Difference between revisions of "Part:BBa K782001"
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<partinfo>BBa_K782001 short</partinfo> | <partinfo>BBa_K782001 short</partinfo> | ||
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* TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011). | * TALD label represents TAL effector 1295 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 contains | + | Our construct contains [https://parts.igem.org/Part:BBa_K782067 two specific binding sites] for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782007 NicTAL12] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782005 TALD] 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_K782011 NicTAL12:KRAB] or [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782009 TALD:KRAB] on binding sites, a repression of reporter protein mCitrine occurs. | After binding of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782011 NicTAL12:KRAB] or [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782009 TALD:KRAB] on binding sites, a repression of reporter protein mCitrine occurs. | ||
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Single binding sequence for TALD is: TCGTCCAATAGCTTCTC | Single binding sequence for TALD is: TCGTCCAATAGCTTCTC | ||
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upstream of CMV promoter and reporter protein mCitrine. | upstream of CMV promoter and reporter protein mCitrine. | ||
| + | ==Characterization== | ||
| + | Results: Specific TAL [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782024 binding sites] were further characterized with other reporter constructs. | ||
| + | * mCitrine was provided from host lab. | ||
| + | * 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 21:31, 26 September 2012
2x[NicTAL]+2x[TALD] operator_CMV promoter_mCitrine
- TALD label represents TAL effector 1295 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 contains two specific binding sites for NicTAL12 and TALD 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 NicTAL12:KRAB or TALD:KRAB on binding sites, a repression of reporter protein mCitrine occurs.
Single binding sequence for NicTAL12 is: TCTATCAATGATAGA
Single binding sequence for TALD is: TCGTCCAATAGCTTCTC
Figure 1. Shematic representation of two consecutive specific binding sites for NicTAL12:KRAB and TALD:KRAB upstream of CMV promoter and reporter protein mCitrine.
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 140
Illegal XhoI site found at 770 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 35
Illegal AgeI site found at 105 - 1000COMPATIBLE WITH RFC[1000]