Difference between revisions of "Part:BBa K782024"

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2x[NicTAL]+2x[TALD] operator_minimal promoter_mCitrine

• TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011).

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 two 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 two consecutive specific binding sites for NicTAL12 and TALD upstream of minimal promoter and reporter protein mCitrine.

• 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).

Characterization

HEK293T cells were cotransfected with TAL activator constructs, constituitively expressed by the CMV promoter, and mCitrine reporter plasmid, containing two 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 TAL activator.

• 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