Part:BBa_K1941005:Experience
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Applications of BBa_K1941005
Activation of CYC1 promoter:
A) Activation design. scCYC1_c3 construct with MS2 recruits its cognate RNA-binding protein MCP fused to VP64 and targets the region c3 of CYC1 promoter. dCas9 allows the activator complex to move and bind the c3 sequence, then the activator domain VP64 stimulates gene expression.
B) Histogram of FACS results (Relative Fluorescence). The same strains as used in the experiment shown in figure 2B) were grown overnight in 2% glucose selective medium and the GFP fluorescence was measured with FACS (LSRII Snoopy). The data were normalized to report to the basal level, which corresponds to cells single-transfected with the plasmid carrying CYC1-GFP. As in figure 2B), the fluorescence emitted upon activation is more than two times higher than the basal level.
C) Overnight Tecan micro-plate reader (RFU/OD). Cells were grown overnight in 2% glucose in a 96 well plate. Every 5 min, growth and GFP fluorescence were measured using OD390 values and relative fluorescence units (RFU), respectively. The experiments were performed in triplicate. The blue line corresponds to not-transfected cells (W303), which don’t express GFP. The red line represents cells single-transfected with CYC1-GFP. Lastly, the green line represents cells additionally transfected with all the activation components: the scRNA MS2, the fused activator protein MCP-VP64 and dCas9. Activation exceeds more than two times the basal level of GFP expression. We can also suppose that incubating cells longer (e.g. until an OD390 of 1.4-1.5) would induce an even stronger activation.
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