Difference between revisions of "Part:BBa K1150021"

Revision as of 19:39, 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
Backbone	pSB1C3
Organism	Human cytomegalovirus Streptococcus pyogenes Homo sapiens
Source	iGEM Team Freiburg 2013 Feng Zhang, Addgene Konrad Müller, 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 [http://2013.igem.org/Team:Freiburg 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 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.

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

@@ Line 29: / Line 29: @@
 <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>
-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>
+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>
 <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>