Difference between revisions of "Part:BBa K782017"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K782017 short</partinfo> | <partinfo>BBa_K782017 short</partinfo> | ||
| + | * TALA label represents TAL effector 1257 from zebrafish experiments (Sander et al., 2011). | ||
| + | * DNA binding sites for individual TAL effectors are indicated with square brackets [ ]. | ||
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==Introduction== | ==Introduction== | ||
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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. | 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 contain [https://parts.igem.org/Part:BBa_K782070 | + | Our construct contain [https://parts.igem.org/Part:BBa_K782070 10 specific binding sites] for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782004 TALA] upstream of CMV promoter. Downstream of CMV 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 [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782008 TALA:KRAB] on binding sites, a repression of reporter protein mCitrine occurs. | After binding of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782008 TALA:KRAB] on binding sites, a repression of reporter protein mCitrine occurs. | ||
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'''Figure 1.''' Schematic representation of the construct. | '''Figure 1.''' Schematic representation of the construct. | ||
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==Characterization== | ==Characterization== | ||
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* mCitrine was provided from host lab. | * mCitrine was provided from host lab. | ||
| − | * Binding sites for TAL effectors were ordered from | + | * Binding sites for TAL effectors were ordered from GeneArt. |
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==References== | ==References== | ||
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Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53. | Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53. | ||
| − | 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 | + | 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. |
Latest revision as of 21:36, 26 September 2012
10x[TALA] operator_CMV promoter_mCitrine
- TALA label represents TAL effector 1257 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 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 contain 10 specific binding sites for TALA upstream of CMV promoter. Downstream of CMV 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 TALA:KRAB on binding sites, a repression of reporter protein mCitrine occurs.
Figure 1. Schematic representation of the construct.
Characterization
Results: Specific TAL binding sites were further characterized with other reporter constructs.
- mCitrine was provided from host lab.
- Binding sites for TAL effectors were ordered from GeneArt.
References
Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 720
Illegal XhoI site found at 1350 - 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]