Difference between revisions of "Part:BBa K1150024"
Line 1: Line 1:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1150024 short</partinfo>
 
<partinfo>BBa_K1150024 short</partinfo>
 +
 +
 +
 +
 +
 +
{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
 +
! colspan="2" style="background:#FFBF00;"|pCMV:HA-NLS-dCas9-G9a-NLS:tBGH
 +
|-
 +
|'''Function'''
 +
|DNA binding protein fused to a methyl histone transferase
 +
|-
 +
|'''Use in'''
 +
|Mammalian cells
 +
|-
 +
|'''RFC standard'''
 +
|[https://parts.igem.org/Help:Assembly_standard_25 RFC 25]
 +
|-
 +
|'''Backbone'''
 +
|pSB1C3<br>
 +
|-
 +
|'''Organism'''
 +
|<i>Streptococcus pyogenes, Mus musculus</i>
 +
|-
 +
|'''Source'''
 +
|Feng Zhang, Addgene<br> Albert Jeltsch, University of Stuttgart
 +
|-
 +
|'''Submitted by'''
 +
|[http://2013.igem.org/Team:Freiburg Freiburg 2013]
 +
|}
  
 
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.  
 
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.  

Revision as of 09:52, 27 September 2013

uniCAS Histone Modifier (CMV promoter)



pCMV:HA-NLS-dCas9-G9a-NLS:tBGH
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]

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. The usage of the strong CMV promoter enables this device to be expressed in a strong manner. If weaker expression levels are needed, we recommend using our uniCAS Histone Modificator device with an SV40 promoter ([[1]]).

Usage and Biology

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. 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 Streptococcus pyogenes called CRISPR. Hijacking this system leads to a whole new approach for multiple gene targeting. The team Freiburg 2013 combined these two elements to create a transcriptional repressor that is able to repress by a specific mechanism the targeted locus. This approach offers new possibilities for fundamental epigenetic research, tissue engineering 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 576
    Illegal BglII site found at 900
    Illegal BglII site found at 5375
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]