Difference between revisions of "Part:BBa K838000:Experience"

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__NOTOC__
 
__NOTOC__
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In order to fully check that this fusion protein works as expected, we need to check for it's presence, correct folding and import into the nucleus. Here we detail the two first points we managed to prove successfully. The third point was tested as well, but we did not manage to obtain conclusive enough results.
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== Western blot ==
 
== Western blot ==
  
 
=== Idea ===
 
=== Idea ===
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LovTAP-VP16 contains the C domain of VP16, therefore with a VP16 antibody it is possible to detect LovTAP in a Western blot.
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LovTAP-VP16 contains the C domain of VP16, therefore with a VP16 antibody it is possible to detect LovTAP in a Western blot. We can therefore use a commercially available [http://www.abcam.com/VP16-tag-antibody-ab4808.html anti-VP16] antibody and GAL4-VP16 protein as positive control.
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=== Experimental Setup ===
 
=== Experimental Setup ===
  
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We transfected the CHO (strain dg44) cells the evening before the experiment with 100% pcDNA3-LovTAP, turned on the blue light for 2 hours the next morning, took samples and performed the Western blot using [http://2012.igem.org/Team:EPF-Lausanne/Protocol/Western_Blot this] protocol.
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We transfected the CHO (strain DG44) cells the evening before the experiment with 100% pcDNA3-LovTAP, turned on the blue light for 2 hours the next morning, took samples and performed the Western blot using [http://2012.igem.org/Team:EPF-Lausanne/Protocol/Western_Blot this] protocol.
 
We did the same with HEK cells.
 
We did the same with HEK cells.
  
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=== Idea ===
 
=== Idea ===
  
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The photosensitive lov2 domain in LovTAP-VP16 has an exploitable characteristic which is purple autofluorescence. We decided to use this feature to detect by flow cytometry if the lov2 domain is present and folded correctly.
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=== Experimental Setup ===
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 +
CHO cells (strain DG44) were transfected with 100% pcDNA3-LovTAP-VP16 and were sampled 24 hours after transfection.
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The sampling was done using [http://2012.igem.org/Team:EPF-Lausanne/Protocol/FACS this protocol] and brought them to the flow cytometry facility at our school.
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=== Results ===
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[[Image:Lov_auto_fluo.JPG]]
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On the left: Untransfected cells.
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On the right: LovTAP-VP16 transfected cells.
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On the x-axis we see the fluorescence intensity and on the y-axis the number of cells.
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We can clearly see that a second peak appears in the cells transfected with LovTAP-VP16 in contrast to cells which were not transfected. This tells us that in the transfected cells population, a proportion of cells start expression Lov2 domain, which causes this peak.
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=== Conclusion ===
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From these two experiments so far, we know that the VP16 activator domain is present, as well as the very essential lov2 domain (part of the protein which reacts to light) and is also correctly folded.
  
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We could not obtain conclusive data from microscopy regarding the functionality of the Nuclear Localization Signal (NLS), meaning we could not clearly observe lov2 autofluorescence inside the nucleus or outside.
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 06:38, 6 October 2012


In order to fully check that this fusion protein works as expected, we need to check for it's presence, correct folding and import into the nucleus. Here we detail the two first points we managed to prove successfully. The third point was tested as well, but we did not manage to obtain conclusive enough results.


Western blot

Idea

LovTAP-VP16 contains the C domain of VP16, therefore with a VP16 antibody it is possible to detect LovTAP in a Western blot. We can therefore use a commercially available [http://www.abcam.com/VP16-tag-antibody-ab4808.html anti-VP16] antibody and GAL4-VP16 protein as positive control.

Experimental Setup

We transfected the CHO (strain DG44) cells the evening before the experiment with 100% pcDNA3-LovTAP, turned on the blue light for 2 hours the next morning, took samples and performed the Western blot using [http://2012.igem.org/Team:EPF-Lausanne/Protocol/Western_Blot this] protocol. We did the same with HEK cells.

Results

Lane 1: VP16 positive control (VP16 protein) Lane 3: Untransfected CHO cells Lane 4-5: LovTAP-VP16 transfected CHO cells Lane 6: Untransfected HEK cells Lane 7-8: LovTAP-VP16 transfected HEK cells.


WB.JPG

We can clearly see that the VP16 antibody recognized a VP16 domain in lanes 4-5 and 7-8, which are the LovTAP-VP16 transfected cells. We can thus say that the cells expressed a protein with a VP16 domain.

Flow cytometry

Idea

The photosensitive lov2 domain in LovTAP-VP16 has an exploitable characteristic which is purple autofluorescence. We decided to use this feature to detect by flow cytometry if the lov2 domain is present and folded correctly.

Experimental Setup

CHO cells (strain DG44) were transfected with 100% pcDNA3-LovTAP-VP16 and were sampled 24 hours after transfection. The sampling was done using [http://2012.igem.org/Team:EPF-Lausanne/Protocol/FACS this protocol] and brought them to the flow cytometry facility at our school.

Results

Lov auto fluo.JPG

On the left: Untransfected cells. On the right: LovTAP-VP16 transfected cells.

On the x-axis we see the fluorescence intensity and on the y-axis the number of cells.

We can clearly see that a second peak appears in the cells transfected with LovTAP-VP16 in contrast to cells which were not transfected. This tells us that in the transfected cells population, a proportion of cells start expression Lov2 domain, which causes this peak.

Conclusion

From these two experiments so far, we know that the VP16 activator domain is present, as well as the very essential lov2 domain (part of the protein which reacts to light) and is also correctly folded.

We could not obtain conclusive data from microscopy regarding the functionality of the Nuclear Localization Signal (NLS), meaning we could not clearly observe lov2 autofluorescence inside the nucleus or outside.

User Reviews

UNIQa5e0e92407c2517a-partinfo-00000000-QINU UNIQa5e0e92407c2517a-partinfo-00000001-QINU