Difference between revisions of "Part:BBa K782018"

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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K782018 short</partinfo>
 
<partinfo>BBa_K782018 short</partinfo>
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==Introduction==
 
==Introduction==
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Transcription activation like (TAL) effectors are proteins able to specifically bind desired DNA sequence. The central domain of the protein is constructed from variable number of tandem repeats differing only in two amino acids. The 12th and the 13th amino acid are called a “repeat variable diresidue” (RVD) and are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences.
 
Transcription activation like (TAL) effectors are proteins able to specifically bind desired DNA sequence. The central domain of the protein is constructed from variable number of tandem repeats differing only in two amino acids. The 12th and the 13th amino acid are called a “repeat variable diresidue” (RVD) and are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences.
  
Our construct contain [https://parts.igem.org/Part:BBa_K782071 ten specific binding sites] for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782006 TALB] upstream of CMV promoter. Downstream of CMV promoter we cloned yellow fluorescent protein mCitrine, an easy detectable monomer with excitation maximum at 516 nm and emission maximum at 529 nm. (Figure 1).  
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Our construct contain [https://parts.igem.org/Part:BBa_K782071 10 specific binding sites] for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782006 TALB] upstream of CMV promoter. Downstream of CMV promoter we cloned yellow fluorescent protein mCitrine, an easy detectable monomer with excitation maximum at 516 nm and emission maximum at 529 nm. (Figure 1).  
 
After binding of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782010 TALB:KRAB] on binding sites, a repression of reporter protein mCitrine occurs.  
 
After binding of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782010 TALB:KRAB] on binding sites, a repression of reporter protein mCitrine occurs.  
  
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'''Figure 1.''' Schematic representation of the construct.  
 
'''Figure 1.''' Schematic representation of the construct.  
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==Characterization==
 
==Characterization==
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[[Image:Svn_12_10xB-CMV-mCit.png‎]]
 
[[Image:Svn_12_10xB-CMV-mCit.png‎]]
  
'''Figure 2.''' Repression reporter construct by TALB:KRAB.  
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'''Figure 2.''' Repression this reporter construct by TALB:KRAB.  
  
  
 
* mCitrine was provided from host lab.
 
* mCitrine was provided from host lab.
* Binding sites for TAL effectors were ordered from IDT.  
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* Binding sites for TAL effectors were ordered from GeneArt.  
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==References==
 
==References==

Revision as of 16:41, 26 September 2012

10x[TALB] operator_CMV promoter_mCitrine


Introduction

Transcription activation like (TAL) effectors are proteins able to specifically bind desired DNA sequence. The central domain of the protein is constructed from variable number of tandem repeats differing only in two amino acids. The 12th and the 13th amino acid are called a “repeat variable diresidue” (RVD) and are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences.

Our construct contain 10 specific binding sites for TALB upstream of CMV promoter. Downstream of CMV promoter we cloned yellow fluorescent protein mCitrine, an easy detectable monomer with excitation maximum at 516 nm and emission maximum at 529 nm. (Figure 1). After binding of TALB:KRAB on binding sites, a repression of reporter protein mCitrine occurs.



10xB-pCMV-mCit.png

Figure 1. Schematic representation of the construct.


Characterization

Results: Specific TAL binding sites were further characterized.

Svn 12 10xB-CMV-mCit.png

Figure 2. Repression this reporter construct by TALB:KRAB.


  • mCitrine was provided from host lab.
  • Binding sites for TAL effectors were ordered from GeneArt.


References

Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.

Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nature Biotechnology 29, 697–698



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 720
    Illegal XhoI site found at 1350
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 147
    Illegal NgoMIV site found at 507
    Illegal AgeI site found at 12
    Illegal AgeI site found at 347
    Illegal AgeI site found at 372
    Illegal AgeI site found at 707
  • 1000
    COMPATIBLE WITH RFC[1000]