Part:BBa_K782031

12x[TALD] operator_minimal promoter_mCitrine

• TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011)
• DNA binding sites for individual TAL effectors are indicated with square brackets [ ].

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 minimal promoter. Downstream of the promoter we cloned the yellow fluorescent protein mCitrine an easy detectable monomer with excitation maximum at 516 nm and emission maximum at 529 nm.

Figure 1: Schematic representation of our construct

Characterisation

HEK293T cells were cotransfected with 10x[TALA] operator_minimal promoter_mCitrine and TALD:VP16 (Figure 2). All experiments were executed in 3 biological replicates and repeated over 3 times with similar results.

Figure 2: Schematic representation of activation experiments. A: in the absence of a TAL activator, the expression of the reporter gene is repressed. B: when TAL activator is present, it binds to its binding site upstream of the minimal promoter and activates transcription of the reporter gene with the VP16 domain.

Figure 3: Testing activation of reporter gene transcription by addition of TAL activator.

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 XhoI site found at 503
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 214
  Illegal AgeI site found at 446
  
• 1000
  COMPATIBLE WITH RFC[1000]