Difference between revisions of "Part:BBa K4917011"

 
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Starting position for shRNA ver10 in DWV is 2104
 
Starting position for shRNA ver10 in DWV is 2104
  
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===Usage and Biology===
 
===Usage and Biology===
  
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<table style = "border-collapse: collapse">
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    <tr>
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        <td style = "border: 1px solid black"><b>Name&nbsp;&nbsp;</b></td><td style = "border: 1px solid black"><b>Backbone/Plasmids used for GG assembly&nbsp;&nbsp;</b></td><td style = "border: 1px solid black"><b>Content&nbsp;&nbsp;</b></td><td style = "border: 1px solid black"><b>Description&nbsp;&nbsp;</b></td>
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    </tr>
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    <tr>
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        <td style = "border: 1px solid black">p207</td><td style = "border: 1px solid black">pRS304</td><td style = "border: 1px solid black">pGAL1_EGFP*siRNAv10_tCYC1</td><td style = "border: 1px solid black">Plasmid containing GFP sensor fused to siRNA target</td>
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    </tr>
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    <tr>
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        <td style = "border: 1px solid black">p222</td><td style = "border: 1px solid black">Golden Gate assembly</td><td style = "border: 1px solid black"><i>shRNA_v10</i></td><td style = "border: 1px solid black">shRNA expression vectors</td>
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    </tr>
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    <tr>
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        <td style = "border: 1px solid black">p224</td><td style = "border: 1px solid black">Golden Gate assembly</td><td style = "border: 1px solid black"><i>pPGK1-AGO1-tPGK1</i> + <i> pTEF1-DCR1-tPGK1</i></td><td style = "border: 1px solid black">Plasmid containing <i>Ago1</i> and <i>Dcr1</i> transcriptional units</td>
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    </tr>
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</table>
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===Yeast strains used in the study===
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<table style = "border-collapse: collapse">
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    <tr>
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        <td style = "border: 1px solid black"><b>Strain name&nbsp;&nbsp;</b></td><td style = "border: 1px solid black"><b>Genotype&nbsp;&nbsp;</b></td><td style = "border: 1px solid black"><b>Description&nbsp;&nbsp;</b></td>
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    </tr>
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    <tr>
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        <td style = "border: 1px solid black"><b>DOM90</b></td>
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<td style = "border: 1px solid black"><i>w303 MATa {leu2-3,112 trp1-1 can1-100<br />ura3-1 ade2-1 his3-11,15 bar1::hisG}[phi+]</i></td>
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<td style = "border: 1px solid black">Background strain</td>
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    </tr>
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    <tr>
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        <td style = "border: 1px solid black"><b>I63</b></td>
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<td style = "border: 1px solid black"><i>DOM90 Leu2::Ago1+Dcr1</i></td>
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<td style = "border: 1px solid black">Strain expressing Ago1 and Dcr1. It was used to transform<br />with vectors
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expressing shRNA and target sequences</td>
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    </tr>
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    <tr>
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        <td style = "border: 1px solid black"><b>I73</b></td>
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<td style = "border: 1px solid black"><i>DOM90 Leu2::Ago1+Dcr1 Trp1::GFP-Target_v10</i></td>
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<td style = "border: 1px solid black">Strain expressing Ago1, Dcr1, and GFP_V10 target</td>
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    </tr>
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    <tr>
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        <td style = "border: 1px solid black"><b>I83</b></td>
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<td style = "border: 1px solid black"><i>DOM90 Leu2::Ago1+Dcr1 Trp1::GFP-Target_v10 URA3::shRNA V10&nbsp;&nbsp;</i></td>
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<td style = "border: 1px solid black">Strain expressing of shRNA_V10 and its GFP_V10 target&nbsp;&nbsp;</td>
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    </tr>
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</table>
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 +
 
 +
===TEST the Effect of siRNA on GFP Expression===
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 +
To assess the efficiency of the siRNA we designed a sensor that consisted of GFP fused with the viral target sequence for the siRNA. If the siRNA is active and efficient, the mRNA will be degraded leading to no or decreased GFP fluorescence signal compared to cells without siRNA treatment.
 +
 
 +
===Flow cytometry reveals suppression of EGFP expression by siRNA induction in yeast===
 +
 
