Difference between revisions of "Part:BBa K782083"
 
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<partinfo>BBa_K782083 short</partinfo>
 
<partinfo>BBa_K782083 short</partinfo>
  
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TALB and TALA labels represents TAL effectors 1297 and 1257 respectively from zebrafish experiments (Sander et al., 2011)
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* 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==
 
==Introduction==
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This part combined with 10x[TALA] operator_CMV promoter_TALB:NLS:KRAB_t2a_mNeptune is the key element of the mutual repressor switch.  
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This part combined with 10x[TALA] operator_CMV promoter_TALB:NLS:KRAB_t2a_mNeptune is the key element of the [http://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch mutual repressor switch].  
  
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The part contains 10 repeats of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782071 TALB binding sites] upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter, there is the TAL repressor [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782008 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.
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The part contains 10 repeats of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782071 TALB binding sites] upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter, there is the TAL repressor [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782008 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.
  
 
[[Image:10×-B-_PCMV_TAK_Cit.png]]
 
[[Image:10×-B-_PCMV_TAK_Cit.png]]
  
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'''Figure 1:''' Schematic representation of the construct
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'''Figure 1: ''' Schematic representation of the construct.
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==Characterization==
 
==Characterization==
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[[Image:SVN12_registry_mikroskop_citrin.jpg]]
 
[[Image:SVN12_registry_mikroskop_citrin.jpg]]
  
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'''Figure 2:'''Cells transfected with 10×[B]_PCMV_TALA:KRAB_mCitrine, fluorescing with mCitrine
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'''Figure 2: '''Cells transfected with 10×[B]_PCMV_TALA:KRAB_mCitrine, fluorescing with mCitrine.
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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.
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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.
  
 
[[Image:SVN12 stikalo luc za registri.png | 600 px]]
 
[[Image:SVN12 stikalo luc za registri.png | 600 px]]
  
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'''Figure 3:'''Repression of TALA:KRAB, by TALB:KRAB
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'''Figure 3: '''Repression of TALA:KRAB, by TALB:KRAB.
  
 
==References==
 
==References==
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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  
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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.  
 
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.  

Latest revision as of 02:24, 27 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 [http://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch 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.

10×-B- PCMV TAK Cit.png

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.

SVN12 registry mikroskop citrin.jpg

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.

SVN12 stikalo luc za registri.png

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


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 SapI.rc site found at 4103