Difference between revisions of "Part:BBa K1150021"
 
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|'''RFC standard'''
 
|'''RFC standard'''
 
|[https://parts.igem.org/Help:Assembly_standard_25 RFC 25]
 
|[https://parts.igem.org/Help:Assembly_standard_25 RFC 25]
−
|-
 
−
|'''Backbone'''
 
−
|pSB1C3<br>
 
 
|-
 
|-
 
|'''Organism'''
 
|'''Organism'''
−
|<i>Human cytomegalovirus<br> Streptococcus pyogenes<br> Homo sapiens</i>
+
|<i>Simian virus 40<br> Streptococcus pyogenes<br> Homo sapiens</i>
 
|-
 
|-
 
|'''Source'''
 
|'''Source'''
−
|iGEM Team Freiburg 2013<br>Feng Zhang, MIT <br> Konrad Müller, University of Freiburg
+
|iGEM Team Freiburg 2013<br>Feng Zhang, MIT <br> AG Weber, University of Freiburg
 
|-
 
|-
 
|'''Submitted by'''
 
|'''Submitted by'''
 
|[http://2013.igem.org/Team:Freiburg Freiburg 2013]
 
|[http://2013.igem.org/Team:Freiburg Freiburg 2013]
 
|}
 
|}
−
 
 
<div align="justify"; margin-right:10px>Krüppel-associated Box repressor domains - commonly termed as KRAB - are highly conserved polypeptide motifs and were first functionally characterized in 1991 (<i>Rosati et al.</i>, 1991). As they constitute about one third of all human zinc finger transcription factors, key regulatory features in higher eukaryotic transcriptomics are suggested (<i>Witzgall et al.</i>, 1994).  Even in terms of tetrapod evolution, the role of their great abundance has been extensively discussed (<i>Birtle</i>, 2006). Even though KRAB minimal domains are usually no longer than 50-75 amino acids, their mechanism of function remains complex. </div><br>
 
<div align="justify"; margin-right:10px>Krüppel-associated Box repressor domains - commonly termed as KRAB - are highly conserved polypeptide motifs and were first functionally characterized in 1991 (<i>Rosati et al.</i>, 1991). As they constitute about one third of all human zinc finger transcription factors, key regulatory features in higher eukaryotic transcriptomics are suggested (<i>Witzgall et al.</i>, 1994).  Even in terms of tetrapod evolution, the role of their great abundance has been extensively discussed (<i>Birtle</i>, 2006). Even though KRAB minimal domains are usually no longer than 50-75 amino acids, their mechanism of function remains complex. </div><br>
−
<div align="justify"; margin-right:10px>Common biochemical models suggest a key role in epigenetic silencing, by recruiting a scaffold of diverse proteins - amongst others histone deacetylases and histone methyltransferases (<i>Urrutia</i>, 2003). Til date in 2013, KRAB repressor domains were attached to several DNA binding proteins such as tetR, TAL effectors and [http://2013.igem.org/Team:Freiburg dCas9] - thereby efficiently silencing gene expression downstream of desired target promoters.<br><br>
+
<div align="justify"; margin-right:10px>Common biochemical models suggest a key role in epigenetic silencing, by recruiting a scaffold of diverse proteins - amongst others histone deacetylases and histone methyltransferases (<i>Urrutia</i>, 2003). Til date in 2013, KRAB repressor domains were attached to several DNA binding proteins such as tetR, TAL effectors and [https://parts.igem.org/Part:BBa_K1150000 dCas9] - thereby efficiently silencing gene expression downstream of desired target promoters.<br><br>
−
In this attempt, an [https://parts.igem.org/Part:BBa_K1150011 SV40] promoter was cloned in front of a RFC25-conformal dCas9. Flanked by two [https://parts.igem.org/Part:BBa_K1150010 NLS], complemented with an [https://parts.igem.org/Part:BBa_K1150016 HA-Tag] for Western blot detections and fused to [https://parts.igem.org/Part:BBa_K1150002 KRAB] with a net 7 amino acid [https://parts.igem.org/Part:BBa_K1150013 linker], this resulted in the here-shown composite part.<br><br><br><br>[[File:Freiburg2013 Plasmid Cas9-KRAB-1.jpg|800px|thumb|left|<b>Fig. 1</b> Schematic overview of the dCas9-KRAB composite part with all features.]]</div> <br><br><br><br><br><br><br>
+
In this attempt, an [https://parts.igem.org/Part:BBa_K1150011 SV40] promoter was cloned in front of a RFC25-conformal dCas9. Flanked by two [https://parts.igem.org/Part:BBa_K1150010 NLS], complemented with an [https://parts.igem.org/Part:BBa_K1150016 HA-Tag] for Western blot detections and fused to [https://parts.igem.org/Part:BBa_K1150002 KRAB] with a net 7 amino acid [https://parts.igem.org/Part:BBa_K1150013 linker], this resulted in the here-shown composite part.<br><br>[[File:Freiburg2013 Plasmid Cas9-KRAB-1.jpg|800px|thumb|left|<b>Fig. 1</b> Schematic overview of the dCas9-KRAB composite part with all features.]]</div> <br><br><br><br><br><br><br>
−
<div align="justify"; margin-right:10px>Theoretically, this functional repressor can be directed towards almost any gene or promoter of interest, by harnessing a specifically designed [http://2013.igem.org/Team:Freiburg/Project/crrna#rnaimer RNAimer plasmid]. Two corresponding 30-basepair oligos, corresponding to the desired target, must therefore be annealed and inserted into the RNAimer plasmid. Team Freiburg 2013 provides an easy [http://2013.igem.org/Team:Freiburg/Project/crrna#design_tool designing tool] for such an attempt. Furthermore, [http://2013.igem.org/Team:Freiburg/Project/crrna#multiple_targeting multiple transcriptional regulation] can be achieved by an idempotent cloning strategy, utilizing various crRNA plasmids and co-transfecting this final vector with dCas9-effectors.</div>
+
<div align="justify"; margin-right:10px>Theoretically, this functional repressor can be directed towards almost any gene or promoter of interest, by harnessing a specifically designed [http://2013.igem.org/Team:Freiburg/Project/crrna#rnaimer RNAimer plasmid]. Two 30-basepair oligos, corresponding to the desired target, must therefore be annealed and inserted into the RNAimer plasmid. Team Freiburg 2013 provides an easy [http://2013.igem.org/Team:Freiburg/Project/crrna#design_tool designing tool] for such an attempt. Furthermore, [http://2013.igem.org/Team:Freiburg/Project/crrna#multiple_targeting multiple transcriptional regulation] can be achieved by an idempotent cloning strategy, utilizing various crRNA plasmids and co-transfecting this final vector with dCas9-effectors.</div>
  
