Difference between revisions of "Part:BBa K2403004"
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<partinfo>BBa_K2403004 short</partinfo> | <partinfo>BBa_K2403004 short</partinfo> | ||
| − | This parts can be used for expressing dsRNA in Saccharomyces | + | This parts can be used for expressing dsRNA in <i>Saccharomyces cerevisiae</i>. |
<!-- 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 | + | 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== | ==Characterization== | ||
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==Reference== | ==Reference== | ||
For the sequence of the loop part used for Hairpin loop-dsRNA expression, refer to the following paper. | For the sequence of the loop part used for Hairpin loop-dsRNA expression, refer to the following paper. | ||
| − | [1] | + | <li>[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 <i>nihms</i>, 516215</li> |
| − | + | <li>[2]Alla Sigova, Nicholas Rhind1, and Phillip D. Zamore (2004) | |
| + | A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in <i>Schizosaccharomyces pombe</i> <i>Genes and Development</i>, 18: 2359-2367 </li> | ||
Latest revision as of 02:47, 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 113
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]