Part:BBa_K782084

10x[TALA] operator_CMV promoter_TALB:NLS:KRAB_t2a_mNeptune

• 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[TALB] operator_CMV promoter_TALA:KRAB:NLS_t2a_mCitrine is the key element of the [http://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch mutual repressor switch].

The part contains 10 repeats of TALA binding sites upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter, there is the TAL repressor TALB:KRAB linked to the red fluorescent protein mNeptune, by a t2A sequence. The t2A sequence, causes the ribosome to skip the formation of a peptide bond during protein translation, producing TALB:KRAB and mNeptune as separate proteins in equimolar amounts (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

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 720
  Illegal BamHI site found at 3773
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 147
  Illegal NgoMIV site found at 507
  Illegal AgeI site found at 12
  Illegal AgeI site found at 347
  Illegal AgeI site found at 372
  Illegal AgeI site found at 707
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 4139
  Illegal BsaI.rc site found at 4688
  Illegal BsaI.rc site found at 4877
  Illegal SapI.rc site found at 4103
  Illegal SapI.rc site found at 4259