Difference between revisions of "Part:BBa K782016"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K782016 short</partinfo>
 
<partinfo>BBa_K782016 short</partinfo>
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* TALA and TALB labels represents TAL effector 1257 and 1297 respectively from zebrafish experiments (Sander et al., 2011).
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* DNA binding sites for individual TAL effectors are indicated with square brackets [ ].
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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.
  
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Our construct contain [https://parts.igem.org/Part:BBa_K782068 twelve specific binding sites] for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782007 NicTAL12] 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_K782069 10 specific binding sites] for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782004 TALA] and [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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After binding of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782011 NicTAL12:KRAB] con binding sites, a repression of reporter protein mCitrine occurs.  
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After binding of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782008 TALA:KRAB] or [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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Single binding sequence for NicTAL12 is: TCTATCAATGATAGA
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Single binding site sequence for TALA: TTTACTGCTGCTCCCGCT
  
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Single binding site sequence for TALB: TCTTCCGTTTCCACATCT
  
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[[Image:10xab.png]]
 
  
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'''Figure 1.''' Shematic representation of twelve specific binding site for TALD:KRAB
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[[Image:10ab.png]]
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'''Figure 1.''' Shematic representation of ten alternating specific binding site for TALA and TALB
 
upstream of CMV promoter and reporter protein mCitrine.  
 
upstream of CMV promoter and reporter protein mCitrine.  
  
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Results: Specific TAL binding sites were further characterized with other reporter constructs.  
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[[Image:Svn_12_10XAB_pCMV_mCit.png]]
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* mCitrine was provided from host lab.
 
* mCitrine was provided from host lab.
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* 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==
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Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.
 
Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.
  
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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  
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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.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 21:35, 26 September 2012

10x[TALA+TALB] operator_CMV promoter_mCitrine

  • TALA and TALB labels represents TAL effector 1257 and 1297 respectively from zebrafish experiments (Sander et al., 2011).
  • DNA binding sites for individual TAL effectors are indicated with square brackets [ ].


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 TALA and 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 TALA:KRAB or TALB:KRAB on binding sites, a repression of reporter protein mCitrine occurs.

Single binding site sequence for TALA: TTTACTGCTGCTCCCGCT

Single binding site sequence for TALB: TCTTCCGTTTCCACATCT


10ab.png

Figure 1. Shematic representation of ten alternating specific binding site for TALA and TALB upstream of CMV promoter and reporter protein mCitrine.


Characterization

Svn 12 10XAB pCMV mCit.png


  • 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 730
    Illegal XhoI site found at 1360
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 149
    Illegal NgoMIV site found at 514
    Illegal AgeI site found at 12
    Illegal AgeI site found at 352
    Illegal AgeI site found at 377
    Illegal AgeI site found at 717
  • 1000
    COMPATIBLE WITH RFC[1000]