Part:BBa_K782040

TALA based fast degradable KRAB (CL1-PEST tag)

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). On N-terminal end of TALE repressor degradation sequences CL1 and PEST that increases protein degradation were added. PEST, is a forty-amino acid sequence isolated from the C-terminal region of mouse ornithine decarboxylase, CL1 was isolated from yeast, and has also been shown to increase protein degradation.

Figure 1: Schematic representation of the repressor construct.

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 2619
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 129
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2985
  Illegal SapI.rc site found at 2949