Difference between revisions of "Part:BBa K747100"
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<partinfo>BBa_K747100 short</partinfo>
 
<partinfo>BBa_K747100 short</partinfo>
  
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Precise Gene Knockout
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{{Curation/ccdb}}
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Precise Gene Knockout:
  
 
TALENs are a very powerful tool for efficient gene knockout. In order to prove that our TALEN construct was functional, we decided to simply knock out a destabilized GFP gene on a plasmid, which we co-transfected with our TALEN plasmids into HEK cells. Moreover, we also transfected our cells with an mCherry vector to normalize for transfection efficiency. TAL constructs were designed to bind to opposite strands of the target plasmid in a way that the FokI monomers of each TALEN construct would be able to dimerize in the spacer region between the TALEN binding sites. 48 hours after transfection, gene knock-out efficiency was evaluated by FACS analysis.  
 
TALENs are a very powerful tool for efficient gene knockout. In order to prove that our TALEN construct was functional, we decided to simply knock out a destabilized GFP gene on a plasmid, which we co-transfected with our TALEN plasmids into HEK cells. Moreover, we also transfected our cells with an mCherry vector to normalize for transfection efficiency. TAL constructs were designed to bind to opposite strands of the target plasmid in a way that the FokI monomers of each TALEN construct would be able to dimerize in the spacer region between the TALEN binding sites. 48 hours after transfection, gene knock-out efficiency was evaluated by FACS analysis.  
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The FokI-nuclease was inserted into the Plug and Play Effecor Cassette of the Eukaryotic TAL expression vector via golden gate cloning.
  
  
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<img src="https://static.igem.org/mediawiki/2012/7/76/Xx.png" width="450px" style="margin-left:150px"/>
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"https://static.igem.org/mediawiki/2012/7/76/Xx.png"
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 18:48, 9 July 2015

pTALEN

All ccdb parts have been withdrawn from the Registry. Samples of parts containing the ccdB gene cannot be requested. - iGEM HQ

Precise Gene Knockout:

TALENs are a very powerful tool for efficient gene knockout. In order to prove that our TALEN construct was functional, we decided to simply knock out a destabilized GFP gene on a plasmid, which we co-transfected with our TALEN plasmids into HEK cells. Moreover, we also transfected our cells with an mCherry vector to normalize for transfection efficiency. TAL constructs were designed to bind to opposite strands of the target plasmid in a way that the FokI monomers of each TALEN construct would be able to dimerize in the spacer region between the TALEN binding sites. 48 hours after transfection, gene knock-out efficiency was evaluated by FACS analysis.

The FokI-nuclease was inserted into the Plug and Play Effecor Cassette of the Eukaryotic TAL expression vector via golden gate cloning.


"Xx.png"

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 966
    Illegal suffix found in sequence at 4462
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 966
    Illegal SpeI site found at 4463
    Illegal PstI site found at 4477
    Illegal NotI site found at 972
    Illegal NotI site found at 2146
    Illegal NotI site found at 4470
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 966
    Illegal BglII site found at 1563
    Illegal BglII site found at 2914
    Illegal BamHI site found at 2217
    Illegal BamHI site found at 2920
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 966
    Illegal suffix found in sequence at 4463
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 966
    Illegal XbaI site found at 981
    Illegal SpeI site found at 4463
    Illegal PstI site found at 4477
  • 1000
    COMPATIBLE WITH RFC[1000]