Difference between revisions of "Part:BBa K782020"

Revision as of 07:53, 26 September 2012

10x[TALA] operator_CMV promoter_fLuciferase

TALA label represents TAL effector 1257 from zebrafish experiments (Sander et al., 2011)

Introduction

Transcription activation like (TAL) effectors are DNA binding proteins with a high specifity, built from tandem repeats, with near identical seqences, differing only in two amino acids in each repeat called “repeat variable diresidue” (RVD), which determine the specifity for a single nucleotide.(Scholze and Boch, 2011)

We designed our construct with 10 binding sites for TALA, upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter we cloned fLuciferase (firefly luciferase) to function as a reporter.

Figure1:Schematic representation of our construct

Characterisation

HEK293T cells were transfected with the 10x[TALA] operator_CMV promoter_fLuciferase reporter and TALA:NLS:KRAB (Figure 2). Tests showed, successful repression of fLuciferase with either TAL repressor.

Figure 3:Testing repression of reporter luciferase gene. White column is showing constantly expressed reporter (fLuc), blue column is showing repression with TALA:KRAB.

Sequence and Features