Difference between revisions of "Part:BBa K782011"

(Introduction)
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K782011 short</partinfo>
 
<partinfo>BBa_K782011 short</partinfo>
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==Introduction==
 
==Introduction==
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The Kruppel-associated box (KRAB) domain is a transcriptional repressor module commonly found in eukaryotic zinc finger proteins. When KRAB containing protein binds to corresponding DNA sequence it triggers recruitment of Kap1 corepressor. KRAB domain directly interacts with a RING-B-box-coiled-coil (RBCC) domain of corepressor protein Kap1. When Kap1 binds to a KRAB domain it functions as a scaffold and starts to recruits heterocromatin protein 1 isoforms (HP1-α HP1-β HP1-γ), histone deacetylases (HDACs) and Setdb1. This complex forms facultative heterocromatin environment on a target promoter and mediates transcriptional repression (Urrutia, 2003).  
 
The Kruppel-associated box (KRAB) domain is a transcriptional repressor module commonly found in eukaryotic zinc finger proteins. When KRAB containing protein binds to corresponding DNA sequence it triggers recruitment of Kap1 corepressor. KRAB domain directly interacts with a RING-B-box-coiled-coil (RBCC) domain of corepressor protein Kap1. When Kap1 binds to a KRAB domain it functions as a scaffold and starts to recruits heterocromatin protein 1 isoforms (HP1-α HP1-β HP1-γ), histone deacetylases (HDACs) and Setdb1. This complex forms facultative heterocromatin environment on a target promoter and mediates transcriptional repression (Urrutia, 2003).  
 
   
 
   
We discovered that [https://parts.igem.org/wiki/index.php?title=Part:BBa_K323214 NicTAL part] deposited in the Registry by the Slovenian iGEM2010 team  was missing subdomain in DNA-binding domain, so our team added this missing part[https://parts.igem.org/wiki/index.php?title=Part:BBa_K782007] along with the KRAB domain.  
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NicTAL was originally designed by iGEM 2010 team Slovenia. But we discovered that [https://parts.igem.org/wiki/index.php?title=Part:BBa_K323214 NicTAL10] deposited in the Registry by the Slovenian iGEM2010 team  was missing subdomain in DNA-binding domain and was non functional. Our team added this missing amino acids [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782007] along with the KRAB domain.  
  
  
[[Image:SVN_12_NicTAL12K.png]]
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[[Image:Svn_12_NicTAL12K--.png]]
  
 
'''Figure 1:''' Schematic representation of the repressor construct.
 
'''Figure 1:''' Schematic representation of the repressor construct.
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Single binding sequence for NicTAL: TCTATCAATGATAGA
 
Single binding sequence for NicTAL: TCTATCAATGATAGA
 +
  
 
==Characterization==
 
==Characterization==
  
HEK293T cells were cotransfected with NicTAL repressor constructs, constituively expressed by the CMV promoter, and mCitrine [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782015 reporter plasmids] (Figure 2), containing 12 binding sites for the designated TAL repressor upstream of the CMV promoter. All experiments were executed in 3 biological replicates and repeated over 3 times with similar results. (Figure 3).
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HEK293T cells were cotransfected with NicTAL repressor construct, constituively expressed by the CMV promoter, and fLuciferase [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782023 reporter plasmid] (Figure 2), containing 12 binding sites for the designated TAL repressor upstream of the CMV promoter. All experiments were executed in 3 biological replicates and repeated over 3 times with similar results. (Figure 3).
 
Results demonstrate excellent repression ability by NicTAL12:KRAB, improved by the 2012 team (Figure 3).  
 
Results demonstrate excellent repression ability by NicTAL12:KRAB, improved by the 2012 team (Figure 3).  
  
[[Image:SVN_12_reporter_citrin.png | 300 px]]
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[[Image:Svn_12_represija-NicAl_K-shema.png | 300 px]]
  
 
'''Figure 2:''' Schematic representation of repression experiments. A: in the absence of a TAL repressor, the reporter gene is constituitively expressed. B: when a TAL repressor is present, it binds to its respective binding site upstream of the CMV promoter and represses transcription of the reporter gene with the KRAB domain.
 
