Difference between revisions of "Part:BBa K782030"
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<partinfo>BBa_K782030 short</partinfo> | <partinfo>BBa_K782030 short</partinfo> | ||
| − | TALB label represents TAL effector 1297 from zebrafish experiments (Sander et al., 2011) | + | * TALB label represents TAL effector 1297 from zebrafish experiments (Sander et al., 2011). |
| + | * DNA binding sites for individual TAL effectors are indicated with square brackets [ ]. | ||
| + | |||
==Introduction== | ==Introduction== | ||
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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: | + | [[Image:10×B_pMIN_mCit_shema1.png]] |
| + | |||
| + | '''Figure 1: '''Schematic representation of our construct. | ||
| − | |||
==Characterisation== | ==Characterisation== | ||
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: | + | [[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. |
[[Image:Svn_12_TALB-VP16-graf_.png | 300 px]] | [[Image:Svn_12_TALB-VP16-graf_.png | 300 px]] | ||
| − | '''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. |
| − | |||
| − | + | ==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. | ||
Latest revision as of 22:00, 26 September 2012
10x[TALB] operator_minimal promoter_mCitrine
- TALB label represents TAL effector 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 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] 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.
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 758
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]