Part:BBa_K782011

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.

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).

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 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