BME494s2013 Project Team2: Difference between revisions
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'''DEVICE STRUCTURE'''<br> | '''DEVICE STRUCTURE'''<br> | ||
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:<br><br> | 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:<br><br> | ||
<b>Brick 1: IPTG-Inducible Lac Promoter Brick </b | <b>Brick 1: IPTG-Inducible Lac Promoter Brick </b> | ||
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. <br> | 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. <br> | ||
<br> This BioBrick consists of: <br> | <br> This BioBrick consists of: <br> | ||
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* The gene for the <b>Lac-I repressor protein</b>: This gene is what will be transcribed to create the protein of interest, the Lac-I repressor. <br> | * The gene for the <b>Lac-I repressor protein</b>: This gene is what will be transcribed to create the protein of interest, the Lac-I repressor. <br> | ||
* <b>Terminators:</b> These signal the end of the transcription process. <br> | * <b>Terminators:</b> These signal the end of the transcription process. <br> | ||
* <b>Lac-I regulated promoter:</b> 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. <br> | * <b>Lac-I regulated promoter:</b> 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. <br><br> | ||
<b>Brick 2: GFP Production Brick</b | <b>Brick 2: GFP Production Brick</b> | ||
The second BioBrick includes the parts necessary to produce an output of Green Fluorescence Protein (GFP). This output is regulated by the previous stage. <br> | The second BioBrick includes the parts necessary to produce an output of Green Fluorescence Protein (GFP). This output is regulated by the previous stage. <br> | ||
<br> This BioBrick consists of: <br> | <br> This BioBrick consists of: <br> |
Revision as of 17:42, 25 April 2013
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Overview & Purpose
Background: The Lac OperonThe 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
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).
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.
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 REGULATION: CAP-cAMP Complex
SUMMARY
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.
Design: Our genetic circuitOUR 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.
DEVICE STRUCTURE
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.
How it Works: The Role of IPTG and Lac-I Building: Assembly Scheme
Testing: Modeling and GFP Imaging
Human Practices
Our Team
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.
[4] Insert Shay's Microbiology textbook here. [5] Insert Shay's other Lac Operon image information here. |