Description
From 2006.igem.org
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The Cell-See-Us thermometer is a gene construct that produces differing amounts of green and red fluorescing proteins in response to temperature changes. | The Cell-See-Us thermometer is a gene construct that produces differing amounts of green and red fluorescing proteins in response to temperature changes. | ||
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== GFP vs. mRFP expression == | == GFP vs. mRFP expression == | ||
The functionality of the Cell-See-Us thermometer is based on the dimerization of a temperature sensitive LacI protein (<bbpart>BBa_J06501</bbpart>). This protein is produced in dimer form, which exists in equilibrium with its tetramer form. Modifying the environmental temperature (ie. of the cellular host) will cause a noticeable shift in this equilibrium. | The functionality of the Cell-See-Us thermometer is based on the dimerization of a temperature sensitive LacI protein (<bbpart>BBa_J06501</bbpart>). This protein is produced in dimer form, which exists in equilibrium with its tetramer form. Modifying the environmental temperature (ie. of the cellular host) will cause a noticeable shift in this equilibrium. | ||
| - | At low temperatures, there is a high ratio of LacI ts tetramers to dimers. In the thermometer construct, the tetrameric form of the LacI binds to and represses the pLac promoter. This prevents the transcription of the tetR gene. In turn, the lack of tetR protein production will allow the pTet promoter immediately downstream to upregulate the expression of green fluorescent protein (GFP). At the same time, the second pLac in the construct will also be repressed, and since it drives monomeric red fluorescent protein (mRFP) production, there will be a decrease in intracellular levels of mRFP. The amount of GFP produced relative to the amount of mRFP will result in net green fluorescence. | + | At low temperatures, there is a high ratio of LacI ts tetramers to dimers. In the thermometer construct, the tetrameric form of the LacI binds to and represses the pLac promoter (which is actually a pL promoter with LacI sites, forming an effective pLac-like repressor). This prevents the transcription of the tetR gene. In turn, the lack of tetR protein production will allow the pTet promoter immediately downstream to upregulate the expression of green fluorescent protein (GFP). At the same time, the second pLac in the construct will also be repressed, and since it drives monomeric red fluorescent protein (mRFP) production, there will be a decrease in intracellular levels of mRFP. The amount of GFP produced relative to the amount of mRFP will result in net green fluorescence. |
Conversely, when cells containing the thermometer are exposed to high temperatures, LacI will predominately exist in the dimer form. With a lower intracellular concentration of tetramers, repression of the pLac promoters is relieved. The first pLac will cause tetR to be expressed, which will bind to and repress the pTet promoter controlling GFP expression. The second pLac will promote the transcription and expression of mRFP. This results in net red fluorescence. | Conversely, when cells containing the thermometer are exposed to high temperatures, LacI will predominately exist in the dimer form. With a lower intracellular concentration of tetramers, repression of the pLac promoters is relieved. The first pLac will cause tetR to be expressed, which will bind to and repress the pTet promoter controlling GFP expression. The second pLac will promote the transcription and expression of mRFP. This results in net red fluorescence. | ||
| - | In a nutshell: | + | In a nutshell: Low temperatures make the cells glow green, high temperatures make the cells glow red, and temperatures in between result in a combination of the two (yellow). |
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[References for LacI dimerization etc etc] | [References for LacI dimerization etc etc] | ||
| - | + | LacI: see Chao et al. (2002) Biotechnology and Bioengineering, 79(1):1-8. | |
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| + | == Regulation of thermometer expression using arabinose == | ||
| + | The thermometer construct is induced through the addition of L-arabinose to the cell culture. L-arabinose enters the cells and binds to an endogenous E. coli protein, AraC. AraC is a repressive regulator of gene transcription, so the thermometer starts off in the "off" state and no fluorescent proteins are produced. Its mode of repression is through DNA binding near the pBad promoter at the beginning of the construct and so preventing the host cellular transcription machinery from transcribing genes controlled by pBad. Binding of the L-arabinose to AraC allows expression from the pBad promoter, and in the thermometer pBad drives the production of temperature sensitive LacI. Having the pBad promoter creates another level of regulation, allowing the entire thermometer to be turned on and off using arabinose. | ||
| + | pBad: see Gallegos et al. (1997) and Schleif (1996), as cited in Newman and Fuqua (1999). Gene, 227:197-203. | ||
== Creation of the Cell-See-Us thermometer == | == Creation of the Cell-See-Us thermometer == | ||
| - | The Cell-See-Us thermometer was designed and constructed by the Blue Water iGEM 2006 team. For details of the design, construction, and testing phases, please see the [[ | + | The Cell-See-Us thermometer was designed and constructed by the Blue Water iGEM 2006 team. For details of the design, construction, and testing phases, please see the [[Construction|Lab Notebook]]. |
Latest revision as of 08:00, 2 November 2006
Contents |
Cell-See-Us Thermometer
This construct can be viewed in the Registry at BBa_J5530.
