Difference between revisions of "Part:BBa K2403004"

 
Line 6: Line 6:
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
−
===Usage and Biology===
+
==Usage and Biology==
 +
In order to kill pine wood nematodes by feeding RNAi, we made hairpin-dsRNA targeted to the essential genes in the nematode, and expressed them in budding yeast provided as food. We used Gal1promoter (''' [https://parts.igem.org/Part:BBa_J63006  BBa_J63006] ''' ) as a promoter in that case. This part was used to express hairpin loop-dsRNA in budding yeast by adding loop [1], [2] to ''' [https://parts.igem.org/Part:BBa_J63006  BBa_J63006] '''  You can create a plasmid transcribing dsRNA with a hairpin loop which can be expressed in budding yeast by cleaving with the restriction enzyme NotI, connecting the sense part between the promoter and loop parts, then cleaving with the restriction enzyme HindIII for ligating the antisense part.
 +
 
 +
==Characterization==
 +
The expression level of dsRNA was assayed using Gal1 promoter (''' [https://parts.igem.org/Part:BBa_J63006  BBa_J63006] ''' ) and GPD promoter (''' [https://parts.igem.org/Part:BBa_K517001  BBa_K517001] ''' ) to characterize the RNA expression ability of these promoter parts.
 +
 
 +
As noted in ''' [https://parts.igem.org/Part:BBa_K517001  BBa_K517001] ''' , strong expression was not confirmed for this part. For aiming at strong expression in budding yeast, we recommend using the conditional Gal1 promoter (''' [https://parts.igem.org/Part:BBa_J63006  BBa_J63006] ''' ) or the full length TDH3 promoter (''' [https://parts.igem.org/Part:BBa_K530008  BBa_K530008] ''') instead of this promoter. Since this plasmid was weakly expressed, it was used as a negative control ('''[http://2017.igem.org/Team:Kyoto/Results  iGEM kyoto 2017 result]''').
 +
 
 +
==Reference==
 +
　For the sequence of the loop part used for Hairpin loop-dsRNA expression, refer to the following paper.
 +
　[1]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3786161/pdf/nihms-516215.pdf
 +
　[2]http://genesdev.cshlp.org/content/18/19/2359.long
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
  
 
<!-- -->
 
<!-- -->

Revision as of 09:46, 1 November 2017


GPD promoter>loop for dsRNA

This parts can be used for expressing dsRNA in Saccharomyces Cerevisiae.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 113
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]