Difference between revisions of "Part:BBa K782008"
 
(14 intermediate revisions by 4 users not shown)
Line 3: Line 3:
  
  
−
TALA label represents TAL efector 1257 from zebrafish experiments (Sander et al., 2011)
+
TALA label represents TAL effector 1257 from zebrafish experiments (Sander et al., 2011).
 +
 
  
 
==Introduction==
 
==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.  
+
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).
 
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 designed TALE-based repressors for specific gene repression, by fusing TAL effectors with the KRAB transcriptional repression domain downstream of the CMV promoter. KRAB was placed on the C-terminal ends of the TALE DNA-binding domain (Figure 1)
+
We designed TALE-based repressors for specific gene repression, by fusing TAL effectors with the KRAB transcriptional repression domain downstream of the CMV promoter. KRAB was placed on the C-terminal ends of the TALE DNA-binding domain (Figure 1).
  
−
[[Image:SVN_12_TALA_KRAB.png]]
+
[[Image:TALA_KRAB--.png]]
 +
 
 +
'''Figure 1:''' Schematic representation of the repressor construct.
 +
 
 +
 
 +
Single binding sequence for TALA:tTTACTGCTGCTCCCGCT
  
−
'''Figure 1:''' Schematic representation of the repressor construct
 
  
 
==Characterization==
 
==Characterization==
 
   
 
   
−
HEK293T cells were cotransfected with TAL repressor constructs, constituively expressed by the CMV promoter, and firefly luciferase reporter plasmids (Figure 2), containing 10 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.Tests showed that tested construct exhibited over 90% repression of the reporter plasmid (Figure 3).
+
HEK293T cells were cotransfected with TAL repressor construct, constituively expressed by the CMV promoter, and firefly luciferase [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782020 reporter plasmid] (Figure 2), containing 10 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.Tests showed that tested construct exhibited over 90% repression of the reporter plasmid (Figure 3).
  
 
[[Image:Luciferaza_reporter.png | 300 px]]
 
[[Image:Luciferaza_reporter.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.
  
  
 +
[[Image:Svn_12_talakgraf.png]]
  
 +
'''Figure 3: '''Repression of the reporter luciferase gene by TALA:KRAB repressor. White column shows the constitutively expressed reporter (fLuc - [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782020 BBa_K782020]), blue column shows the decreased reporter caused by the coexpression of TALA:KRAB repressor.
  
−
'''Figure 3:'''Testing repression of reporter gene transcription by addition of TAL repressors
 
  
  
−
+
 
 +
*KRAB domain was contributed by the host lab.
  
 
==References==
 
==References==
  
−
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  
+
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.
 +
 
 +
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 23:43, 26 September 2012

TALA:NLS:KRAB


TALA label represents TAL effector 1257 from zebrafish experiments (Sander et al., 2011).


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).

We designed TALE-based repressors for specific gene repression, by fusing TAL effectors with the KRAB transcriptional repression domain downstream of the CMV promoter. KRAB was placed on the C-terminal ends of the TALE DNA-binding domain (Figure 1).

TALA KRAB--.png

Figure 1: Schematic representation of the repressor construct.


Single binding sequence for TALA:tTTACTGCTGCTCCCGCT


Characterization

HEK293T cells were cotransfected with TAL repressor construct, constituively expressed by the CMV promoter, and firefly luciferase reporter plasmid (Figure 2), containing 10 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.Tests showed that tested construct exhibited over 90% repression of the reporter plasmid (Figure 3).

Luciferaza reporter.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 talakgraf.png

Figure 3: Repression of the reporter luciferase gene by TALA:KRAB repressor. White column shows the constitutively expressed reporter (fLuc - BBa_K782020), blue column shows the decreased reporter caused by the coexpression of TALA:KRAB repressor.



  • KRAB domain was contributed by the host lab.

References

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.

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 2439
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2805
    Illegal SapI.rc site found at 2769