitrary unit, Relative Luminescence Units / sec can be normalized from expression at pH 7. Expected Observations A plasmid containing Firefly Luciferase as a reporter was cultured in Luria Bertani media and used to innoculate pH adjusted media in duplicates. An arbitrary unit, RLU/s (Relative Luminescence Units/per sec) was taken with a luminometer. A control population is maintained by not introducing the plasmid - no luminescence should be observed. Ever. Expression should be most prominent below the 5.5 pH threshold. As the table demonstrates, luminescence is increased fourfold as pH decreases from 7 to 4.5. Tuning Our System As the environment becomes more acidic (decreasing pH), luminescence noticeably increases. Expression should cease at extreme acidity (stomach acid pH = 1.5-3) - As in, luminescence should drop. Expression is independent of heat. HUMAN PRACTICES Some future applications of such a project would be to add something to breast milk to ensure that it has not spoiled. Human breast milk often does not last longer than 8 days, but there is enough variation between women in respect to time of spoilage that such a visual indicator would be very useful to many mothers who do not wish to feed their younglings spoiled milk. Past the various milk-applications, this could be used in other food products likely to expire. Once the actual idea is conceptualized, the applications are infinite. OUR TEAM Kris Guiang Major: Biomedical Engineering. Lacking a sense of humor and a full schedule, Kris signed onto the class because he finds synthetic biology interesting. Sunny Begala Major: Biomedical Engineering. She is taking BME 494 to improve her knowledge on Synthetic Biology. Paul Kurywchak Major: Molecular biosciences and biotechnology. I'm taking BME494 because synthetic biology will eventually be a lucrative field that develops many useful products. I want to learn more about the field and may attempt to join the 2012 ASU iGEM team. Tiffany Juan Major: Biomedical Engineering. Synthetic biology sounded interesting and is becoming a more prominent field in BME. She likes to crochet in her free time. WORKS CITED Banner Picture: http://www.institut-rosell-lallemand.com/uploads/images/souches/lactobacillus-R52_big.jpg Glowing Milk: http://gadgets.kenxu.com/assets/uploads/2008/12/milk-light-glass.jpg Bacillus Cell Structure: <http://cahoney-l-acidophilus.pbworks.com/f/lactobacilli.gif Smelly Milk: http://www.featurepics.com/FI/Thumb300/20100421/Spoiled-Milk-1514977.jpg Anderson, John. Part:BBa_J23119:Experience. Part:BBa J23119:Experience. IGEM2006_Berkeley, 24 Aug. 2006. Web. 10 Mar. 2012. <http://partsregistry.org/Part:BBa_J23119:Experience>. "Dairy Microbiology." University of Guelph. Web. 06 Mar. 2012. <http://www.foodsci.uoguelph.ca/dairyedu/micro.html>. Vector Database PGL3-Basic Vector Sequence. LabLife, 2011. Web. 10 Mar. 2012. <https://www.lablife.org/g?a=seqa&id=vdb_g2.CXFAGM7ed2MOaGd0Jm_mXPNC4wA-_sequence_13b4b2b5e6163a3ede94f32bf942bdf0c47b3633_10>.