Difference between revisions of "Part:BBa K782024"

Revision as of 14:26, 26 September 2012

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:KRAB 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 CMV 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

Sequence and Features