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Demonstrate - Fluorescence Polarization

We set out to determine whether or not protein Hfq is required for sRNA-mRNA hybridization in Staphylococcus aureus. Our FP assay results suggest that Hfq does not bind to the sRNA binding region we synthesized based on results from the literature, and therefore does not effect sRNA-mRNA hybridization in Staphylococcus aureus. When the protein concentration was then increased 100 fold and tested with only the AUAUAUA oligo reported in the literature to bind strongly to Hfq, with no significant difference between wells without protein. Therefore, we concluded that sRNA mediated gene silencing is possible in Staphylococcus species without the inclusion of Hfq on the vector delivered to target cells.

Demonstrate - qPCR Assay

To test the efficacy of gene silencing in vivo, we designed a qPCR assay around the target genes of interest (secA, mecA, ddl), Hfq, and two reporter genes: 6-phosphofructokinase (6-PFK) and acetyl-CoA carboxylase (ACC). These reporters were chosen as they are part of crucial metabolic function, and expression levels are expected to be constant, even if gene silencing is occurring at other sites. Furthermore, these genes are genetically distant from the genes of interest, and thus have no probability of being expressed on a polycistronic mRNA. We successfully demonstrated that the assay is highly sensitive and produces linear results using a block of DNA containing each element of interest. Note the figures plot copy number of the genes of interest against fluorescence emission data.