Difference between revisions of "Part:BBa K782022"
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<partinfo>BBa_K782022 short</partinfo> | <partinfo>BBa_K782022 short</partinfo> | ||
| − | + | * TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011). | |
| + | * DNA binding sites for individual TAL effectors are indicated with square brackets [ ]. | ||
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| + | ==Introduction== | ||
| + | Transcription activation like (TAL) effectors are DNA binding proteins with a high specifity, built from tandem repeats, with near identical seqences, differing only in two amino acids in each repeat called “repeat variable diresidue” (RVD), which determine the specifity for a single nucleotide.(Scholze and Boch, 2011). | ||
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| + | We designed our construct with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782072 12] binding sites for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782005 TALD], upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter we cloned fLuciferase (firefly luciferase) to function as a reporter. | ||
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| + | [[Image:12×D_CMV_fLuc_shema1.png]] | ||
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| + | '''Figure 1:'''Schematic representation of our construct. | ||
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| + | ==Characterisation== | ||
| + | HEK293T cells were transfected with the 12x[TALD] operator_CMV promoter_fLuciferase reporter and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782009 TALD:KRAB] (Figure 2). Tests showed, successful repression of fLuciferase with TAL repressor. Tests showed sucessful repression of the fLuciferase reporter (Figure 3). | ||
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| + | [[Image:12×D_CMV_fLuc_shema2.png]] | ||
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| + | '''Figure 2:'''Schematic representation of repression experiments. A: in the absence of a TALD:KRAB, fLuciferase is constituitively expressed. B: when a TALD:KRAB is present, it binds to its binding site upstream of the CMV promoter and represses transcription of fLuciferase with the KRAB domain. | ||
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| + | [[Image:12×D_CMV_fLuc_graf1.png]] | ||
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| + | '''Figure 3:'''Testing repression of reporter luciferase gene. White column is showing constantly expressed reporter (fLuc), blue column is showing repression with TALD:KRAB. | ||
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| + | *fLuciferase was contributed by host lab. | ||
| + | *Binding sites for TAL effectors were ordered from GeneArt. | ||
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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 | <!-- Add more about the biology of this part here | ||
Latest revision as of 22:05, 26 September 2012
12x[TALD] operator_CMV promoter_fLuciferase
- TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011).
- DNA binding sites for individual TAL effectors are indicated with square brackets [ ].
Introduction
Transcription activation like (TAL) effectors are DNA binding proteins with a high specifity, built from tandem repeats, with near identical seqences, differing only in two amino acids in each repeat called “repeat variable diresidue” (RVD), which determine the specifity for a single nucleotide.(Scholze and Boch, 2011).
We designed our construct with 12 binding sites for TALD, upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter we cloned fLuciferase (firefly luciferase) to function as a reporter.
Figure 1:Schematic representation of our construct.
Characterisation
HEK293T cells were transfected with the 12x[TALD] operator_CMV promoter_fLuciferase reporter and TALD:KRAB (Figure 2). Tests showed, successful repression of fLuciferase with TAL repressor. Tests showed sucessful repression of the fLuciferase reporter (Figure 3).
Figure 2:Schematic representation of repression experiments. A: in the absence of a TALD:KRAB, fLuciferase is constituitively expressed. B: when a TALD:KRAB is present, it binds to its binding site upstream of the CMV promoter and represses transcription of fLuciferase with the KRAB domain.
Figure 3:Testing repression of reporter luciferase gene. White column is showing constantly expressed reporter (fLuc), blue column is showing repression with TALD:KRAB.
- fLuciferase was contributed by host lab.
- Binding sites for TAL effectors were ordered from GeneArt.
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.
Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 659
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1371
Illegal NgoMIV site found at 2715
Illegal NgoMIV site found at 2736
Illegal NgoMIV site found at 3051
Illegal AgeI site found at 408
Illegal AgeI site found at 640
Illegal AgeI site found at 2439 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 2621