Difference between revisions of "Part:BBa K1150023"
 
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__NOTOC__
 
__NOTOC__
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{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
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! colspan="2" style="background:#FFBF00;"|SV40:HA-NLS-dCas9-Linker-G9a-NLS:BGH
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|-
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|'''Function'''
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|DNA binding protein fused to a methyl histone transferase
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|-
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|'''Use in'''
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|Mammalian cells
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|-
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|'''RFC standard'''
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|[https://parts.igem.org/Help:Assembly_standard_25 RFC 25]
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|-
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|'''Backbone'''
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|pSB1C3<br>
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|-
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|'''Organism'''
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|<i>Streptococcus pyogenes, Mus musculus</i>
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|-
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|'''Source'''
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|Feng Zhang, Addgene<br> Albert Jeltsch, University of Stuttgart
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|-
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|'''Submitted by'''
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|[http://2013.igem.org/Team:Freiburg Freiburg 2013]
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|}
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<partinfo>BBa_K1150023 short</partinfo>
 
<partinfo>BBa_K1150023 short</partinfo>
  
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This device is combining the dCAS9 protein, that enables multiple gene targeting with the set-domain of the murine G9a. dCAS9 is working as a carrier for this histone methyltransferase and enables specific methylation of histone 3 Lysin 9 (H3K9me2/3) when targeted to a histone locus that is accessible for DNA binding proteins. Literature indicates that targeting the G9a Set-Domain to an open locus leads to a transcriptionally inactive state.
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This device is combining the [https://parts.igem.org/Part:BBa_K1150000 dCas9] protein, that enables multiple gene targeting with the set-domain of the murine [https://parts.igem.org/Part:BBa_K1150003 G9a]. dCas9 is working as a carrier for this histone methyltransferase and enables specific methylation of histone 3 lysine 9 (H3K9me2/3) when targeted to a histone locus that is accessible for DNA binding proteins. Literature indicates that targeting the G9a set-domain to an open locus leads to a transcriptionally inactive state [1].
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The usage of the medium strong SV40 promoter optimizes this device for means were a strong expression is suboptimal. If a strong expression is desired check [[https://parts.igem.org/Part:BBa_K1150024]].
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===Usage and Biology===
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H3K9 methylation is a hallmark of repressed transcriptional states. The murine G9a-Set domain is able to transfer methyl groups to H3K9 when targeting it to the DNA (see Snowden et.al., 2003) and repress transcription. G9a is also known to be involved in downstream signalling, but by targeting it to a specific locus we reduce the functionality to its histone modification ability.
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The dCAS9 protein is able to be targeted to several loci at once as it interacts with small RNAs to build up a complex that will interact with complementary DNA strands. Its origin is the adaptive immune system of <i> Streptococcus pyogenes </i> called CRISPR. Hijacking this system leads to a whole new approach for multiple gene targeting.
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The iGEM team Freiburg 2013 combined these two elements to create a transcriptional repressor that is able to repress by a specific mechanism the targeted locus.
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This approach offers new possibilities for fundamental epigenetic research, tissue engineering and cancer research.
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The usage of the medium strong [https://parts.igem.org/Part:BBa_K1150011 SV40 promoter] optimizes this device for means were a strong expression is suboptimal. If a strong expression is desired check our uniCAS Histone Modifier [https://parts.igem.org/Part:BBa_K1150024 device with CMV promoter].
  
