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Protein Expression and Purification

The gene for Hfq (plus His tag) was inserted into the suffix region of BBa_K314103, a lac inducible expression cassette constructed by Washington iGEM 2010, and the entire construct was simultaneously inserted into the pSB1K3.m1 plasmid backbone using the Anza enzyme system protocol. See agarose gel below for confirmation of construct.

The resulting plasmid was purified and transformed into Rosetta 2 pLySs cells and grown in LB broth with a 30μg/mL concentration of kanamycin. Initially, 3mL of a 10mL starter culture was used to expand to a 300mL culture, which was grown until OD600 = 0.82 before being induced with 0.8mM IPTG. Cells were taken at t = 0h, 1h, 2h, 12h, 14h, and 18h, before being harvested after 18h. Protein expression levels over time are shown below in an SDS-PAGE gel below; protein expression levels appear to be highest at 18h (see SDS-PAGE gel below). Hfq was purified using a Ni-NTA column, and the resulting concentration in fraction 1 totaled 0.31mg/mL.

Fluorescence Polarization

Fresh, pure Hfq was subsequently used in a fluorescence polarization (FP) assay to assess protein binding to sRNA-mRNA duplexes. Plates were read in the Biotek Synergy 2 plate reader with default FP settings and gain adjusted to 25, with each concentration of protein repeated in triplicate on two separate plates. 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. 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. Wells containing only sRNA compared to wells containing both sRNA and mRNA successfully showed that the RNA was hybridizing; 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.

Fluorescence polarization assay of mecA sRNA (right) and AUAUAUA oligo (left). Florescence was measured by 5’ fluorescein label on sRNA. sRNA and mRNA pairs were incubated with varying concentrations of staphylococcal Hfq protein. Wells contained fixed 0.5nM siRNA, 0.5nM mRNA, 25mM TRIS-HCl, 50mM NaCl, 0.5mM EDTA at pH 8. No trend is observed.

Fluorescence polarization assay of secA sRNA (left) and ddl sRNA (right). Florescence was measured by 5’ fluorescein label on sRNA. sRNA and mRNA pairs were incubated with varying concentrations of staphylococcal Hfq protein. Wells contained fixed 0.5nM siRNA, 0.5nM mRNA, 25mM TRIS-HCl, 50mM NaCl, 0.5mM EDTA at pH 8. No trend is observed.

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.