Difference between revisions of "Part:BBa K782019"
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<partinfo>BBa_K782019 short</partinfo> | <partinfo>BBa_K782019 short</partinfo> | ||
| − | + | * TALA and TALB labels represents TAL effectors 1257 and 1297 respectively 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). | ||
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| + | We designed our construct with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782069 10 alterations] of binding sites for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782004 TALA] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782006 TALB], 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:10×AB_CMV_fLuc_shema1.png]] | ||
| + | |||
| + | '''Figure 1:'''Schematic representation of our construct. | ||
| + | |||
| + | ===Characterisation=== | ||
| + | HEK293T cells were transfected with the 10x[TALA+TALB] operator_CMV promoter_fLuciferase reporter and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782008 TALA:NLS:KRAB] or [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782010 TALB:NLS:KRAB] (Figure 2). Test showed successful repression with either TAL repressor (Figure 3). | ||
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| + | [[Image:10×AB_CMV_fLuc_shema2.png]] | ||
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| + | '''Figure 2:'''Schematic representation of repression experiments. A: in the absence of a TAL repressor, the reporter gene is constituitively expressed. B: when a TALA:NLS: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:10×AB_CMV_fLuc_graf.png]] | ||
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| + | '''Figure 3:''' Repression of luciferase reporter. Control: 10x[TALA+TALB] operator_CMV promoter_fLuciferase without TAL:NLS:KRAB. | ||
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| + | *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. | ||
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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 22:01, 26 September 2012
10x[TALA+TALB] operator_CMV promoter_fLuciferase
- TALA and TALB labels represents TAL effectors 1257 and 1297 respectively 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 10 alterations of binding sites for TALA and TALB, 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 10x[TALA+TALB] operator_CMV promoter_fLuciferase reporter and TALA:NLS:KRAB or TALB:NLS:KRAB (Figure 2). Test showed successful repression with either TAL repressor (Figure 3).
Figure 2:Schematic representation of repression experiments. A: in the absence of a TAL repressor, the reporter gene is constituitively expressed. B: when a TALA:NLS: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: Repression of luciferase reporter. Control: 10x[TALA+TALB] operator_CMV promoter_fLuciferase without TAL:NLS: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 924
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 343
Illegal NgoMIV site found at 708
Illegal NgoMIV site found at 1636
Illegal NgoMIV site found at 2980
Illegal NgoMIV site found at 3001
Illegal NgoMIV site found at 3316
Illegal AgeI site found at 206
Illegal AgeI site found at 546
Illegal AgeI site found at 571
Illegal AgeI site found at 911
Illegal AgeI site found at 2704 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 2886