Difference between revisions of "Part:BBa K782019"
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===Introduction===
 
===Introduction===
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Transcription activation like (TAL) effectors are bacterial plant pathogen transcription factors that bind to DNA by recognizing a specific DNA sequence in which each base pair is bound by a single tandem repeat in the TAL DNA-binding domain. A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD) are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). All TAL repeats have almost identical sequences, differing only in the RVDs. This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences by designing specific binding domains for a selected TAL effector.
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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 ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K782069 10×[A + B]), 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.
 
We designed our construct with 10 alterations of binding sites for TALA and TALB ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K782069 10×[A + B]), 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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===Characterisation===
 
===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). Tests showed, successful repression of fLuciferase with either TAL repressor.
 
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). Tests showed, successful repression of fLuciferase with either TAL repressor.
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[[Image:10×AB_CMV_fLuc_shema2.png]]
 
[[Image:10×AB_CMV_fLuc_shema2.png]]
  
 
'''Figure 2:'''Schematic representation of repression experiments. A: in the absence of a TAL repressor, the reporter gene is constituitively expressed. B: when a TAL repressor is present, it binds to its respective binding site upstream of the CMV promoter and represses transcription of the reporter gene with the KRAB domain.
 
'''Figure 2:'''Schematic representation of repression experiments. A: in the absence of a TAL repressor, the reporter gene is constituitively expressed. B: when a TAL repressor is present, it binds to its respective binding site upstream of the CMV promoter and represses transcription of the reporter gene with the KRAB domain.
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[[Image:10×AB_CMV_fLuc_graf.png]]  
 
[[Image:10×AB_CMV_fLuc_graf.png]]  
  
 
'''Figure 3:''' Repression of luciferase reporter. Control: 10x[TALA+TALB] operator_CMV promoter_fLuciferase without TAL:NLS:KRAB
 
'''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
 
*fLuciferase was contributed by host lab

Revision as of 07:40, 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)

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 (10×[A + B), 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.

10×AB CMV fLuc shema1.png

Figure1: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). Tests showed, successful repression of fLuciferase with either TAL repressor.


10×AB CMV fLuc shema2.png

Figure 2:Schematic representation of repression experiments. A: in the absence of a TAL repressor, the reporter gene is constituitively expressed. B: when a TAL repressor is present, it binds to its respective binding site upstream of the CMV promoter and represses transcription of the reporter gene with the KRAB domain.


10×AB CMV fLuc graf.png

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 IDT.

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 924
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2886