 +
Flow cytometry offers a means for efficient and precise evaluation of GFP expression at the individual cell level. In our experimental setup, we cultivated genetically modified yeast strains under tightly regulated environmental conditions. Subsequently, upon initiation of siRNA and GFP production, we subjected each yeast cell culture to flow cytometry. This method enables accurate measurement of any changes in GFP signal. A reduction in GFP fluorescence signifies the efficacy of the siRNA.
 +
We used flow cytometry to measure the GFP fluorescence intensities in yeast cultures 24h after inducing the expression of shRNA and the GFP-target sequence reporter. In the absence of shRNA expression, the GFP-reporter-containing cultures showed at least 3 times higher GFP fluorescence signal, confirming sufficient expression of the reporter protein (<b>Fig. 1A</b>). Additionally, we observed variations in fluorescence intensities among different GFP reporter constructs, suggesting that the viral sequence introduced into the 3'-UTR of the transcript may influence mRNA stability. Reduced mRNA stability, in turn, leads to impaired translation and decreased GFP fluorescence. For this reason, to compare the impact of the shRNA on GFP reporter expression, we normalized the GFP fluorescence data for each strain to the data obtained for its parent strain without shRNA expression (<b>Fig. 1B</b>). Interestingly, the anti-DWV shRNA V10 caused a drop in the GFP reporter fluorescence signal to background level (<b>Fig. 1B</b>).
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<html>
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<center>
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<figure>
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<img alt=""
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style="width: 800px"
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src="https://static.igem.wiki/teams/4917/wiki/contribution/shrna-v10.png">
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<figcaption><b>Figure 1. Expressing shRNA in the engineered RNAi-capable yeast enables testing of siRNA activities. (A)</b> Plot showing the mean GFP fluorescence intensities of a population of cells expressing shRNA v10 and the GFP reporters, measured by flow cytometry 24h after induction. <b>(B)</b> The GFP fluorescence data presented in panel <b>(A)</b> was normalized to cells expressing the GFP reporter, but not shRNA. The mean with standard deviation from 3 biological replicates for shRNA v10 is shown. </figcaption>
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</figure>
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</center>
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</html>
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===References:===
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Drinnenberg, I. A., Weinberg, D. E., Xie, K. T., Mower, J. P., Wolfe, K. H., Fink, G. R., & Bartel, D. P. (2009). RNAi in Budding Yeast. <i>Science, 326</i>(5952), 544–550. <a href="https://doi.org/10.1126/science.1176945">https://doi.org/10.1126/science.1176945</a>
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4917011 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4917011 SequenceAndFeatures</partinfo>

Latest revision as of 05:39, 12 October 2023


shRNA against DWV virus ver10

Starting position for shRNA ver10 in DWV is 2104

Usage and Biology

Name  Backbone/Plasmids used for GG assembly  Content  Description  
p207pRS304pGAL1_EGFP*siRNAv10_tCYC1Plasmid containing GFP sensor fused to siRNA target
p222Golden Gate assemblyshRNA_v10shRNA expression vectors
p224Golden Gate assemblypPGK1-AGO1-tPGK1 + pTEF1-DCR1-tPGK1Plasmid containing Ago1 and Dcr1 transcriptional units


Yeast strains used in the study

Strain name  Genotype  Description  
DOM90 w303 MATa {leu2-3,112 trp1-1 can1-100
ura3-1 ade2-1 his3-11,15 bar1::hisG}[phi+]		 Background strain
I63 DOM90 Leu2::Ago1+Dcr1 Strain expressing Ago1 and Dcr1. It was used to transform
with vectors expressing shRNA and target sequences
I73 DOM90 Leu2::Ago1+Dcr1 Trp1::GFP-Target_v10 Strain expressing Ago1, Dcr1, and GFP_V10 target
I83 DOM90 Leu2::Ago1+Dcr1 Trp1::GFP-Target_v10 URA3::shRNA V10   Strain expressing of shRNA_V10 and its GFP_V10 target  


TEST the Effect of siRNA on GFP Expression

To assess the efficiency of the siRNA we designed a sensor that consisted of GFP fused with the viral target sequence for the siRNA. If the siRNA is active and efficient, the mRNA will be degraded leading to no or decreased GFP fluorescence signal compared to cells without siRNA treatment.

Flow cytometry reveals suppression of EGFP expression by siRNA induction in yeast

Flow cytometry offers a means for efficient and precise evaluation of GFP expression at the individual cell level. In our experimental setup, we cultivated genetically modified yeast strains under tightly regulated environmental conditions. Subsequently, upon initiation of siRNA and GFP production, we subjected each yeast cell culture to flow cytometry. This method enables accurate measurement of any changes in GFP signal. A reduction in GFP fluorescence signifies the efficacy of the siRNA. We used flow cytometry to measure the GFP fluorescence intensities in yeast cultures 24h after inducing the expression of shRNA and the GFP-target sequence reporter. In the absence of shRNA expression, the GFP-reporter-containing cultures showed at least 3 times higher GFP fluorescence signal, confirming sufficient expression of the reporter protein (Fig. 1A). Additionally, we observed variations in fluorescence intensities among different GFP reporter constructs, suggesting that the viral sequence introduced into the 3'-UTR of the transcript may influence mRNA stability. Reduced mRNA stability, in turn, leads to impaired translation and decreased GFP fluorescence. For this reason, to compare the impact of the shRNA on GFP reporter expression, we normalized the GFP fluorescence data for each strain to the data obtained for its parent strain without shRNA expression (Fig. 1B). Interestingly, the anti-DWV shRNA V10 caused a drop in the GFP reporter fluorescence signal to background level (Fig. 1B).

Figure 1. Expressing shRNA in the engineered RNAi-capable yeast enables testing of siRNA activities. (A) Plot showing the mean GFP fluorescence intensities of a population of cells expressing shRNA v10 and the GFP reporters, measured by flow cytometry 24h after induction. (B) The GFP fluorescence data presented in panel (A) was normalized to cells expressing the GFP reporter, but not shRNA. The mean with standard deviation from 3 biological replicates for shRNA v10 is shown.

References:

Drinnenberg, I. A., Weinberg, D. E., Xie, K. T., Mower, J. P., Wolfe, K. H., Fink, G. R., & Bartel, D. P. (2009). RNAi in Budding Yeast. Science, 326(5952), 544–550. <a href="https://doi.org/10.1126/science.1176945">https://doi.org/10.1126/science.1176945</a>


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 66