BME494s2013 Project Team2: Difference between revisions
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* The gene for the <b>LacI repressor protein</b>: This gene is what will be transcribed to create the protein of interest, the LacI repressor. <br> | * The gene for the <b>LacI repressor protein</b>: This gene is what will be transcribed to create the protein of interest, the LacI 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> | * <b>LacI regulated promoter:</b> This promoter will cause the next stage of transcription to begin, and is negatively regulated (repressed) by the LacI protein. Therefore, transcription will occur only in the absence of the LacI protein. <br><br> | ||
<b>Brick 2: GFP Production Brick</b><br> | <b>Brick 2: GFP Production Brick</b><br> | ||
The second BioBrick includes the parts necessary to produce an output of Green Fluorescence Protein (GFP). This output is regulated by the parts from the previous stage. This BioBrick consists of: <br> | The second BioBrick includes the parts necessary to produce an output of Green Fluorescence Protein (GFP). This output is regulated by the parts from the previous stage. This BioBrick consists of: <br> | ||
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* <b>Terminators</b>: These signal the end of the transcription process. <br><br> | * <b>Terminators</b>: These signal the end of the transcription process. <br><br> | ||
'''How it Works: The Role of IPTG and Lac-I'''<br> | '''How it Works: The Role of IPTG and Lac-I'''<br> | ||
The switch response of this device is due to the relationships it creates between IPTG, the | The switch response of this device is due to the relationships it creates between IPTG, the LacI 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 LacI repressor protein. Presence of the LacI protein inhibits the promoter, which turns off GFP production. The LacI protein is created in stage 1 of the genetic circuit. In its default state, the mechanism would operate as follows: <br><br> | ||
LacI protein is created -> LacI regulated promoter is inhibited -> Transcription of GFP is inhibited -> <b>No Output</b> <br><br> | |||
On the other hand, when an IPTG input is added to the system, results in the following: <br><br> | |||
IPTG is added -> LacI protein is created -> IPTG binds to LacI -> Conformational change in LacI protein -> LacI can no longer bind to the stage 2 promoter -> Transcription of GFP is no longer inhibited -> <b>GFP Is Produced</b> | |||
==Building: Assembly Scheme== | ==Building: Assembly Scheme== |
Revision as of 18:48, 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
How it Works: The Role of IPTG and Lac-I LacI protein is created -> LacI regulated promoter is inhibited -> Transcription of GFP is inhibited -> No Output On the other hand, when an IPTG input is added to the system, results in the following: IPTG is added -> LacI protein is created -> IPTG binds to LacI -> Conformational change in LacI protein -> LacI can no longer bind to the stage 2 promoter -> Transcription of GFP is no longer inhibited -> GFP Is Produced 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. [3] Registry of Standard Biological Parts. Web. 25 Apr 2013. <partsregistry.org>. [4] Slonczewski, Joan, and John Watkins. Foster. Microbiology: An Evolving Science. New York: W.W. Norton &, 2009. Print. [5] Insert Shay's other Lac Operon image information here. |