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| − | __NOTOC__
| + | 0605 http://manualss7iy.pp.ua/bnwrwn1.html http://manualss7iy.pp.ua/xpem2.html http://documentsoex.pp.ua/tsq1.html http://documentsoex.pp.ua/kvxtcu2.html http://instructions9rxg.pp.ua/iiejn2.html http://instructions9rxg.pp.ua/lddpim4.html http://instruktsiya721.pp.ua/olkkpx2.html http://instruktsiya721.pp.ua/vrol1.html http://rukovodstnnr.pp.ua/mwcfk2.html http://rukovodstnnr.pp.ua/xuykyh3.html http://rukovodstnnr.pp.ua/juqpxs2.html http://rukovodstnnr.pp.ua/wil3.html http://instruktsiya5nd.pp.ua/mvcgo2.html http://instruktsiya5nd.pp.ua/ytjmtg3.html http://rukovodstvqk.pp.ua/tqu2.html http://rukovodstvqk.pp.ua/kjanan3.html |
| − | <partinfo>BBa_K782084 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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| − | * DNA binding sites for individual TAL effectors are indicated with square brackets [ ].
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| − | ==Introduction==
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| − | 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].
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| − | The part contains 10 repeats of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782070 TALA 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_K782010 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.
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| − | [[Image:10×-A-_PCMV_TBK_Nep.png]]
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| − | '''Figure 1: ''' Schematic representation of the construct.
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| − | ==Characterization==
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| − | 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.
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| − | [[Image:SVN12_registry_mikroskop_neptun.jpg]]
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| − | '''Figure 2: ''' Cells transfected with 10×[A]_PCMV_TALB:KRAB_mNeptune, fluorescing with mNeptune.
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| − | 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.
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| − | [[Image:SVN12_mreps_repr_tala.png | 600 px]]
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| − | '''Figure 3: '''Repression of TALB:KRAB, by TALA:KRAB.
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| − | ==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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| − | 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.
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| − | <!-- Add more about the biology of this part here
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| − | ===Usage and Biology===
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| − | <!-- -->
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| − | <span class='h3bb'>Sequence and Features</span>
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| − | <partinfo>BBa_K782084 SequenceAndFeatures</partinfo>
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| − | <!-- Uncomment this to enable Functional Parameter display
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| − | ===Functional Parameters===
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| − | <partinfo>BBa_K782084 parameters</partinfo>
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| − | <!-- -->
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0605 http://manualss7iy.pp.ua/bnwrwn1.html http://manualss7iy.pp.ua/xpem2.html http://documentsoex.pp.ua/tsq1.html http://documentsoex.pp.ua/kvxtcu2.html http://instructions9rxg.pp.ua/iiejn2.html http://instructions9rxg.pp.ua/lddpim4.html http://instruktsiya721.pp.ua/olkkpx2.html http://instruktsiya721.pp.ua/vrol1.html http://rukovodstnnr.pp.ua/mwcfk2.html http://rukovodstnnr.pp.ua/xuykyh3.html http://rukovodstnnr.pp.ua/juqpxs2.html http://rukovodstnnr.pp.ua/wil3.html http://instruktsiya5nd.pp.ua/mvcgo2.html http://instruktsiya5nd.pp.ua/ytjmtg3.html http://rukovodstvqk.pp.ua/tqu2.html http://rukovodstvqk.pp.ua/kjanan3.html
