Difference between revisions of "Part:BBa K782028"
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[[Image:10×AB_pMIN_mCit_shema1.png]] | [[Image:10×AB_pMIN_mCit_shema1.png]] | ||
| − | '''Figure 1:'''Schematic representation of our construct. | + | '''Figure 1: '''Schematic representation of our construct. |
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[[Image:10×AB_pMIN_mCit_shema2.png]] | [[Image:10×AB_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. |
[[Image:Svn_12_10A10B-pmin-mCit.png]] | [[Image:Svn_12_10A10B-pmin-mCit.png]] | ||
| − | '''Figure 3:'''Testing activation of reporter gene transcription by addition of TAL activator. | + | '''Figure 3: '''Testing activation of reporter gene transcription by addition of TAL activator. |
Latest revision as of 21:53, 26 September 2012
10x[TALA+TALB] operator_minimal promoter_mCitrine
- TALA and TALB labels represents TAL effectors 1257 and 1297 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 binding sites for TALA and 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.
Figure 1: Schematic representation of our construct.
Characterisation
HEK293T cells were cotransfected with 10x[TALA+TALB] operator_minimal promoter_mCitrine and either TALA:VP16 or TALB:VP16 (Figure 2). All experiments were executed in 3 biological replicates and repeated over 3 times with similar results.
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 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 768
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 149
Illegal NgoMIV site found at 514
Illegal AgeI site found at 12
Illegal AgeI site found at 352
Illegal AgeI site found at 377
Illegal AgeI site found at 717 - 1000COMPATIBLE WITH RFC[1000]