Difference between revisions of "Part:BBa K782001"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K782001 short</partinfo>
 
<partinfo>BBa_K782001 short</partinfo>
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* TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011).
 
* TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011).
 +
* DNA binding sites for individual TAL effectors are indicated with square brackets [ ].
 +
  
 
==Introduction==
 
==Introduction==
  
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Transcription activation like (TAL) effectors are bacterial plant pathogen transcription factors that bind to DNA by recognizing a specific DNA sequence in which each base pair binds a single tandem repeat in in the TAL DNA-binding domain. A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD) are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011).  All TAL repeats have almost identical sequences, differing only in the RVDs. This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences by designing specific binding domains for a selected TAL effector. We designed two consecutive specific binding sites for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782011 NicTAL12:KRAB] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782009 TALD:KRAB] upstream of CMV promoter (Figure 1), that cause a repression of reporter protein mCitrine, after binding of NicTAL12:KRAB or TALD:KRAB. mCitrine is yellow fluorescent protein.  
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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.
  
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Single binding sequence for NicTAL is: TCTATCAATGATAGA
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Our construct contains [https://parts.igem.org/Part:BBa_K782067 two specific binding sites] for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782007 NicTAL12] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782005 TALD]  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] or [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782009 TALD:KRAB] on binding sites, a repression of reporter protein mCitrine occurs.
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Single binding sequence for NicTAL12 is: TCTATCAATGATAGA  
  
 
Single binding sequence for TALD is: TCGTCCAATAGCTTCTC
 
Single binding sequence for TALD is: TCGTCCAATAGCTTCTC
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[[Image:11xTALD.png]]
 
[[Image:11xTALD.png]]
  
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Figure 1. Shematic representation of two consecutive specific binding sites for NicTAL and TALD  
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'''Figure 1.''' Shematic representation of two consecutive specific binding sites for NicTAL12:KRAB and TALD:KRAB
 
upstream of CMV promoter and reporter protein mCitrine.  
 
upstream of CMV promoter and reporter protein mCitrine.  
  
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==Characterization==
  
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Results: Specific TAL [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782024 binding sites] were further characterized with other reporter constructs.
  
 
* 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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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.
  
  

Latest revision as of 21:31, 26 September 2012

2x[NicTAL]+2x[TALD] operator_CMV promoter_mCitrine


  • TALD label represents TAL effector 1295 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 contains two specific binding sites for NicTAL12 and TALD 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 NicTAL12:KRAB or TALD:KRAB on binding sites, a repression of reporter protein mCitrine occurs.

Single binding sequence for NicTAL12 is: TCTATCAATGATAGA

Single binding sequence for TALD is: TCGTCCAATAGCTTCTC


11xTALD.png

Figure 1. Shematic representation of two consecutive specific binding sites for NicTAL12:KRAB and TALD:KRAB upstream of CMV promoter and reporter protein mCitrine.

Characterization

Results: Specific TAL binding sites were further characterized with other reporter constructs.

  • 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 140
    Illegal XhoI site found at 770
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 35
    Illegal AgeI site found at 105
  • 1000
    COMPATIBLE WITH RFC[1000]