Difference between revisions of "Part:BBa K782026"

Latest revision as of 21:43, 26 September 2012

7x[TALA+TALB] operator_minimal promoter_mCitrine

• TALA and TALB labels represents TAL effector 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 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 seven specific binding sites for TALA and TALB 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 TALA:VP16 or TALB:VP16 on binding sites, an activation of reporter protein mCitrine occurs.

Single binding site sequence for TALA: TTTACTGCTGCTCCCGCT

Single binding site sequence for TALB: TCTTCCGTTTCCACATCT

Figure 1: Shematic representation of our construct.

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

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