Difference between revisions of "Part:BBa K782018"

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10x[TALB] operator_CMV promoter_mCitrine

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 contain ten specific binding sites for TALB upstream of CMV promoter. Downstream of CMV 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 TALB:KRAB on binding sites, a repression of reporter protein mCitrine occurs.

Figure 1. Schematic representation of the construct.

Characterization

Results: Specific TAL binding sites were further characterized.

Figure 2. Repression of TALB:KRAB. 

• mCitrine was provided from host lab.
• Binding sites for TAL effectors were ordered from IDT.

References

Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.

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

Sequence and Features