Part:BBa_K782022

12x[TALD] operator_CMV promoter_fLuciferase

TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011)

Introduction

Transcription activation like (TAL) effectors are DNA binding proteins with a high specifity, built from tandem repeats, with near identical seqences, differing only in two amino acids in each repeat called “repeat variable diresidue” (RVD), which determine the specifity for a single nucleotide.(Scholze and Boch, 2011)

We designed our construct with 12 binding sites for TALD, upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter we cloned fLuciferase (firefly luciferase) to function as a reporter.

Figure1:Schematic representation of our construct

Characterisation

HEK293T cells were transfected with the 12x[TALD] operator_CMV promoter_fLuciferase reporter and TALD:KRAB (Figure 2). Tests showed, successful repression of fLuciferase with TAL repressor. Tests showed sucessful repression of the fLuciferase reporter (Figure 3).

Figure 2:Schematic representation of repression experiments. A: in the absence of a TALD:KRAB, fLuciferase is constituitively expressed. B: when a TALD:KRAB is present, it binds to its binding site upstream of the CMV promoter and represses transcription of fLuciferase with the KRAB domain.

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

• fLuciferase was contributed by host lab
• Binding sites for TAL effectors were ordered from IDT.

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

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 659
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1371
  Illegal NgoMIV site found at 2715
  Illegal NgoMIV site found at 2736
  Illegal NgoMIV site found at 3051
  Illegal AgeI site found at 408
  Illegal AgeI site found at 640
  Illegal AgeI site found at 2439
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2621