Difference between revisions of "Part:BBa K1347003"
 
(2 intermediate revisions by the same user not shown)
Line 3: Line 3:
  
 
COI1 - COI1 is an E3 ligase that interacts with Jaz proteins (leading to their subsequent ubiquitination and degradation) after coronatin is introduced to the system. It is a system found in plants that is analogous to the auxin-TIR1-AID system studied by the Evry 2012 team.  
 
COI1 - COI1 is an E3 ligase that interacts with Jaz proteins (leading to their subsequent ubiquitination and degradation) after coronatin is introduced to the system. It is a system found in plants that is analogous to the auxin-TIR1-AID system studied by the Evry 2012 team.  
−
 
−
The 2014 Nevada iGEM team cloned this part into a yeast integrating vector and observed its expression and functionality.
 
  
  
Line 11: Line 9:
 
<img " src="https://static.igem.org/mediawiki/2014/c/c6/Auxin-and-Coronitine-System-Gif_small_2.gif" />           
 
<img " src="https://static.igem.org/mediawiki/2014/c/c6/Auxin-and-Coronitine-System-Gif_small_2.gif" />           
  
 +
<br>
 +
 +
The 2014 Nevada iGEM team cloned this part into a yeast integrating vector and observed its expression and functionality. An HA-tagged COI1 under the control of a pADH1 promoter was integrated into yeast and expression was observed via western blot with an anti-HA antibody.
 +
 +
<img src="http://www.matthewbrianhawn.com/jpg/western_blot_of_coi1">
 +
</br>
  
 +
After showing expression of the COI1 E3 ligase, a yeast 2u plasmid containing the Jaz6 degron fused to GFP was introduced to the same strain to see if coronatine inducible degradation of the Jaz6-GFP fusion by COI1  mediated ubiquitination could be achieved. The results (show below) prove that this system is capable of functioning in yeast and COI1 is active in the presence of coronatine.
 +
</br>
  
 +
<img src="http://www.matthewbrianhawn.com/jpg/western_blot_coronatine" width="200px" height="96px">
 
</html>
 
</html>
  

Latest revision as of 16:03, 1 November 2014

COI1

COI1 - COI1 is an E3 ligase that interacts with Jaz proteins (leading to their subsequent ubiquitination and degradation) after coronatin is introduced to the system. It is a system found in plants that is analogous to the auxin-TIR1-AID system studied by the Evry 2012 team.



The 2014 Nevada iGEM team cloned this part into a yeast integrating vector and observed its expression and functionality. An HA-tagged COI1 under the control of a pADH1 promoter was integrated into yeast and expression was observed via western blot with an anti-HA antibody.
After showing expression of the COI1 E3 ligase, a yeast 2u plasmid containing the Jaz6 degron fused to GFP was introduced to the same strain to see if coronatine inducible degradation of the Jaz6-GFP fusion by COI1 mediated ubiquitination could be achieved. The results (show below) prove that this system is capable of functioning in yeast and COI1 is active in the presence of coronatine.

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 1260
    Illegal BglII site found at 1590
    Illegal BamHI site found at 6
    Illegal XhoI site found at 967
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 826