Description
From 2006.igem.org
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[[Image:CellSeeUs.Thermometer.jpg]] | [[Image:CellSeeUs.Thermometer.jpg]] | ||
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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. | ||
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| + | == 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. 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 | + | 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. |
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| + | In a nutshell: | ||
| + | - see green fluorescence at low temperatures | ||
| + | - see red fluorescence at high temperatures | ||
| + | - see a combination of the two (ie. yellow) between the extremes | ||
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| + | [References for LacI dimerization etc etc] | ||
| + | |||
| + | == 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. This is a repressive regulator of gene transcription, so the thermometer starts off in the "off" state and no fluorescent proteins are produced. | ||
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| - | + | == 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 Blue Water wiki. [link this to wiki page] | ||
Revision as of 03:47, 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. 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: - see green fluorescence at low temperatures - see red fluorescence at high temperatures - see a combination of the two (ie. yellow) between the extremes
[References for LacI dimerization etc etc]
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. This is a repressive regulator of gene transcription, so the thermometer starts off in the "off" state and no fluorescent proteins are produced.
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 Blue Water wiki. [link this to wiki page]