The Cell-See-Us thermometer is a gene construct that produces differing amounts of green and red fluorescing proteins in response to temperature changes.
GFP vs. mRFP expression
The functionality of the Cell-See-Us thermometer is based on the dimerization of a temperature sensitive LacI protein (BBa_J06501). This protein is produced in dimer form, which exists in equilibrium with its tetramer form. Modifying the environmental temperature (ie. of the cellular host) will cause a noticeable shift in this equilibrium.
At low temperatures, there is a high ratio of LacI ts tetramers to dimers. In the thermometer construct, the tetrameric form of the LacI binds to and represses the pLac promoter (which is actually a pL promoter with LacI sites, forming an effective pLac-like repressor). This prevents the transcription of the tetR gene. In turn, the lack of tetR protein production will allow the pTet promoter immediately downstream to upregulate the expression of green fluorescent protein (GFP). At the same time, the second pLac in the construct will also be repressed, and since it drives monomeric red fluorescent protein (mRFP) production, there will be a decrease in intracellular levels of mRFP. The amount of GFP produced relative to the amount of mRFP will result in net green fluorescence.
Conversely, when cells containing the thermometer are exposed to high temperatures, LacI will predominately exist in the dimer form. With a lower intracellular concentration of tetramers, repression of the pLac promoters is relieved. The first pLac will cause tetR to be expressed, which will bind to and repress the pTet promoter controlling GFP expression. The second pLac will promote the transcription and expression of mRFP. This results in net red fluorescence.
In a nutshell: Low temperatures make the cells glow green, high temperatures make the cells glow red, and temperatures in between result in a combination of the two (yellow).
[References for LacI dimerization etc etc]
LacI: see Chao et al. (2002) Biotechnology and Bioengineering, 79(1):1-8.
Regulation of thermometer expression using arabinose
The thermometer construct is induced through the addition of L-arabinose to the cell culture. L-arabinose enters the cells and binds to an endogenous E. coli protein, AraC. AraC is a repressive regulator of gene transcription, so the thermometer starts off in the "off" state and no fluorescent proteins are produced. Its mode of repression is through DNA binding near the pBad promoter at the beginning of the construct and so preventing the host cellular transcription machinery from transcribing genes controlled by pBad. Binding of the L-arabinose to AraC allows expression from the pBad promoter, and in the thermometer pBad drives the production of temperature sensitive LacI. Having the pBad promoter creates another level of regulation, allowing the entire thermometer to be turned on and off using arabinose.
pBad: see Gallegos et al. (1997) and Schleif (1996), as cited in Newman and Fuqua (1999). Gene, 227:197-203.
Creation of the Cell-See-Us thermometer
The Cell-See-Us thermometer was designed and constructed by the Blue Water iGEM 2006 team. For details of the design, construction, and testing phases, please see the Lab Notebook.
