Difference between revisions of "Part:BBa K782084"

Revision as of 14:55, 25 September 2012

10x[TALA] operator_CMV promoter_TALB:NLS:KRAB_t2a_mNeptune

Introduction

This part combined with 10x[TALB] operator_CMV promoter_TALA:KRAB:NLS_t2a_mCitrine is the key element of the mutual repressor switch.

The part contains 10 repeats of TALA binding sites [1] upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter, there is the TAL repressor TALB:KRAB [2] linked to the red fluorescent protein mNeptune, by a t2A sequence. The t2A sequence interrupts protein translation and causes TALB:KRAB and mNeptune to be expressed in equimolar amounts as separate proteins (Garg et.al, 2012). mNeptune functions as a reporter for the expression of TALB:KRAB, it is a red fluorescent protein with an excitation peak at 600 nm and emission peak at 650 nm and brightness of 13.4.

Figure 1: Schematic representation of the construct

Characterization

HEK293T cells were transfected with 10×[A]_PCMV_TALB:KRAB_mNeptune and visualized under confocal microscope. Cells were shown to fluoresce with mNeptune, showing that mNeptune is expressed.

Figure 2: Cells transfected with 10×[A]_PCMV_TALB:KRAB_mNeptune, fluorescing with mNeptune

HEK293T cells were transfected with 10×[A]_PCMV_TALB:KRAB_mNeptune, 10×[B]_PCMV_TALA:KRAB_mCitrine, 10×[B]_PCMV_fLuciferase reporter (firefly luciferase) and different amounts of PCMV_TALA:KRAB. TALA:KRAB represses TALB:KRAB causing derepression of fLuciferase, showing that the construct is repressed by TALA:KRAB.

Figure 3:Repression of TALB:KRAB, by TALA:KRAB

References

Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T. Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. Nucleic Acids Res. 40, 7584-7595.

Sequence and Features