Difference between revisions of "Part:BBa K782023"

Latest revision as of 22:06, 26 September 2012

12x[NicTAL] operator_CMV promoter_fLuciferase

• DNA binding sites for individual TAL effectors are indicated with square brackets [ ].

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 12 binding sites for NicTAL (as described here), 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.

Figure 1:Schematic representation of our construct.

Characterisation

HEK293T cells were transfected with the 12x[NicTAL] operator_CMV promoter_fLuciferase reporter and NicTAL:KRAB (Figure 2). Tests showed, successful repression of fLuciferase with TAL repressor. Tests showed sucessful repression of the fLuciferase reporter (Figure 3).

Figure 2:Schematic representation of repression experiments. A: in the absence of a NicTAL:KRAB, fLuciferase is constituitively expressed. B: when a NicTAL:KRAB is present, it binds to its binding site upstream of the CMV promoter and represses transcription of fLuciferase with the KRAB domain.

Figure 3:Testing repression of reporter luciferase gene. Light-blue column is showing constantly expressed reporter (fLuc), dark-blue column is showing repression of fLuc reporter with NicTAL:KRAB.

• fLuciferase was contributed by 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.

Sequence and Features