Part:BBa_K782028

10x[TALA+TALB] 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 and TALB, 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.

Figure 1:Schematic representation of our construct.

Characterisation

HEK293T cells were cotransfected with 10x[TALA+TALB] operator_minimal promoter_mCitrine and either TALA:VP16 or TALB: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 768
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 149
  Illegal NgoMIV site found at 514
  Illegal AgeI site found at 12
  Illegal AgeI site found at 352
  Illegal AgeI site found at 377
  Illegal AgeI site found at 717
  
• 1000
  COMPATIBLE WITH RFC[1000]