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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_K782025 short</partinfo>
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| − | * TALD label represents TAL effector 1295 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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| − | Transcription activation like (TAL) effectors are proteins able to specifically bind desired DNA sequence. The central domain of the protein is constructed from variable number of tandem repeats differing only in two amino acids. The 12th and the 13th amino acid are called a “repeat variable diresidue” (RVD) and are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences.
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| − | Our construct contains [https://parts.igem.org/Part:BBa_K782068 four specific binding sites] for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782007 NicTAL12] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782005 TALD] upstream of minimal promoter. Downstream of minimal promoter we cloned yellow fluorescent protein mCitrine an easy detectable monomer with excitation maximum at 516 nm and emission maximum at 529 nm (Figure 1).
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| − | After binding of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782066 NicTAL12:VP16] or [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782012 TALD:VP16] on binding sites, an activation of reporter protein mCitrine occurs.
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| − | Single binding sequence for NicTAL12 is: TCTATCAATGATAGA
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| − | Single binding sequence for TALD is: TCGTCCAATAGCTTCTC
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| − | [[Image:4xN+4xD-pMIN-mCit.png]]
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| − | '''Figure 1:''' Shematic representation of four consecutive specific binding sites for NicTAL12 and TALD upstream of minimal promoter and reporter protein mCitrine.
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| − | ==Characterization==
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| − | HEK293T cells were cotransfected with TAL activator construct, constituitively expressed by the CMV promoter, and mCitrine reporter plasmid, containing four binding sites for the designated TAL activator upstream of a minimal promoter (Figure 2). All experiments were executed in 3 biological replicates and repeated over 3 times with similar results.
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| − | [[Image:Svn_12_PMIN_sistem.png |300 px]]
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| − | '''Figure 2:''' Schematic representation of activation experiments. A: in the absence of a TAL activator, the expression of the reporter gene is repressed. B: when TAL activator is present, it binds to its respective binding site upstream of the minimal promoter and activates transcription of the reporter gene with the VP16 domain.
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| − | [[Image:Svn_12_4N4D_pmin-mCit.png]]
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| − | '''Figure 3:''' Testing activation of reporter gene transcription by addition of NicTAL12:VP16 or TALD:VP16 .
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| − | * mCitrine was provided from host lab.
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| − | * Binding sites for TAL effectors were ordered from GeneArt.
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| − | * Minimal promotor is taken from pGL4 vector series (promotor is described [http://www.promega.com/~/media/files/resources/protocols/technical%20manuals/0/pgl4%20luciferase%20reporter%20vectors%20protocol.pdf?la=en here] on the page 18).
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| − | * VP16 domain was contributed by the host lab
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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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| − | Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.
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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_K782025 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_K782025 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
