Part:BBa_K782010

TALB:NLS:KRAB

TALB label represents TAL effector 1297 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).

Figure 1: Schematic representation of the repressor construct

Single binding sequence for TALB: tCTTCCGTTTCCACATCT

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).

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 3:Testing repression of reporter luciferase gene. White column is showing constantly expressed reporter (fLuc - BBa_K782021), blue column is showing repression with TALB:KRAB.

• 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