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

Figure 1 : Results of detection by qRT-PCR (qRT-PCR targeted to the loop portion of hairpin. Data was normalized with 25S rRNA. n=3 )

As a result, it was revealed that RNA expression from the conditional Gal1promoter was high in the presence of galactose, and repressed in the presence of glucose, as we expected. The constitutive GPD promoter had an RNA expression level even lower than from the conditional Gal1 promoter ( BBa_J63006 ) in Glucose (Glu) medium, a condition which suppresses expression from Gal1promoter.

Reference

For the sequence of the loop part used for Hairpin loop-dsRNA expression, the following paper was referred to.

　

[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 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]