Difference between revisions of "Part:BBa K782083"

Revision as of 22:58, 26 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).
• DNA binding sites for individual TAL effectors are indicated with square brackets [ ].

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, causes the ribosome to skip the formation of a peptide bond during protein translation, producing TALA:KRAB and mCitrine as separate proteins in equimolar amounts (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×[B]_PCMV_TALA:KRAB_mCitrine, 10×[A]_PCMV_TALB:KRAB_mNeptune, 10×[A]_PCMV_fLuciferase reporter (firefly luciferase) and different amounts of PCMV_TALB:KRAB. TALB:KRAB represses TALA:KRAB causing derepression of fLuciferase, showing that the construct is repressed by TALB:KRAB.

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

References

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.

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