Difference between revisions of "Part:BBa K782083"

Revision as of 16:55, 25 September 2012

10x[TALB] operator_CMV promoter_TALA:KRAB:NLS_t2a_mCitrine

TALB and TALA labels represents TAL effectors 1297 and 1257 respectively from zebrafish experiments (Sander et al., 2011)

Introduction

This part combined with 10x[TALA] operator_CMV promoter_TALB:NLS:KRAB_t2a_mNeptune is the key element of the mutual repressor switch.

The part contains 10 repeats of TALB binding sites upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter, there is the TAL repressor TALA:KRAB linked to the yellow fluorescent protein mCitrine, by a t2A sequence. The t2A sequence interrupts protein translation and causes TALA:KRAB and mCitrine to be expressed in equimolar amounts as separate proteins (Garg et.al, 2012). mCitrine functions as a reporter for the expression of TALA:KRAB, it is a yellow fluorescent protein with an excitation peak at 516 nm, an emission peak at 529 nm and brightness of 58.5.

Figure 1: Schematic representation of the construct

Characterization

HEK293T cells were transfected with 10×[B]_PCMV_TALA:KRAB_mCitrine and visualized under confocal microscope. Cells were shown to fluoresce with mCitrine, showing that mCitrine is expressed.

Figure 2:Cells transfected with 10×[B]_PCMV_TALA:KRAB_mCitrine, fluorescing with mCitrine

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

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

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

Sequence and Features