Difference between revisions of "Part:BBa K1067009"

(→‎Usage and Biology)
(→‎Test in E.coli)
 
Line 19: Line 19:
  
 
===Test in ''E.coli''===
 
===Test in ''E.coli''===
 +
After constructing the brick we made a [http://2013.igem.org/Team:DTU-Denmark/Methods/Visualizing_GFP_in_the_periplasm protocol] for directing and visualizing GFP SF in the periplasm of ''E.coli''. As proof of the experiment and to show that the part is working we here include some images we produced with this protocol:
 +
 
[[File:GFP in perimplasm RFP in cytoplasm.png|thumbnail|upright=4|left|alt=Alt text|Transformed E. coli taken with a Fluorescence microscope]]
 
[[File:GFP in perimplasm RFP in cytoplasm.png|thumbnail|upright=4|left|alt=Alt text|Transformed E. coli taken with a Fluorescence microscope]]
 
[[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]]
 
[[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]]
 
[[File:Graf.PNG|thumbnail|upright=2|left|alt=Alt text|Line profile of the cross section of the cell seen in the above close up]]
 
[[File:Graf.PNG|thumbnail|upright=2|left|alt=Alt text|Line profile of the cross section of the cell seen in the above close up]]

Latest revision as of 16:16, 4 October 2013

Periplasm transport visualization device


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.


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.

For better characterization follow this [http://2013.igem.org/Team:DTU-Denmark/HelloWorld link] to DTU iGEM 2013.

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

After constructing the brick we made a [http://2013.igem.org/Team:DTU-Denmark/Methods/Visualizing_GFP_in_the_periplasm protocol] for directing and visualizing GFP SF in the periplasm of E.coli. As proof of the experiment and to show that the part is working we here include some images we produced with this protocol:

Alt text
Transformed E. coli taken with a Fluorescence microscope
Alt text
Close up of several cells, showing GFP expression in the periplasm
Alt text
Line profile of the cross section of the cell seen in the above close up