BME494s2013 Project Team2

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Overview & Purpose Text describing the image Background: The Lac Operon The Lac Operon is a gene specific to E. Coli that controls the cell's digestion of lactose. It consists of a promoter, an operator, three structural genes, and a terminator. It is both positively and negatively regulated, allowing expression to be contingent on the concentrations of glucose and lactose in the cell. Structure of the Lac Operon (1) STRUCTURE The Lac Operon encodes three structural genes: LacZ: The Lac Z structural region, or β-galactosidase, hydrolyzes the disaccharide lactose into glucose and galactose, sugars that are smaller and easier for the cell to digest. However, in low concentrations of lactose, β-galactosidase cleaves and rearranges lactose into allolactose, which acts as an inducer for the LacI repressor (see Positive Regulation). LacY: LacY, or lactose permease, is a transmembrane protein that transports lactose into the cell. LacA: LacA is a transacetylase. While it has functionality, it has little effect on the function of our design, so it will not be discussed. In addition to the structural genes, the Lac Operon includes a promoter and an operator region. The promoter region is the area to which the Lac I repressor and the CAP-cAMP complex bind, the mechanics of which will be discussed later (see Positive Regulation and Negative Regulation). PURPOSE: Efficiency Expression of the Lac Operon is determined jointly by the levels of glucose and lactose in the cell. Being a monosaccharide, glucose is easier (i.e., takes less energy) to digest; therefore, if glucose is present, the cell will prefer to use it as an energy source. However, if glucose is not available as an energy source, the cell will use lactose instead. A table describing this relationship is below: Glucose/Lactose Relationship to Lac Operon Transcription (2) Carbohydrates CAP-cAMP Complex LacI Repressor RNA Polymerase Transcription of Lac Operon + Glucose, + Lactose Not bound to DNA Lifted off operator site Keeps falling off promoter site Very low transcription + Glucose, - Lactose Not bound to DNA Bound to operator site Blocked by repressor No transcription - Glucose, - Lactose Bound to DNA Bound to operator site Blocked by the repressor No transcription - Glucose, + Lactose Bound to DNA Lifted off operator site Sits on promoter site TRANSCRIPTION From this table, we can observe a multitude of things: Transcription only occurs when lactose, but not glucose, is present. When glucose is not present, the CAP-cAMP complex (or the activator protein) is not bound to DNA. Thus, the absence of glucose promotes transcription. When lactose is not present, the LacI repressor is bound to the operator site. When lactose is absent, the repressor is NOT bound to the operator site. Thus, the presence of lactose promotes transcription. For the RNA Polymerase to properly attach to the Lac Operon, the CAP-cAMP complex must be attached to the DNA, and the LacI repressor must not be attached to the operator site. Therefore, transcription only occurs when lactose, but not glucose, is present. see that transcription of the Lac Operon occurs only when the cell is in the presence of lactose, but not glucose. To digest lactose, the cell needs β-galactosidase, which can only be produced by initiating transcription of the Lac Operon. POSITIVE REGULATION: The LacI Repressor Explain here NEGATIVE REGULATION: CAP-cAMP Complex Explain here Insert crap about the Lac Operon. Insert crap about the Lac Operon. Insert crap about the Lac Operon. Insert crap about the Lac Operon. Insert crap about the Lac Operon. Insert crap about the Lac Operon. Insert crap about the Lac Operon. Insert crap about the Lac Operon. 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Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Keep talking about the Lac Operon. Design: Our genetic circuit OUR GENE SWITCH: Device design. Image adapted from [3]. Our design incorporates BioBrick parts from the Registry of Standard Biological Parts. Brick 1: IPTG-Inducible Lac Promoter Brick The first BioBrick includes parts which will work together to create the Lac-I repressor protein, and also includes the promoter which is regulated by it. This BioBrick consists of: A consitutive promoter: Causes transcription to begin. A ribosome binding site: Ribosomes will attach here during transcription. The gene for the Lac-I repressor protein: This gene is what will be transcribed to create the protein of interest, the Lac-I repressor. Terminators: End the transcription process. Lac-I regulated promoter: This promoter will cause the next stage of transcription to begin, and is negatively regulated (repressed) by the Lac-I protein. This means that it will promote the next stage only in the absence of the Lac-I protein. Building: Assembly Scheme Testing: Modeling and GFP Imaging A LAC SWITCH MODEL We used a previously published synthetic switch, developed by Ceroni et al., to understand how our system could potentially be modeled and simulated. AN INTERACTIVE MODEL We used a model of the natural Lac operon to understand how changing the parameter values changes the behavior of the system. COLLECTING IMPERICAL VALUES TO IMPROVE THE MODEL We explored how one technique, imaging via microscopy could be used to determine the production rate of an output protein, in this case GFP in yeast, could be used to determine a "real" value for maximum GFP production rate under our own laboratory conditions. Ideally, the GFP production rate measured by this method could be entered as a value for [which parameter] in the Ceroni et al. model. Human Practices Danger of Chemicals in Farmlands Our Team Shay Ravacchioli My name is Shay Ravacchioli, and I am a Junior majoring in Biomedical Engineering with minors in Biological Sciences and Psychology. I am taking BME 494 because I think Synthetic Biology is fascinating. An interesting fact about me is that I play piano and guitar. Jenessa Lancaster My name is Jenessa Lancaster, and I am a Junior majoring in Biomedical Engineering with a minor in Psychology. I am taking BME 494 because I have always wanted to learn more about Synthetic Biology and Genetic Engineering. An interesting fact about me is that I write songs. Michael Rose My name is ###, and I am a ### majoring in ###. I am taking BME 494 because ###. An interesting fact about me is that ###. Your Name My name is ###, and I am a ### majoring in ###. I am taking BME 494 because ###. An interesting fact about me is that ###. Works Cited [1] Heller, H. Craig., David M. Hillis, Gordon H. Orians, William K. Purves, and David Sadava. Life: The Science of Biology. Sunderland, MA,: Sinauer Ass., W.H. Freeman and, 2008. N. pag. Print. [2] Escalante, Ananias. "Regulation I." Class Notes. University of Arizona. 20 February 2013. Read more: How to Cite Lecture Notes in MLA Format | eHow.com http://www.ehow.com/how_8780129_cite-lecture-notes-mla-format.html#ixzz2RVPioFII [3] partsregistry.org