Difference between revisions of "Part:BBa K782008"

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[[Image:Svn_12_talakgraf.png]]
 
[[Image:Svn_12_talakgraf.png]]
  
'''Figure 3:'''Testing repression of reporter Luciferase gene. White column shows expression of reporter without repressor, blue column shows expression of reporter after addition of TAL repressor.
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'''Figure 3:'''Testing repression of reporter luciferase gene. White column is showing constantly expressed reporter (fLuc), blue column is showing repression with TALA:KRAB.
  
  

Revision as of 14:42, 25 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)

SVN 12 TALA KRAB.png

Figure 1: Schematic representation of the repressor construct

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).

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:Testing repression of reporter luciferase gene. White column is showing constantly expressed reporter (fLuc), blue column is showing repression with TALA:KRAB.


Single binding sequence for TALA:tTTACTGCTGCTCCCGCT


  • 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