Difference between revisions of "Part:BBa K782024"

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==Characterization==
 
==Characterization==
  
HEK293T cells were cotransfected with TAL activator constructs, constituitively expressed by the CMV promoter, and mCitrine [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782025 reporter plasmids], containing 2 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. Tested TAL activator exhibited over 500-fold activation of the mCitrine reporter (Figure 3).
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HEK293T cells were cotransfected with TAL activator constructs, constituitively expressed by the CMV promoter, and mCitrine [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782025 reporter plasmids], containing 2 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.
  
 
[[Image:Svn_12_PMIN_sistem.png |300 px]]
 
[[Image:Svn_12_PMIN_sistem.png |300 px]]
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[[Image:Svn_12_2n-2d-pmin-mCit.png‎]]
 
[[Image:Svn_12_2n-2d-pmin-mCit.png‎]]
  
'''Figure 3:''' Testing activation of reporter gene transcription by addition of TAL activator
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'''Figure 3:''' Testing activation of reporter gene transcription by addition of TAL activator.
  
  

Revision as of 14:42, 26 September 2012

2x[NicTAL]+2x[TALD] operator_minimal promoter_mCitrine

  • TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011).

Introduction

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.

Our construct contains two specific binding sites for NicTAL12 and 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). After binding of NicTAL12:VP16 or TALD:KRAB on binding sites, an activation of reporter protein mCitrine occurs.

Single binding sequence for NicTAL12 is: TCTATCAATGATAGA

Single binding sequence for TALD is: TCGTCCAATAGCTTCTC


7xN+7xD-pMIN-mCit.png

Figure 1: Shematic representation of two consecutive specific binding sites for NicTAL12 and TALD upstream of minimal promoter and reporter protein mCitrine.


  • mCitrine was provided from host lab.
  • Binding sites for TAL effectors were ordered from GeneArt.
  • 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).


Characterization

HEK293T cells were cotransfected with TAL activator constructs, constituitively expressed by the CMV promoter, and mCitrine reporter plasmids, containing 2 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.

Svn 12 PMIN sistem.png

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.


Svn 12 2n-2d-pmin-mCit.png

Figure 3: Testing activation of reporter gene transcription by addition of TAL activator.


  • VP16 domain was contributed by the host lab


References

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 XhoI site found at 178
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 35
    Illegal AgeI site found at 105
  • 1000
    COMPATIBLE WITH RFC[1000]