Difference between revisions of "Part:BBa K782030"

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We designed our construct with 10 binding sites for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782006 TALB], upstream of a minimal  promoter (like [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782088 here]). Downstream of the promoter we cloned the yellow fluorescent protein mCitrine an easy detectable monomer with excitation maximum at 516 nm and emission maximum at 529 nm.
 
We designed our construct with 10 binding sites for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782006 TALB], upstream of a minimal  promoter (like [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782088 here]). Downstream of the promoter we cloned the yellow fluorescent protein mCitrine an easy detectable monomer with excitation maximum at 516 nm and emission maximum at 529 nm.
  
[[Image:10×A_pMIN_mCit_shema1.png]]
+
[[Image:10×B_pMIN_mCit_shema1.png]]
  
 
'''Figure1:'''Schematic representation of our construct
 
'''Figure1:'''Schematic representation of our construct
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HEK293T cells were cotransfected with 10x[TALA] operator_minimal promoter_mCitrine and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782013 TALB:VP16] (Figure 2). All experiments were executed in 3 biological replicates and repeated over 3 times with similar results.
 
HEK293T cells were cotransfected with 10x[TALA] operator_minimal promoter_mCitrine and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782013 TALB:VP16] (Figure 2). All experiments were executed in 3 biological replicates and repeated over 3 times with similar results.
  
[[Image:10×A_pMIN_mCit_shema2.png]]
+
[[Image:10×B_pMIN_mCit_shema2.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 binding site upstream of the minimal promoter and activates transcription of the reporter gene with the VP16 domain.
 
'''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 binding site upstream of the minimal promoter and activates transcription of the reporter gene with the VP16 domain.

Revision as of 15:37, 26 September 2012

10x[TALB] operator_minimal promoter_mCitrine

TALB label represents TAL effector 1297 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 TALB, upstream of a minimal promoter (like here). Downstream of the promoter we cloned the yellow fluorescent protein mCitrine an easy detectable monomer with excitation maximum at 516 nm and emission maximum at 529 nm.

10×B pMIN mCit shema1.png

Figure1:Schematic representation of our construct

Characterisation

HEK293T cells were cotransfected with 10x[TALA] operator_minimal promoter_mCitrine and TALB:VP16 (Figure 2). All experiments were executed in 3 biological replicates and repeated over 3 times with similar results.

10×B pMIN mCit shema2.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 binding site upstream of the minimal promoter and activates transcription of the reporter gene with the VP16 domain.

Svn 12 TALB-VP16-graf .png

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

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 XhoI site found at 758
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 147
    Illegal NgoMIV site found at 507
    Illegal AgeI site found at 12
    Illegal AgeI site found at 347
    Illegal AgeI site found at 372
    Illegal AgeI site found at 707
  • 1000
    COMPATIBLE WITH RFC[1000]