Part:BBa_K782025

4x[NicTAL]+4x[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 proteins able to specifically bind desired DNA sequence. The central domain of the protein is constructed from variable number of tandem repeats differing only in two amino acids. The 12th and the 13th amino acid are called a “repeat variable diresidue” (RVD) and are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences.

Our construct contains four specific binding sites for NicTAL12 and TALD upstream of minimal promoter. Downstream of minimal promoter we cloned yellow fluorescent protein mCitrine an easy detectable monomer with excitation maximum at 516 nm and emission maximum at 529 nm (Figure 1). After binding of NicTAL12:VP16 or TALD:VP16 on binding sites, an activation of reporter protein mCitrine occurs.

Single binding sequence for NicTAL12 is: TCTATCAATGATAGA

Single binding sequence for TALD is: TCGTCCAATAGCTTCTC

Figure 1: Shematic representation of four consecutive specific binding sites for NicTAL12 and TALD upstream of minimal promoter and reporter protein mCitrine.

Characterization

HEK293T cells were cotransfected with TAL activator construct, constituitively expressed by the CMV promoter, and mCitrine mCitrine reporter plasmid, containing four binding sites for the designated TAL activator upstream of a minimal promoter (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 respective 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 NicTAL12:VP16 or TALD:VP16 .

• mCitrine was provided from host lab.
• Binding sites for TAL effectors were ordered from GeneArt.
• Minimal promotor is taken from pGL4 vector series (promotor is described [http://www.promega.com/~/media/files/resources/protocols/technical%20manuals/0/pgl4%20luciferase%20reporter%20vectors%20protocol.pdf?la=en here] on the page 18).
• VP16 domain was contributed by the host lab

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 308
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 35
  Illegal AgeI site found at 235
  
• 1000
  COMPATIBLE WITH RFC[1000]