Difference between revisions of "Part:BBa K1067001"

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(→‎Functional Parameters)
 
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This biobrick is a part composed of GFP SF exported to the periplasm by twin-arginine translocation (TAT) signal peptide. In addition this is transcriptionally fused to a RFP with RBS associated.  
 
This biobrick is a part composed of GFP SF exported to the periplasm by twin-arginine translocation (TAT) signal peptide. In addition this is transcriptionally fused to a RFP with RBS associated.  
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This brick have been inspired by [http://2011.igem.org/Team:Cambridge/Experiments/Periplasmic_Export#/Experiments/Periplasmic_Export Cambridge iGEM team 2011].
  
  
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The TAT pathway can also be used to export fluorescence protein to the periplasm. Advantages when using TAT pathway instead of Sec to export fluorescence protein is that the fluorophore cannot form under the redox state in the periplasm. While the TAT pathway transport an already folded protein that have formed a fluorophore the Sec pathway transport an unfolded state of the protein and the fluorophore will have to form inside the periplasm.   
 
The TAT pathway can also be used to export fluorescence protein to the periplasm. Advantages when using TAT pathway instead of Sec to export fluorescence protein is that the fluorophore cannot form under the redox state in the periplasm. While the TAT pathway transport an already folded protein that have formed a fluorophore the Sec pathway transport an unfolded state of the protein and the fluorophore will have to form inside the periplasm.   
  
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The ration of expression between GFP SF and RFP can be tuned by switching out the RBS sequence upstream GFP SF.
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The ration of expression between GFP SF and RFP can be tuned by switching out the RBS sequence upstream GFP SF.  
 +
 
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The brick have been tested with both a constitutive promoter and a inducible one. It seems like the ability to visualize,on a fluorescence microscope, that the GFP SF is exported to the periplasm, requires that there is only a very low concentration i GFP SF inside the cytoplasm. So when putting the brick downstream a constitutive promoter we could not distinguish between periplasm and cytoplasm on the fluorescence microscope. The same thing was apparent when a leaky inducible promoter (like [[Part:BBa_K808000]]) was used. First after we used an optimized "tight" pBAD promoter (like [[Part:BBa_K1067007]]) we where able to distinguish clearly between cytoplasm and periplasm. See Team DTU 2013 [http://2013.igem.org/Team:DTU-Denmark wiki].
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1067001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1067001 SequenceAndFeatures</partinfo>
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===Test in ''E.coli''===
 
===Test in ''E.coli''===
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The brick was tested by setting it downstream our engineered [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1067007 araBAD system]. This can also be found as a biobrick ([[part:BBa_K1067009]]).
  
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[[File:GFP in perimplasm RFP in cytoplasm.png|frame|600px|alt=Alt text|Caption text]]
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Following is an image taken with fluorescence microscope that show how the part works under optimal conditions.  
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[[File:GFP in perimplasm RFP in cytoplasm close up.png|600px]]
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[[File:GFP in perimplasm RFP in cytoplasm close up.png|thumbnail|upright=2|left|alt=Alt text|Close up of several cells, showing GFP expression in the periplasm]]
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[[File:Graf.PNG|600px]]
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frame|none|alt=Alt text|Caption text]].
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===Functional Parameters===
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Latest revision as of 16:26, 4 October 2013

Twin-arginine translocation reporter

This biobrick is a part composed of GFP SF exported to the periplasm by twin-arginine translocation (TAT) signal peptide. In addition this is transcriptionally fused to a RFP with RBS associated.

This brick have been inspired by [http://2011.igem.org/Team:Cambridge/Experiments/Periplasmic_Export#/Experiments/Periplasmic_Export Cambridge iGEM team 2011].


Usage and Biology

The twin-arginine translocation is a pathway for secretion of proteins in prokaryotes but can also be found in plants and archaea. The TAT pathway unlike the Sec pathway in E.coli transport the proteins after folding. This can be practical when working with proteins that cannot fold properly in the periplasm because of the different redox state compared to the cytoplasm.

The TAT pathway can also be used to export fluorescence protein to the periplasm. Advantages when using TAT pathway instead of Sec to export fluorescence protein is that the fluorophore cannot form under the redox state in the periplasm. While the TAT pathway transport an already folded protein that have formed a fluorophore the Sec pathway transport an unfolded state of the protein and the fluorophore will have to form inside the periplasm.

The ration of expression between GFP SF and RFP can be tuned by switching out the RBS sequence upstream GFP SF.

The brick have been tested with both a constitutive promoter and a inducible one. It seems like the ability to visualize,on a fluorescence microscope, that the GFP SF is exported to the periplasm, requires that there is only a very low concentration i GFP SF inside the cytoplasm. So when putting the brick downstream a constitutive promoter we could not distinguish between periplasm and cytoplasm on the fluorescence microscope. The same thing was apparent when a leaky inducible promoter (like Part:BBa_K808000) was used. First after we used an optimized "tight" pBAD promoter (like Part:BBa_K1067007) we where able to distinguish clearly between cytoplasm and periplasm. See Team DTU 2013 [http://2013.igem.org/Team:DTU-Denmark wiki].

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1225
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1225
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1225
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1225
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 149

Test in E.coli

The brick was tested by setting it downstream our engineered araBAD system. This can also be found as a biobrick (part:BBa_K1067009).

Following is an image taken with fluorescence microscope that show how the part works under optimal conditions.

Alt text
Close up of several cells, showing GFP expression in the periplasm