Part:BBa_K782029

10x[TALA] operator_minimal promoter_mCitrine

• TALA and TALB labels represents TAL effectors 1257 and 1297 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 10 binding sites for TALA, upstream of a minimal promoter (like here). 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.

Figure1:Schematic representation of our construct

Characterisation

HEK293T cells were cotransfected with 10x[TALA] operator_minimal promoter_mCitrine and TALA: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 758
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 147
  Illegal NgoMIV site found at 507
  Illegal AgeI site found at 12
  Illegal AgeI site found at 347
  Illegal AgeI site found at 372
  Illegal AgeI site found at 707
  
• 1000
  COMPATIBLE WITH RFC[1000]