 
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<partinfo>BBa_K1150021 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1150021 SequenceAndFeatures</partinfo>
 +
 +
==References==
 +
<small>
 +
Rosati, M. <i>et al.</i> (1991). Members of the zinc finger protein gene family sharing a conserved N-terminal module. Nucleic acids research 19, <i>5661-5667</i>. <br>
 +
Witzgall, R. <i>et al.</i> (1994). The Krüppel-associated box-A domain of zinc finger proteins mediates transcriptional repression. Proc Nati Acad Sci 91, <i>4514-4518</i>. <br>
 +
Birtle, Z. and Ponting, C. (2006). Meisetz and the birth of the KRAB motif. Bioinformatics 22, <i>2841-2845</i>. <br>
 +
Urrutia, R. (2003). KRAB-containing zinc-finger repressor proteins. Genome Biology 4, <i>4:231</i>.</small>

Latest revision as of 23:35, 4 October 2013

uniCAS Repressor (SV40 promoter)

SV40:HA-NLS-dCas9-Linker-KRAB-NLS:BGH
Function Transcriptional Repression
Use in Mammalian cells
RFC standard RFC 25
Organism Simian virus 40
Streptococcus pyogenes
Homo sapiens
Source iGEM Team Freiburg 2013
Feng Zhang, MIT
AG Weber, University of Freiburg
Submitted by [http://2013.igem.org/Team:Freiburg Freiburg 2013]
Krüppel-associated Box repressor domains - commonly termed as KRAB - are highly conserved polypeptide motifs and were first functionally characterized in 1991 (Rosati et al., 1991). As they constitute about one third of all human zinc finger transcription factors, key regulatory features in higher eukaryotic transcriptomics are suggested (Witzgall et al., 1994). Even in terms of tetrapod evolution, the role of their great abundance has been extensively discussed (Birtle, 2006). Even though KRAB minimal domains are usually no longer than 50-75 amino acids, their mechanism of function remains complex.

Common biochemical models suggest a key role in epigenetic silencing, by recruiting a scaffold of diverse proteins - amongst others histone deacetylases and histone methyltransferases (Urrutia, 2003). Til date in 2013, KRAB repressor domains were attached to several DNA binding proteins such as tetR, TAL effectors and dCas9 - thereby efficiently silencing gene expression downstream of desired target promoters.

In this attempt, an SV40 promoter was cloned in front of a RFC25-conformal dCas9. Flanked by two NLS, complemented with an HA-Tag for Western blot detections and fused to KRAB with a net 7 amino acid linker, this resulted in the here-shown composite part.

Fig. 1 Schematic overview of the dCas9-KRAB composite part with all features.







Theoretically, this functional repressor can be directed towards almost any gene or promoter of interest, by harnessing a specifically designed [http://2013.igem.org/Team:Freiburg/Project/crrna#rnaimer RNAimer plasmid]. Two 30-basepair oligos, corresponding to the desired target, must therefore be annealed and inserted into the RNAimer plasmid. Team Freiburg 2013 provides an easy [http://2013.igem.org/Team:Freiburg/Project/crrna#design_tool designing tool] for such an attempt. Furthermore, [http://2013.igem.org/Team:Freiburg/Project/crrna#multiple_targeting multiple transcriptional regulation] can be achieved by an idempotent cloning strategy, utilizing various crRNA plasmids and co-transfecting this final vector with dCas9-effectors.

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
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 4784
    Illegal SapI.rc site found at 4748

References

Rosati, M. et al. (1991). Members of the zinc finger protein gene family sharing a conserved N-terminal module. Nucleic acids research 19, 5661-5667.
Witzgall, R. et al. (1994). The Krüppel-associated box-A domain of zinc finger proteins mediates transcriptional repression. Proc Nati Acad Sci 91, 4514-4518.
Birtle, Z. and Ponting, C. (2006). Meisetz and the birth of the KRAB motif. Bioinformatics 22, 2841-2845.
Urrutia, R. (2003). KRAB-containing zinc-finger repressor proteins. Genome Biology 4, 4:231.