Team:Dundee Schools/Results

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Dundee Schools

Results

We have characterised two synthetic devices: the Secretion of RNA Device (S.O.R.D.) and its partner RNA silencing agent, spiRNA.

S.O.R.D. is a fusion protein consisting of Hfq and OsmY. Hfq is an RNA binding protein found in many pathogenic bacteria. OsmY is a protein readily secreted by E. coli. By fusing these proteins we can theoretically create a device that secretes RNA from the cell. Our spiRNA is made from the expression systems, Ec-sRNA using an Hfq binding site from E. coli and Sma-sRNA using one from Serratia marcesens. Once the spiRNA is outside of the E. coli we want the target bacterium to take up the sRNA so that it can block a vital DNA sequence from being translated. In our proof of concept experiments detailed here we have targetted the flagellar subunit gene fliC. So in order to see if RNA interference would work in the treatment of bacterial infections, we ran some experiments on our BioBricks. First of all we ran numerous western blots to characterise SORD and see if they can be secreted. We then performed motility assays to see if our spiRNA works as we expect it to.

Western Blots

Figure 1: Western blots of our whole cell (A) and supernatant (B) samples of rhamnose induced OsmY-HA (C) expressing cultures.

We ran a western blot on our rhamnose induced OsmY-HA expressing cultures. We used OsmY-HA as a positive control to make sure OsmY is being secreted. First we added 500 ul overnight cultures into 50 ml of LB, we incubated the cultures at 37^oC until we reached an OD_600nm of 0.4. We followed this by adding different concentrations of rhamnose; 0.1%, 0.2%, 0.4% and 0.5% (v/v). We took a 1ml sample from the induced culture and measured the OD_600nmand then the samples from the cultures were spun down to separate the cells and supernatant. Through running both samples on a western blot we were able to determine whether; the protein was being expressed inside of the cell, and if the protein was being secreted outside of the cell. On the whole cell western blot, we can see that our OsmY-HA is being expressed in the presence of rhamnose (Fig. 1A). On the supernatant western blot, we can see traces of our OsmY-HA outside of the cell which shows that it is being secreted (Fig. 1B).

Figure 2: Western blots showing the whole cell samples of rhamnose induced OsmY-Hfq-HA fusion proteins with Hfq from E. coli (A) and Serratia marcesens (B). Western blot showing supernatant samples of both fusion proteins along with OsmY-HA and empty vector. (C) Western blots showing the supernatant samples of OsmY-HA, OsmY-Hfq E. coli and Osmy-Hfq serratia.

Once we knew our OsmY-HA was working and being secreted out of the cell we then proceeded to the next stage, we had to make sure that our fusion protein which was made up of the OsmY-Ha and Hfq from E. coli was also being secreted to test this we ran an identical experiment with cells expressing our OsmY-Hfq-HA constructs (Fig. 2A). The same had to be performed on the fusion containing the Hfq protein from Serratia marcesens and it can be seen that once again the fusion is being expressed when in the presence of the Rhamnose (Fig. 2B). Once we knew that the cells were being expressed we had to perform a western blot on the supernatant of the cells; this was done on the supernatant of each of the fusion proteins as well as the OsmY-HA and on the empty vector to make sure it’s not interfering with any of the results. From the results (Fig. 2C) we can see that both of the fusions are secreting our protein however the Serratia version seems to be more limited in comparison to the E. coli. We can also see that the OsmY-HA is also being secreted out of the cell very well which is to be expected.

Figure 3: Western blots showing the whole cell (A) and supernatant samples (B) of both our fusion proteins along with OsmY-HA and empty vector using a RNAP antibody

However, we had to make sure that our cells were not lysing in the process and releasing their contents into their surroundings; thus we once again performed a western blot, this time using a different anti-body used to check the presence of RNA polymerase which is only found inside of the cell. From the results we can see that each different cell was very rich in RNA polymerase, however we could not detect it in the supernatant (Fig. 3).

Motility Assays

Figure 4: Bar graph showing average colony area and standard deviation of replicates (A) and diagrams of relevant spiRNA constructs below (B). Photographs representative of motility assays of expressed above (C)

We created two spiRNA agents specific for fliC in E. coli using BBa_K1963004 and transformed these plasmids in to MG1655 E. coli cells. We ran motility assays on these strains to see if our spiRNA was binding to the fliC mRNA, preventing translation and inhibiting the ability to swim. We used non taregtting spiRNA on a plate as a positive control and compared it to spiRNA with a 24 bp sequence of CDS which is part of fliC and on spiRNA-RBS-CDS which also covered the ribosome binding site.

For our non-targeting spiRNA plate, we saw a larger colony size than the two other plates which showed a very small amount of growth. This indicates that our spiRNA containing a micC Hfq binding site from E. coli is successfully binding to the CDS and stopping the bacteria from swimming (Fig. 4A). We then worked out the average colony area. We got a larger average colony size for our empty vector than our two plates with our spiRNA. This again infers that our spiRNA is indeed working in the way that we expected. The spiRNA seems to work better for the CDS than the RBS-CDS as we got a smaller average colony size and a smaller standard deviation, however, we would need to run some further experiments to see if this is truly the case.

Looking at these results, it can be seen that we have managed to prove that our S.O.R.D. can be secreted and that our spiRNA from E. coli is able to affect the motility of bacteria. Some further experiments we could have ran would be to test our spiRNA containing a chiA Hfq binding site from Serratia marcesens to see if it also works at reducing bacteria motility. We know Hfq is an RNA binding protein but we don’t know how this function could be affect once it is fused together with OsmY. In future, we could run an experiment called an electrophoretic mobility shift assay to see if it can still bind to the spiRNA.