Difference between revisions of "Part:BBa K782009"
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<partinfo>BBa_K782009 short</partinfo> | <partinfo>BBa_K782009 short</partinfo> | ||
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| + | TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011). | ||
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==Introduction== | ==Introduction== | ||
| − | TAL effectors (TALEs) are bacterial plant pathogen transcription factors, that bind to DNA by specifically recognizing one base pair with a single tandem repeat in their DNA-binding domain. A tandem TALE repeat contains 33 to 35 amino acids, where the 12th and 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair. | + | TAL effectors (TALEs) are bacterial plant pathogen transcription factors, that bind to DNA by specifically recognizing one base pair with a single tandem repeat in their DNA-binding domain. A tandem TALE repeat contains 33 to 35 amino acids, where the 12th and 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). |
| − | The Kruppel-associated box (KRAB) domain is a transcriptional repressor module commonly found in eukaryotic zinc finger proteins. When KRAB containing protein binds to corresponding DNA sequence it triggers recruitment of Kap1 corepressor. KRAB domain directly interacts with a RING-B-box-coiled-coil (RBCC) domain of corepressor protein Kap1. When Kap1 binds to a KRAB domain it functions as a scaffold and starts to recruits heterocromatin protein 1 isoforms (HP1-α HP1-β HP1-γ), histone deacetylases (HDACs) and Setdb1. This complex forms facultative heterocromatin environment on a target promoter and mediates transcriptional repression (Urrutia | + | The Kruppel-associated box (KRAB) domain is a transcriptional repressor module commonly found in eukaryotic zinc finger proteins. When KRAB containing protein binds to corresponding DNA sequence it triggers recruitment of Kap1 corepressor. KRAB domain directly interacts with a RING-B-box-coiled-coil (RBCC) domain of corepressor protein Kap1. When Kap1 binds to a KRAB domain it functions as a scaffold and starts to recruits heterocromatin protein 1 isoforms (HP1-α HP1-β HP1-γ), histone deacetylases (HDACs) and Setdb1. This complex forms facultative heterocromatin environment on a target promoter and mediates transcriptional repression (Urrutia, 2003). |
We designed TALE-based repressors for specific gene repression, by fusing TAL effectors with the KRAB transcriptional repression domain downstream of the CMV promoter. KRAB was placed on the C-terminal ends of the TALE DNA-binding domain (Figure 1). | We designed TALE-based repressors for specific gene repression, by fusing TAL effectors with the KRAB transcriptional repression domain downstream of the CMV promoter. KRAB was placed on the C-terminal ends of the TALE DNA-binding domain (Figure 1). | ||
| − | '''Figure 1:''' Schematic representation of the repressor construct | + | [[Image:TALD_KRAB--.png]] |
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| + | '''Figure 1:''' Schematic representation of the repressor construct. | ||
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| + | Single binding sequence for TALD: tCGTCCAATAGCTTCTC | ||
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==Characterization== | ==Characterization== | ||
| − | HEK293T cells were cotransfected with TAL repressor | + | HEK293T cells were cotransfected with TAL repressor construct, constituively expressed by the CMV promoter, and firefly luciferase [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782022 reporter plasmid] (Figure 2), containing 12 binding sites for the designated TAL repressor upstream of the CMV promoter. All experiments were executed in 3 biological replicates and repeated over 3 times with similar results. Tests showed that tested construct exhibited over 90% repression of the reporter plasmid (Figure 3). |
| − | [[Image:Luciferaza_reporter.png]] | + | [[Image:Luciferaza_reporter.png | 300 px]] |
'''Figure 2:''' Schematic representation of repression experiments. A: in the absence of a TAL repressor, the reporter gene is constituitively expressed. B: when a TAL repressor is present, it binds to its respective binding site upstream of the CMV promoter and represses transcription of the reporter gene with the KRAB domain. | '''Figure 2:''' Schematic representation of repression experiments. A: in the absence of a TAL repressor, the reporter gene is constituitively expressed. B: when a TAL repressor is present, it binds to its respective binding site upstream of the CMV promoter and represses transcription of the reporter gene with the KRAB domain. | ||
| − | '''Figure 3:'''Testing repression of reporter gene | + | [[Image:Svn_12_talD_graf.png]] |
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| + | '''Figure 3:'''Testing repression of reporter luciferase gene. White column is showing constantly expressed reporter (fLuc - [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782022 BBa_K782022]), blue column is showing repression with TALD:KRAB. | ||
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| + | *KRAB domain was contributed by the host lab. | ||
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| + | ==References== | ||
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| + | 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. | ||
| + | Urrutia R. (2003) KRAB-containing zinc-finger repressor proteins. Genome Biology 4, 231. | ||
Latest revision as of 23:42, 26 September 2012
TALD:NLS:KRAB
TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011).
Introduction
TAL effectors (TALEs) are bacterial plant pathogen transcription factors, that bind to DNA by specifically recognizing one base pair with a single tandem repeat in their DNA-binding domain. A tandem TALE repeat contains 33 to 35 amino acids, where the 12th and 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011).
The Kruppel-associated box (KRAB) domain is a transcriptional repressor module commonly found in eukaryotic zinc finger proteins. When KRAB containing protein binds to corresponding DNA sequence it triggers recruitment of Kap1 corepressor. KRAB domain directly interacts with a RING-B-box-coiled-coil (RBCC) domain of corepressor protein Kap1. When Kap1 binds to a KRAB domain it functions as a scaffold and starts to recruits heterocromatin protein 1 isoforms (HP1-α HP1-β HP1-γ), histone deacetylases (HDACs) and Setdb1. This complex forms facultative heterocromatin environment on a target promoter and mediates transcriptional repression (Urrutia, 2003).
We designed TALE-based repressors for specific gene repression, by fusing TAL effectors with the KRAB transcriptional repression domain downstream of the CMV promoter. KRAB was placed on the C-terminal ends of the TALE DNA-binding domain (Figure 1).
Figure 1: Schematic representation of the repressor construct.
Single binding sequence for TALD: tCGTCCAATAGCTTCTC
Characterization
HEK293T cells were cotransfected with TAL repressor construct, constituively expressed by the CMV promoter, and firefly luciferase reporter plasmid (Figure 2), containing 12 binding sites for the designated TAL repressor upstream of the CMV promoter. All experiments were executed in 3 biological replicates and repeated over 3 times with similar results. Tests showed that tested construct exhibited over 90% repression of the reporter plasmid (Figure 3).
Figure 2: Schematic representation of repression experiments. A: in the absence of a TAL repressor, the reporter gene is constituitively expressed. B: when a TAL repressor is present, it binds to its respective binding site upstream of the CMV promoter and represses transcription of the reporter gene with the KRAB domain.
Figure 3:Testing repression of reporter luciferase gene. White column is showing constantly expressed reporter (fLuc - BBa_K782022), blue column is showing repression with TALD:KRAB.
- KRAB domain was contributed by the host lab.
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.
Urrutia R. (2003) KRAB-containing zinc-finger repressor proteins. Genome Biology 4, 231.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2337
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2703
Illegal SapI.rc site found at 2667