Difference between revisions of "Part:BBa K533005:Experience"

 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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===Applications of BBa_K533005===
 
===Applications of BBa_K533005===
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This is a surface marker for E. coli, although it can serve other purposes.
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We verified its membrane location by different means.
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====Protease digestion====
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In order to determine whether GFP is presented outside the membrane, cells were digested with Protease K at 50 centigrade for 30min.
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{| align="center" style="border: none; background: none; border-spacing: 0;text-align:center;margin-left: 100px;"
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! style="border: 1px solid #39F;padding: 5px;" |  Color Channel
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! style="border: 1px solid #39F;padding: 5px;" |  Post-digest
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! style="border: 1px solid #39F;padding: 5px;" |  Pre-digest
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|-
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|  style="border: 1px solid #39F;padding: 5px;" | GFP
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| style="border: 1px solid #39F;padding: 5px;" |  [[Image:Thuexp_gn.png|140px]]
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| style="border: 1px solid #39F;padding: 5px;" |  [[Image:Thuexp_gp.png|140px]]
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|-
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| style="border: 1px solid #39F;padding: 5px;" |  DAPI
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| style="border: 1px solid #39F;padding: 5px;" |  [[Image:Thuexp_gn_d.png|140px]]
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| style="border: 1px solid #39F;padding: 5px;" |  [[Image:Thuexp_gp_d.png|140px]]
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|-
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| style="border: 1px solid #39F;padding: 5px;" |  Merge
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| style="border: 1px solid #39F;padding: 5px;" |  [[Image:Thuexp_gn_m.png|140px]]
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| style="border: 1px solid #39F;padding: 5px;" |  [[Image:Thuexp_gp_m.png|140px]]
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|}
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The fluorescence disappeared after digesion, suggesting that the majority of GFP is on the outside of E. coli.
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====Fractionation and Western blotting====
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The cell components were first fractionated before blotting.
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1. Sonicate the bacteria culture.
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2. Centrifuge 13000rpm, 30min to separate soluble protein and membrane components from the cell debris and the non-soluble protein.
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3. 51000rpm 1h to separate soluble protein from membrane components.
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[[Image:Thuwb.png]]
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The majority of the OmpA-GFP protein is in the pellet after the first centrifuge, indicating incomplete cell lysis or inefficient protein folding. Nonetheless, from the later result, we can see that it is exclusively in the membrane and not in the soluble proteins.
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 15:41, 5 October 2011

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K533005

This is a surface marker for E. coli, although it can serve other purposes.

We verified its membrane location by different means.

Protease digestion

In order to determine whether GFP is presented outside the membrane, cells were digested with Protease K at 50 centigrade for 30min.

Color Channel Post-digest Pre-digest
GFP Thuexp gn.png Thuexp gp.png
DAPI Thuexp gn d.png Thuexp gp d.png
Merge Thuexp gn m.png Thuexp gp m.png

The fluorescence disappeared after digesion, suggesting that the majority of GFP is on the outside of E. coli.

Fractionation and Western blotting

The cell components were first fractionated before blotting.

1. Sonicate the bacteria culture.

2. Centrifuge 13000rpm, 30min to separate soluble protein and membrane components from the cell debris and the non-soluble protein.

3. 51000rpm 1h to separate soluble protein from membrane components.

Thuwb.png

The majority of the OmpA-GFP protein is in the pellet after the first centrifuge, indicating incomplete cell lysis or inefficient protein folding. Nonetheless, from the later result, we can see that it is exclusively in the membrane and not in the soluble proteins.

User Reviews

UNIQ448b2bdc3c07e6d1-partinfo-00000000-QINU UNIQ448b2bdc3c07e6d1-partinfo-00000001-QINU