Difference between revisions of "Part:BBa K782020"

 
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<partinfo>BBa_K782020 short</partinfo>
 
<partinfo>BBa_K782020 short</partinfo>
  
Long
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TALA label represents TAL effector 1257 from zebrafish experiments (Sander et al., 2011)
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===Introduction===
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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)
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We designed our construct with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782070 10]  binding sites for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782004 TALA], 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×A_CMV_fLuc_shema1.png]]
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'''Figure1:'''Schematic representation of our construct
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===Characterisation===
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HEK293T cells were transfected with the 10x[TALA] operator_CMV promoter_fLuciferase reporter and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782008 TALA:NLS:KRAB] (Figure 2). Tests showed, successful repression of fLuciferase with either TAL repressor.
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[[Image:Svn_12_talakgraf.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 TALA:KRAB.
  
 
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Revision as of 07:53, 26 September 2012

10x[TALA] operator_CMV promoter_fLuciferase

TALA label represents TAL effector 1257 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 binding sites for TALA, 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×A CMV fLuc shema1.png

Figure1:Schematic representation of our construct

Characterisation

HEK293T cells were transfected with the 10x[TALA] operator_CMV promoter_fLuciferase reporter and TALA:NLS:KRAB (Figure 2). Tests showed, successful repression of fLuciferase with either TAL repressor.



Svn 12 talakgraf.png

Figure 3:Testing repression of reporter luciferase gene. White column is showing constantly expressed reporter (fLuc), blue column is showing repression with TALA:KRAB.

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 914
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 341
    Illegal NgoMIV site found at 701
    Illegal NgoMIV site found at 1626
    Illegal NgoMIV site found at 2970
    Illegal NgoMIV site found at 2991
    Illegal NgoMIV site found at 3306
    Illegal AgeI site found at 206
    Illegal AgeI site found at 541
    Illegal AgeI site found at 566
    Illegal AgeI site found at 901
    Illegal AgeI site found at 2694
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2876