'''Figure 2:''' Schematic representation of repression experiments. A: in the absence of a TAL repressor, the reporter gene is constituitively expressed. B: when a TAL repressor is present, it binds to its respective binding site upstream of the CMV promoter and represses transcription of the reporter gene with the KRAB domain.
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[[Image:Svn_12_NicTAL_graf.png]]
 
[[Image:Svn_12_NicTAL_graf.png]]
  
'''Figure 3:''' Testing repression of reporter mCitrin gene. White column is showing constantly expressed reporter (mCit), blue column is showing repression with NicTAL10:KRAB or NicTAL12:KRAB.
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'''Figure 3:''' Testing repression of reporter fLuciferase gene. White column is showing constantly expressed reporter (fLuc - [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782023 BBa_K782023]), blue column is showing repression with NicTAL10:KRAB and NicTAL12:KRAB.
 
+
  
  
  
*KRAB domain was contributed by the host lab
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*KRAB domain was contributed by the host lab.
  
 
==References==
 
==References==
  
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.
  
Urrutia R. (2003) KRAB-containing zinc-finger repressor proteins. Genome Biology 4:231
+
Urrutia R. (2003) KRAB-containing zinc-finger repressor proteins. Genome Biology 4, 231.
  
  

Latest revision as of 23:40, 26 September 2012

NicTAL12:NLS:KRAB


Introduction

TAL effectors (TALEs) are bacterial plant pathogen transcription factors, that bind to DNA by specifically recognizing one base pair with a single tandem repeat in their DNA-binding domain. A tandem TALE repeat contains 33 to 35 amino acids, where the 12th and 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011).

The Kruppel-associated box (KRAB) domain is a transcriptional repressor module commonly found in eukaryotic zinc finger proteins. When KRAB containing protein binds to corresponding DNA sequence it triggers recruitment of Kap1 corepressor. KRAB domain directly interacts with a RING-B-box-coiled-coil (RBCC) domain of corepressor protein Kap1. When Kap1 binds to a KRAB domain it functions as a scaffold and starts to recruits heterocromatin protein 1 isoforms (HP1-α HP1-β HP1-γ), histone deacetylases (HDACs) and Setdb1. This complex forms facultative heterocromatin environment on a target promoter and mediates transcriptional repression (Urrutia, 2003).

NicTAL was originally designed by iGEM 2010 team Slovenia. But we discovered that NicTAL10 deposited in the Registry by the Slovenian iGEM2010 team was missing subdomain in DNA-binding domain and was non functional. Our team added this missing amino acids [1] along with the KRAB domain.


Svn 12 NicTAL12K--.png

Figure 1: Schematic representation of the repressor construct.


Single binding sequence for NicTAL: TCTATCAATGATAGA


Characterization

HEK293T cells were cotransfected with NicTAL repressor construct, constituively expressed by the CMV promoter, and fLuciferase reporter plasmid (Figure 2), containing 12 binding sites for the designated TAL repressor upstream of the CMV promoter. All experiments were executed in 3 biological replicates and repeated over 3 times with similar results. (Figure 3). Results demonstrate excellent repression ability by NicTAL12:KRAB, improved by the 2012 team (Figure 3).

Svn 12 represija-NicAl K-shema.png

Figure 2: Schematic representation of repression experiments. A: in the absence of a TAL repressor, the reporter gene is constituitively expressed. B: when a TAL repressor is present, it binds to its respective binding site upstream of the CMV promoter and represses transcription of the reporter gene with the KRAB domain.


Svn 12 NicTAL graf.png

Figure 3: Testing repression of reporter fLuciferase gene. White column is showing constantly expressed reporter (fLuc - BBa_K782023), blue column is showing repression with NicTAL10:KRAB and NicTAL12:KRAB.


  • KRAB domain was contributed by the host lab.

References

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

Urrutia R. (2003) KRAB-containing zinc-finger repressor proteins. Genome Biology 4, 231.


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 2133
    Illegal XhoI site found at 1224
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2499
    Illegal SapI.rc site found at 2463