Difference between revisions of "Part:BBa K200022"

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This part comprises the heat sensitive Lamda cI Repressor designed by Harvard iGEM 2008 ([[Part:BBa_K098995 |BBa_K098995]]) with a standard GFP reporter output ([[Part:BBa_I13504 |BBa_I13504]]). This part was used as a test construct, to check the functionality of the part.  
 
This part comprises the heat sensitive Lamda cI Repressor designed by Harvard iGEM 2008 ([[Part:BBa_K098995 |BBa_K098995]]) with a standard GFP reporter output ([[Part:BBa_I13504 |BBa_I13504]]). This part was used as a test construct, to check the functionality of the part.  
  
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We have shown BBa_K098995 to be functional and have improved its characterisation through successful testing with this construct containing a GFP reporter. By measuring GFP flourescence at 28 degrees, 42 degrees and with a temperature shift from 28 to 42 degrees, the part showed functionality.  
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We have shown [[Part:BBa_K098995 |BBa_K098995]] to be functional and have improved its characterisation through successful testing with this construct containing a GFP reporter. By measuring GFP flourescence at 28 degrees, 42 degrees and with a temperature shift from 28 to 42 degrees, the part showed functionality.  
  
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K200022 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K200022 SequenceAndFeatures</partinfo>
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<br>
  
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===Characterisation===
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In order to characterize the thermoinducible promoter properly, we have used 2 sets of control cells (not shown in graph): <br>
 +
*Positive control cells: Containing the BBa_I13522, acting as a baseline comparison by constitutively expressing GFP.
 +
*Negative control cells: These contain the thermoinducible promoter on its own ( BBa_K098995) with no GFP attached to it. These serve as cells without any fluorescence.
 +
*Cells shifted from 28 to 42 degrees. This characterises the transition stage.
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 +
<br>
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[[Image:II09_Thermoconstruct_main2.jpg|500px]]<br>
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The characterization of the thermoinducible promoter is as follows:<br>
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*At 28 ºC, there is no fluorescence (GFP) output because its expression is repressed
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*At 42 ºC, we can observe an increase in fluorescence (GFP) output,as its expression is no longer repressed
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Hence, we can conclude that an increase in temperature de-represses the expression of the thermoinducible system.<br>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 01:05, 22 October 2009

Heat-inducible system with GFP reporter

Ligation of heat repressible regulation system to GFP to assay its activity.

This part comprises the heat sensitive Lamda cI Repressor designed by Harvard iGEM 2008 (BBa_K098995) with a standard GFP reporter output (BBa_I13504). This part was used as a test construct, to check the functionality of the part.

We have shown BBa_K098995 to be functional and have improved its characterisation through successful testing with this construct containing a GFP reporter. By measuring GFP flourescence at 28 degrees, 42 degrees and with a temperature shift from 28 to 42 degrees, the part showed functionality.


Usage and Biology

The Imperial iGEM 2009 Team successfully used this construct to show the functionality of this composite part. By measuring the increase in GFP flourescence over a temperature shift, the part was shown to be working successfully. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1599


Characterisation

In order to characterize the thermoinducible promoter properly, we have used 2 sets of control cells (not shown in graph):

  • Positive control cells: Containing the BBa_I13522, acting as a baseline comparison by constitutively expressing GFP.
  • Negative control cells: These contain the thermoinducible promoter on its own ( BBa_K098995) with no GFP attached to it. These serve as cells without any fluorescence.
  • Cells shifted from 28 to 42 degrees. This characterises the transition stage.


II09 Thermoconstruct main2.jpg
The characterization of the thermoinducible promoter is as follows:

  • At 28 ºC, there is no fluorescence (GFP) output because its expression is repressed
  • At 42 ºC, we can observe an increase in fluorescence (GFP) output,as its expression is no longer repressed

Hence, we can conclude that an increase in temperature de-represses the expression of the thermoinducible system.