Difference between revisions of "Part:BBa K4917011"

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
p207	pRS304	pGAL1_EGFP*siRNAv10_tCYC1	Plasmid containing GFP sensor fused to siRNA target
p222	Golden Gate assembly	shRNA_v10	shRNA expression vectors
p224	Golden Gate assembly	pPGK1-AGO1-tPGK1 + pTEF1-DCR1-tPGK1	Plasmid 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

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-===Learn===
-These experiments lead to three important conclusions. Firstly, the presented work confirms the previously published results, demonstrating that introducing Dicer and Argonaute proteins to <i>S. cerevisiae</i> is sufficient to reconstitute RNAi response. Secondly, the experiments show that when viral sequences are introduced into the 3’-UTR of a GFP-coding transcript, they can be targeted by shRNA in yeast, offering a straightforward means to assess the efficacy of various shRNAs. Thirdly, we observed complete suppression on GFP-reporter expression with anti-DWV shRNA v10, making this strong candidate to move further into studies in bees to investigate possible off-target effects.
-We have also observed that the presence of the viral target sequence can influence GFP reporter expression, even in the absence of shRNA. As so far all the tested reporters still showed sufficiently high GFP expression to allow measuring of RNAi, further optimization of the length of the viral sequence introduced and the exact positioning of that sequence should be investigated.
-In the current experimental setup, both shRNA and the GFP reporter are expressed from <i>pGAL1</i> promoter, as was done in Drinnenberg <i>et al.</i> 2009. However, it would be better to use different inducible promoters for shRNA and GFP to enable the flexibility of expressing either one within the same strain. The current single-promoter design necessitates the use of different strains to capture the reporter fluorescence signal in the absence and presence of shRNA. Utilizing distinct promoters would enable the use of a single strain in which reporter expression can be independently induced from shRNA expression, simplifying the process of measuring uninterrupted reporter expression and streamlining the methodology.
-<b>?While the first round of experiments confirmed efficient RNAi by the tested shRNA v10, we were not able to detect quantitative differences in shRNA efficiencies, This limitation arises because all shRNAs led to maximal suppression of the GFP reporter(Fig. 8B, fluorescence dropping to GFP negative background level).?</b> In these strains shRNA v10 is expressed from a high copy number 2<i>μ</i>-plasmid, ensuring extremely high expression levels. In future experiments, we could consider expressing shRNA from a CEN plasmid, which exists in lower numbers within yeast cells, thereby resulting in reduced shRNA expression. Such an adjustment could potentially enhance the method's sensitivity to quantitative differences in shRNA efficiency.
 ===References:===