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Team:Imperial College London/Thermoinduction
From 2009.igem.org
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After encapsulation, it becomes <b>more difficult for normal chemical induction</b> to penetrate the outer protective coating. To tackle this problem, <b>heat induction</b> is used. Heat induction is, in addition, a commonly used and highly reproducible induction method in industrial scale manufacturing processes.<br> | After encapsulation, it becomes <b>more difficult for normal chemical induction</b> to penetrate the outer protective coating. To tackle this problem, <b>heat induction</b> is used. Heat induction is, in addition, a commonly used and highly reproducible induction method in industrial scale manufacturing processes.<br> | ||
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| - | The thermosensitive cI promoter is chosen as it is widely cited in literature for efficient thermoinduction of systems [1], and has been proven to work well in commonly available plasmids. [http://partsregistry.org/Part:BBa_K098995 | + | The thermosensitive cI promoter is chosen as it is widely cited in literature for efficient thermoinduction of systems [1], and has been proven to work well in commonly available plasmids. [http://partsregistry.org/Part:BBa_K098995 BBa_K098995], in particular, is the only construct for the thermosensitive cI system in the registry and has already been characterised previously.<br> |
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[[Image:Ii09 thermo2.jpg|300px|right]] | [[Image:Ii09 thermo2.jpg|300px|right]] | ||
| - | The lambda promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0051 | + | The lambda promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0051 R0051]) is strongly repressed at 28 degrees by the cI repressor([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098997 K098997]). Therefore, at 28 degrees, the thermoinducible system has no activity. <br> |
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However, this cI repressor is unique in that it is <b>temperature-sensitive</b>, and will readily lose its functional structure as the temperature increases. Therefore, when the temperature is raised to 42°C, the lambda promoter is no longer repressed and can trigger <b>Module 3</b>. <br> | However, this cI repressor is unique in that it is <b>temperature-sensitive</b>, and will readily lose its functional structure as the temperature increases. Therefore, when the temperature is raised to 42°C, the lambda promoter is no longer repressed and can trigger <b>Module 3</b>. <br> | ||
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==Characterisation of existing biobrick== | ==Characterisation of existing biobrick== | ||
| - | The thermoinducible construct ([http://partsregistry.org/Part:BBa_K098995 | + | The thermoinducible construct ([http://partsregistry.org/Part:BBa_K098995 BBa_K098995]), an existing biobrick, is characterised by ligating it to GFP. The cells are grown at the low temperature of 28°C where the promoter is inactive. The temperature is then shifted up to 42°C. The GFP fluorescence is monitored throughout as an indicator of promoter activity. <br> |
[[Image:II09_Hvd-GFPfluor2.jpg]]<br> | [[Image:II09_Hvd-GFPfluor2.jpg]]<br> | ||
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The GFP fluorescence is shown to be at baseline when cells are grown at 28°C. However, when the cells undergo a temperature shift to 42°C, there is an almost immediate increase in GFP fluorescence. <br> | The GFP fluorescence is shown to be at baseline when cells are grown at 28°C. However, when the cells undergo a temperature shift to 42°C, there is an almost immediate increase in GFP fluorescence. <br> | ||
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| - | This shows that the activity of the lambda promoter does increase when the temperature is shifted up, so the thermoinducible construct ([http://partsregistry.org/Part:BBa_K098995 | + | This shows that the activity of the lambda promoter does increase when the temperature is shifted up, so the thermoinducible construct ([http://partsregistry.org/Part:BBa_K098995 BBa_K098995]) does become activated by higher temperatures. <br> |
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Revision as of 02:59, 22 October 2009
Contents |
Module Integration - Thermoinduction
Overview
When sufficient protective coating has been made, the temperature of the system is raised to 42°C. This activates the thermosensitive promoter, consequently triggering Module 3.
Rationale
After encapsulation, it becomes more difficult for normal chemical induction to penetrate the outer protective coating. To tackle this problem, heat induction is used. Heat induction is, in addition, a commonly used and highly reproducible induction method in industrial scale manufacturing processes.
The thermosensitive cI promoter is chosen as it is widely cited in literature for efficient thermoinduction of systems [1], and has been proven to work well in commonly available plasmids. [http://partsregistry.org/Part:BBa_K098995 BBa_K098995], in particular, is the only construct for the thermosensitive cI system in the registry and has already been characterised previously.
The system
The lambda promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0051 R0051]) is strongly repressed at 28 degrees by the cI repressor([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098997 K098997]). Therefore, at 28 degrees, the thermoinducible system has no activity.
However, this cI repressor is unique in that it is temperature-sensitive, and will readily lose its functional structure as the temperature increases. Therefore, when the temperature is raised to 42°C, the lambda promoter is no longer repressed and can trigger Module 3.
About the thermoinduction system
Characterisation of existing biobrick
The thermoinducible construct ([http://partsregistry.org/Part:BBa_K098995 BBa_K098995]), an existing biobrick, is characterised by ligating it to GFP. The cells are grown at the low temperature of 28°C where the promoter is inactive. The temperature is then shifted up to 42°C. The GFP fluorescence is monitored throughout as an indicator of promoter activity.
The GFP fluorescence is shown to be at baseline when cells are grown at 28°C. However, when the cells undergo a temperature shift to 42°C, there is an almost immediate increase in GFP fluorescence.
This shows that the activity of the lambda promoter does increase when the temperature is shifted up, so the thermoinducible construct ([http://partsregistry.org/Part:BBa_K098995 BBa_K098995]) does become activated by higher temperatures.
About our wet lab results
References
[1] Wolfgang Jechlingera*, Michael P Szostaka 1 , Angela Wittea, Werner Lubitza (1999) Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene E expression in Escherichia coli. FEMS Microbiology Letters. 173(2), pp 347 - 352
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