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For more information [http://2013.igem.org/Team:Freiburg/Project/effector#epigenetics klick here].
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<span class='h3bb'>Sequence and Features</span>
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[[File:Freiburg2013_Plasmid_Cas9-G9a.png|800px|]] <br>
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'''Figure 1:''' Complete overview on CMV:dCas9-G9a with all features.
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==Usage and Biology==
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H3K9 methylation is a hallmark of repressed transcriptional states. [2] Upon close contact the murine G9a-set domain transfers methyl groups to H3K9 leading to transcriptional repression. [3]
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The dCas9 protein can simultaneously be targeted to several DNA loci as it interacts with small RNAs thereby forming a complex that will interact with complementary DNA strands. It origins from the bacterial adaptive immune system of <i> Streptococcus pyogenes </i> called CRISPR. Hijacking this system leads to a whole new approach for multiple gene targeting.
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The team [http://2013.igem.org/Team:Freiburg Freiburg 2013] combined these two elements to create a transcriptional repressor that can be targeted to any desired locus of interest. <br> This approach offers new possibilities for applied as well as for fundamental research, such as tissue engineering, epigenetics and cancer research.
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<span class='h3bb'>
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==Sequence and Features==
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</span>
 
<partinfo>BBa_K1150023 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1150023 SequenceAndFeatures</partinfo>
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==References==
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[1]Wolffe, A., et al. (1999). Epigenetics: Regulation Through Repression. Science 286169, 481. <br>
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[2]Snowden, A., et al. (2002). Gene-Specific Targeting of H3K9 Methylation Is Sufficient for Initiating Repression In Vivo. Current Biology 12, 2159-2166. <br>
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[3]  Lee, D., et al. (2006). Histone 3 Lysine 9 Methyltransferase G9a Is a Transcriptional Coactivator for Nuclear Receptors. Journal of Biological Chemistry 281, 8476-8485.  <br>
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Latest revision as of 22:18, 4 October 2013



SV40:HA-NLS-dCas9-Linker-G9a-NLS:BGH
Function DNA binding protein fused to a methyl histone transferase
Use in Mammalian cells
RFC standard RFC 25
Backbone pSB1C3
Organism Streptococcus pyogenes, Mus musculus
Source Feng Zhang, Addgene
Albert Jeltsch, University of Stuttgart
Submitted by [http://2013.igem.org/Team:Freiburg Freiburg 2013]

uniCAS Histone Modifier (SV40 promoter)

This device is combining the dCas9 protein, that enables multiple gene targeting with the set-domain of the murine G9a. dCas9 is working as a carrier for this histone methyltransferase and enables specific methylation of histone 3 lysine 9 (H3K9me2/3) when targeted to a histone locus that is accessible for DNA binding proteins. Literature indicates that targeting the G9a set-domain to an open locus leads to a transcriptionally inactive state [1].

The usage of the medium strong SV40 promoter optimizes this device for means were a strong expression is suboptimal. If a strong expression is desired check our uniCAS Histone Modifier device with CMV promoter.

For more information [http://2013.igem.org/Team:Freiburg/Project/effector#epigenetics klick here].

Freiburg2013 Plasmid Cas9-G9a.png
Figure 1: Complete overview on CMV:dCas9-G9a with all features.

Usage and Biology

H3K9 methylation is a hallmark of repressed transcriptional states. [2] Upon close contact the murine G9a-set domain transfers methyl groups to H3K9 leading to transcriptional repression. [3] The dCas9 protein can simultaneously be targeted to several DNA loci as it interacts with small RNAs thereby forming a complex that will interact with complementary DNA strands. It origins from the bacterial adaptive immune system of Streptococcus pyogenes called CRISPR. Hijacking this system leads to a whole new approach for multiple gene targeting. The team [http://2013.igem.org/Team:Freiburg Freiburg 2013] combined these two elements to create a transcriptional repressor that can be targeted to any desired locus of interest.
This approach offers new possibilities for applied as well as for fundamental research, such as tissue engineering, epigenetics and cancer research.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 664
    Illegal BglII site found at 5139
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

References

[1]Wolffe, A., et al. (1999). Epigenetics: Regulation Through Repression. Science 286169, 481.
[2]Snowden, A., et al. (2002). Gene-Specific Targeting of H3K9 Methylation Is Sufficient for Initiating Repression In Vivo. Current Biology 12, 2159-2166.
[3] Lee, D., et al. (2006). Histone 3 Lysine 9 Methyltransferase G9a Is a Transcriptional Coactivator for Nuclear Receptors. Journal of Biological Chemistry 281, 8476-8485.