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 Negative 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: Table 1: 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. Why does this phenomenon occur? Well, like stated before, lactose is the cell's last resort energy source because it requires more energy from the cell to digest than does glucose. The enzyme that digests lactose is β-galactosidase, which can only be produced by initiating transcription of the Lac Operon. Thus, to be able to digest lactose, the cell needs to initiate transcription of the Lac Operon. NEGATIVE REGULATION: The LacI Repressor Example of negative regulation of the LacI repressor [5] The genes encoding the LacI repressor are actually located upstream of the Lac Operon. The LacI gene is not regulated; therefore, it is produced continuously. It binds to the Lac Operon in the promoter region; however, it does not bind if there is lactose in the cell. Why is this? Well, the cell produces very low levels of β-galactosidase even when not in the presence of lactose. In these very low lactose conditions, β-galactosidase has a different function: it cleaves lactose and recombines it to form allolactose, which acts as an inducer for LacI. It binds to LacI and causes a conformational change, which in turn makes LacI unable to bind to the promoter region of the Lac Operon. Positive and negative regulation of the Lac Operon [4] POSITIVE REGULATION: CAP-cAMP Complex Remember from before, the absence of the LacI repressor is not the only factor that allows transcription to occur. There is also a form of positive regulation that occurs via the CAP-cAMP Complex, the formation of which is controlled by the levels of glucose within the cell. As glucose levels in the cell begin to decline, E. Coli responds by beginning to synthesize cyclic adenosine monophosphate, or cAMP. As cAMP concentration increases, it binds to a catabolite activator protein, or CAP. cAMP acts as an inducer for CAP, causing a conformational change that allows CAP to bind to the promoter region of the Lac Operon. This cAMP-CAP complex interacts with the RNA polymerase, increasing its affinity for the Lac promoter. Without attachment of the cAMP-CAP complex, or in high levels of glucose, affinity wouldn't be high enough to cause significant transcription. A circuit diagram illustrating glucose and lactose as inputs. SUMMARY So far, what do we know about the natural Lac Operon? Well, we know that it is a gene that produces a number of structural proteins, including β-galactosidase. We also know that it requires lactose to be present and glucose to be absent for transcription to occur. What we haven't discussed is the function of the operon from an engineering perspective. Table 2: Glucose NOT Gate Input (Glucose) Output 1 0 0 1 Table 3: Glucose and Lactose AND Gate Input 1 (Table 2 Output) Input 2 (Lactose) Output 0 0 0 0 1 0 1 0 0 1 1 1 If we analyze it from a digital logic context, we can describe glucose and lactose as inputs, and the transcription of β-galactosidase as an output. Furthermore, we can build a logic circuit symbolizing the operon's functionality (illustrated in diagram on left). When glucose acts as an input, it produces a NOT gate functionality (See Table 2). When lactose and the NOT gate output of glucose are incorporated as inputs to the system, they produce an AND gate functionality (see Table 3). Furthermore, there are a couple of other other proteins that "mimic" the function of lactose as an input for the natural lac operon. Among these are IPTG (used for our switch), and the previously mentioned allolactose which is an isomer of lactose. From a genetic engineering perspective, the structural genes encoding for β-galactosidase can be replaced with different genes encoding other proteins, therefore creating a designer system that produces proteins when induced by lactose. One can create an insulin-creating system, one that produces heparin, or even one that produces a fluorescent protein, therefore making the system function as a reporter of the presence of lactose. Applications of engineering the Lac Operon are endless, only bounded by the human imagination. Design: Our genetic circuit OUR GENE SWITCH: As described above, the structural protein regions of the natural Lac Operon can be replaced by various other protein coding regions to alter the output of the Lac Operon. In the case of our gene switch, we chose to replace β-galactosidase with a gene coding for GFP, or green fluorescent protein. We used the lactose mimic IPTG as our system's input. Therefore, our switch turns "on" in the presence of IPTG, and produces a green fluorescent color as its output. Also, we chose a promoter that was not sensitive to the CAP-cAMP complex so that our switch would not be influenced by the presence of glucose. Therefore, IPTG would be the only input effecting our system, as it would not be positively regulated by glucose intake. Device design. Image adapted from [3]. DEVICE STRUCTURE Our design incorporates BioBrick parts from the Registry of Standard Biological Parts (see diagram on left for specific part numbers). There are two main BioBricks used in our system: 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: These signal the end of 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. Brick 2: GFP Production Brick The second BioBrick includes the parts necessary to produce an output of Green Fluorescence Protein (GFP). This output is regulated by the previous stage. This BioBrick consists of: A ribosome binding site: This stage involves a second transcription, so it needs its own site for ribosome binding. The GFP gene: This is the gene that will be transcribed to use GFP. Terminators: These signal the end of the transcription process. How it Works: The Role of IPTG and Lac-I The switch response of this device is due to the relationships it creates between IPTG, the Lac-I protein, and the GFP output. Transcription of the GFP output depends on the activity of the stage 2 promoter, the Lac-I regulated promoter. If this promoter is active, GFP will be produced. This promoter is regulated by the Lac-I repressor protein. Presence of the Lac-I protein inhibits the promoter, which turns off GFP production. The Lac-I protein is created in stage 1 of the genetic circuit. In its default state, the reaction would go as follows: 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. 6th degree polynomial fit to the raw GFP output data. 5th degree derivative of the polynomial fit. 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. [3] Registry of Standard Biological Parts. Web. 25 Apr 2013. <partsregistry.org>. [4] Insert Shay's Microbiology textbook here. [5] Insert Shay's other Lac Operon image information here.