Part:BBa_K2403005

Gal1 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 loop-dsRNA targeted to the nematodes expressed in budding yeast serving as feed. We used BBa_J63006 as a promoter in that case. This part was used to express hairpin-dsRNA in budding yeast by adding loop [1], [2] to BBa_J63006 . You can create plasmid transcribing dsRNA with hairpin loop which can be expressed in budding yeast by cleaving with restriction enzyme Not1, connecting sense part between the loop part and the promoter, further cleaving with a restriction enzyme, Hind 3 sand connecting antisense strand.

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.

The results of the detection by qRT-PCR were as follows

Medium　 Stock　　　plasmid　　　Average value　　SD Gal MKY13 WT Gal1p-AK1 3.61 0.98 Glu MKY13 WT Gal1p-AK1 0.11 0.00 Glu MKY13 WT GPD-Ak1 0.07 0.02

(The hairpin loop sequence was targeted for the RT-PCR. It was corrected with 25S rRNA. n=3)

Reference

For the sequence of the loop part used for Hairpin loop-dsRNA expression, the following paper was referred to. 　[1]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3786161/pdf/nihms-516215.pdf 　[2]http://genesdev.cshlp.org/content/18/19/2359.long

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 550
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 150
  
• 1000
  COMPATIBLE WITH RFC[1000]