Difference between revisions of "Part:BBa K2403004"

Revision as of 02:44, 2 November 2017

GPD promoter>loop for dsRNA

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

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 ( 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 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 ( BBa_J63006 ) and GPD promoter ( BBa_K517001 ) to characterize the RNA expression ability of these promoter parts.

As noted in 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 ( BBa_J63006 ) or the full length TDH3 promoter ( 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]Ines A. Drinnenberg, David E. Weinberg, Kathleen T. Xie, Jeffrey P. Mower, Kenneth H. Wolfe, Gerald R. Fink,and David P. Bartel (2009) RNAi in budding yeast nihms, 516215

[2]Alla Sigova, Nicholas Rhind1, and Phillip D. Zamore (2004) A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe Genes and Development, 18: 2359-2367

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]

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 <!-- Add more about the biology of this part here-->
 ==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.
+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 <i>Not</i>I, connecting the sense part between the promoter and loop parts, then cleaving with the restriction enzyme <i>Hind</i>III for ligating the antisense part.
 ==Characterization==